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https://openalex.org/W3179839637	https://iris.uniroma1.it/bitstream/11573/1562347/3/Pascarella_SARS%e2%80%90CoV%e2%80%902%20B.1.617_2021.pdf	English	null	SARS‐CoV‐2 B.1.617 Indian variants: Are electrostatic potential changes responsible for a higher transmission rate?	Journal of medical virology	2,021	cc-by	4,179	J M d Vi l 2021 1 6 il li lib /j l/j \| 1 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. © 2021 The Authors. Journal of Medical Virology Published by Wiley Periodicals LLC Stefano Pascarella and Massimo Ciccozzi contributed equally to this study. SARS‐CoV‐2 B.1.617 Indian variants: Are electrostatic potential changes responsible for a higher transmission rate? Stefano Pascarella1 \| Massimo Ciccozzi2 \| Davide Zella3 \| Martina Bianchi1 \| Francesca Benedetti3 \| Domenico Benvenuto2 \| Francesco Broccolo4 \| Roberto Cauda5 \| Arnaldo Caruso6 \| Silvia Angeletti7 \| Marta Giovanetti8 \| Antonio Cassone9 1Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2Medical Statistic and Molecular Epidemiology Unit, University of Biomedical Campus, Rome, Italy 3Department of Biochemistry and Molecular Biology, Institute of Human Virology and Global Virus Network Center, University of Maryland School of Medicine, Baltimore, Maryland, USA 1Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2Medical Statistic and Molecular Epidemiology Unit, University of Biomedical Campus, Rome, Italy 3Department of Biochemistry and Molecular Biology, Institute of Human Virology and Global Virus of Medicine, Baltimore, Maryland, USA 4Department of Clinical Medicine and Prevention, University of Milano‐Bicocca, Milan, Italy 5Istituto Clinica di Malattie Infettive, Università Cattolica del Sacro Cuore, Rome, Italy 6Department of Microbiology and Virology, Spedali Civili, Brescia, Italy 7Unit of Clinical Laboratory Science, University Campus Bio‐Medico of Rome, Rome, Italy 8Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil 9Center of of Genomics, Genetics and Biology, Siena, Italy Correspondence Massimo Ciccozzi, Medical Statistic and Molecular Epidemiology Unit, University of Biomedical Campus, Via Álvaro del Portillo, 21, 00128 Rome, Italy. Email: M.ciccozzi@unicampus.it Silvia Angeletti, Unit of Clinical Laboratory Science, University Campus Bio‐Medico of Rome, Via Álvaro del Portillo, 21, 00128 Rome, Italy. Email: S.Angeletti@unicampus.it J Med Virol. 2021;1–6. Received: 21 May 2021 \| Revised: 11 June 2021 \| Accepted: 8 July 2021 DOI: 10.1002/jmv.27210 R E S E A R C H A R T I C L E SARS‐CoV‐2 B.1.617 Indian variants: Are electrostatic potential changes responsible for a higher transmission rate? Stefano Pascarella1 \| Massimo Ciccozzi2 \| Davide Zella3 \| Martina Bianchi1 \| Francesca Benedetti3 \| Domenico Benvenuto2 \| Francesco Broccolo4 \| Roberto Cauda5 \| Arnaldo Caruso6 \| Silvia Angeletti7 \| Marta Giovanetti8 \| Antonio Cassone9 1Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2Medical Statistic and Molecular Epidemiology Unit, University of Biomedical Campus, Rome, Italy 3Department of Biochemistry and Molecular Biology, Institute of Human Virology and Global Virus Network Center, University of Maryland School of Medicine, Baltimore, Maryland, USA 4Department of Clinical Medicine and Prevention, University of Milano‐Bicocca, Milan, Italy 5Istituto Clinica di Malattie Infettive, Università Cattolica del Sacro Cuore, Rome, Italy 6Department of Microbiology and Virology, Spedali Civili, Brescia, Italy 7Unit of Clinical Laboratory Science, University Campus Bio‐Medico of Rome, Rome, Italy 8Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil 9Center of of Genomics, Genetics and Biology, Siena, Italy Received: 21 May 2021 \| Revised: 11 June 2021 \| Accepted: 8 July 2021 DOI: 10.1002/jmv.27210 Revised: 11 June 2021 \| Accepted: 8 July 2021 R E S E A R C H A R T I C L E ree energy difference (ΔΔG) between wild‐type and mutant structure. Negative values indicate destabilization. brational entropy difference (ΔΔS) between wild‐type and mutant structure. Positive values indicate increased flex 1 \| INTRODUCTION England). Here, we focus on biochemical and biophysical changes conferred to the B.1.617+ VUI by the P478K and E484Q mutations. We then compare these changes with other VOCs to establish whether and to what extent those amino acid changes can influence the interaction of the spike protein with ACE2, thus potentially af- fecting SARS‐CoV‐2 transmission and immune‐escape properties. The coronavirus disease 19 (COVID‐19), caused by the new cor- onavirus SARS‐CoV‐2, continues to spread worldwide, with more than 163 million infections and about 3.5 million deaths as of May 17, 2021 (www.who.int). To fight this dreadful disease, several safe and efficacious vaccines against SARS‐CoV‐2 are being used with remarkable effectiveness in some countries and limited availability in others. In particular, the capacity of some countries to halt SARS‐ CoV‐2 spread is still limited due to inadequate resources and vac- cination infrastructures.1,2 In this scenario, several SARS‐CoV‐2 variants have been identified and have become a global threat. Some of them have been classified as variants of concern (VOCs) due to their mutations in the S1 subunit of the spike (S) protein, particularly in its receptor‐binding domain (RBD).3–5 One of them, identified as B.1.1.7 (α), also known as the UK variant, bears a substitution of asparagine with tyrosine on the position 501 and deletion of two amino acids in the position 69–70 of the S1 subunit. This variant has quickly spread in several European countries to become globally dominant.5 Other VOCs have been isolated in South Africa and Brazil and have been studied for their enhanced contagiousness and re- sistance to neutralization by antibodies from convalescent and vaccine‐recipient subjects6–8 (Table 1). Quite recently, a new variant under investigation (VUI) has been isolated from Maharashtra, India, in a setting of the highly diffusive epidemic with devastating pro- portions. This variant, identified as B.1.617, carries several non‐ synonymous mutations. Two of them, the E484Q (or the P478K) and the L452R, are located in the RBD region, and they are critical sites for the binding with ACE2. Initial data suggest these mutations could confer increased transmission and immune evasion.9–11 Currently, B.1.617 comprises three subvariants, B.1.617.1‐3, with different distribution of the mutations P478K and E484Q (Public Health Abstract Lineage B.1.617+, also known as G/452R.V3 and now denoted by WHO with the Greek letters δ and κ, is a recently described SARS‐CoV‐2 variant under investigation first identified in October 2020 in India. As of May 2021, three sublineages labeled as B.1.617.1 (κ), B.1.617.2 (δ), and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R, and P681R. Here, we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the receptor‐binding domain (RBD) of the spike protein. Enhanced RBD‐EP is particularly noticeable in the B.1.617.2 (δ) sublineage, which shows multiple replacements of neutral or negatively charged amino acids with positively charged amino acids. We here hy- pothesize that this EP change can favor the interaction between the B.1.617+ RBD and the negatively charged ACE2, thus conferring a potential increase in the virus transmission. wileyonlinelibrary.com/journal/jmv PASCARELLA ET AL. \| 2 K E Y W O R D S B.1.617 δ and κ variants, electrostatics potential changes, SARS‐CoV‐2 2 \| METHODS To perform a robust analysis three different three‐dimensional structures of SARS‐CoV‐2 spike glycoprotein have been downloaded from Protein Data Bank with the following characteristics: 1. SARS‐CoV‐2 RBD in complex with neutralizing antibody CC12.3 (PDB code: 6XC4, chain A), 2. SARS‐CoV‐2 RBD in complex with ACE2 (PDB code: 6M0J, chain E), 3. SARS‐CoV‐2 RBD in complex with ACE2‐B0AT1 (PDB code: 6M17, chain F). The DynaMut server12 has been used to predict the impact of mutations on protein stability analyzing the folding free energy (ΔΔG) and the vibrational movement (ΔΔS), two crucial character- istics of the function and the molecular recognition of the protein. DynaMut automatically provides also results from DUET analysis, a method that combines two complementary approaches (mCSM and SDM) in a consensus prediction of ΔΔG.13 DUET results also have been considered in combination with DynaMut for the character- ization of the mutants. PyMol (PyMol, version 2.4) suite was utilized for in‐silico mutagenesis and its Adaptive Poisson Boltzmann Solver (APBS) plugin14 has been used to calculate the electrostatic potential TABLE 1 Results of DynaMut and DUET analysis Mutant sites DynaMut DUET ΔΔG (kcal/mol)a ΔΔS (kcal/mol K)b ΔΔG (kcal/mol)a 6M17 6M0J 6XC4 6M17 6M0J 6XC4 6M17 6M0J 6XC4 N501Y 0.089 −0.272 0.089 −0.162 0.138 −0.312 −0.297 −0.474 −0.391 K417N −0.399 −0.651 −1.174 0.487 0.659 0.347 −0.513 −1.295 −0.990 K417T −0.152 −0.566 −0.832 0.198 0.507 0.193 −0.854 −1.343 −1.119 E484K 0.087 −0.101 −0.109 −0.075 0.171 0.336 0.656 0.128 0.348 E484Q 0.399 −0.644 −0.755 −0.084 0.151 0.189 0.099 −0.438 −0.319 L452R −0.417 −0.319 −0.462 0.150 0.059 −0161 −0.548 −0.741 −0.661 T478K −0.334 1.003 0.257 0.111 −0.385 −0.152 0.109 −0.024 0.037 aF diff (ΔΔG) b t ild t d t t t t N ti l i di t d t bili ti TABLE 1 Results of DynaMut and DUET analysis 3.1 \| Protein stability In silico prediction of the mutation impact on the RBD stability has been carried out with DynaMut. Three alternative RBD structures denoted by the PDB codes 6M17, 6M0J, and 6XC4 have been tested. These structures display small differences in the conformation of loops, especially in the one inside the receptor‐binding motif (RBM). According to the parameters of our in silico experiments, the output of DynaMut for mutant sites within loops differs depending on the starting loop conformation. For this reason, we used a normalized procedure, whereby the same mutation has been tested in each of the three different RBD structures. The effect on stability, de‐ or stabilization, has been evaluated following a majority criterion over the results of DynaMut and DUET. Detailed results have been re- ported in Table 1. TABLE 1 Results of DynaMut and DUET analysis PASCARELLA ET AL. 3 of the wild‐type and VOCs SARS‐CoV‐2 spike receptor‐binding do- main (RBD) and evaluate potential differences in terms of molecular interaction with ACE2 receptor. The results have been reported within a range between −5 and +5 kT/e. consistently assigns a destabilizing effect. For the majority rule, this mutation should be considered destabilizing (Table 1). Also, position 417 is within the interface α‐helix where Lys interacts with ACE2 Asp30. However, in this case, our results predict that both mutants (K417N and K417T) could destabilize the protein and increase local flexibility. Similarly, Glu484 is in an interfacial loop and interacts with ACE2 Lys31. Our data predict that mutation E484K is stabilizing, although data from DynaMut and DUET do not match. E484Q exerts a destabilizing effect, again with no strong consensus from the two methods. We note that glutamic acid is a polar, negatively charged, hydrophilic amino acid while lysine is a basic, charged and partly aliphatic amino acid, and its ε‐amino group often participates in hy- drogen bonding, salt bridges, and covalent interactions. Both mu- tants E484K and E484Q would likely increase local molecular flexibility. L452 is in a short β‐strand, and it is exposed to the solvent. Apparently, it does not interact directly with ACE2. Mutation L452R is predicted to be destabilizing with increased local flexibility. Posi- tion 478 is in a loop in the proximity of the ACE2 although not in direct contact with it. Mutation T478K is stabilizing and is predicted to decrease local protein flexibility. 484, and 501 are within the RBM, containing residues that bind to ACE2. In contrast, mutant position 417 is located outside the mo- tif.15 According to our analysis, the mutations in positions 417, 484, and 501 might increase the spike binding affinity with the ACE2 receptor. In particular, the Tyr replacing Asn501 may form an aromatic interaction with ACE2 Tyr41, a hydrogen bond with ACE2 D38, and a potential cation–π interaction with ACE2 Lys353. In addition, substitutions of Glu484 with Lys or Gln may form hydro- phobic interactions to Ile472, Gly482, Phe486, Cys488, and Tyr489. Our data also indicate that replacing Lys or Gln with Glu484 abol- ishes the interfacial salt bridge between Glu484 and ACE2 Lys31. Due to the fact that Lys417 is solvent‐exposed and forms salt‐bridge interactions with Asp30 of ACE2, the replacement of Thr/Asn with Lys417 could abolish this interaction. Moreover, although the mutations in positions 452 and 478 are within the receptor‐binding motif, our analysis does not show a direct interaction with ACE2. Glu484 is replaced by the uncharged residue Gln. In contrast, the B.1.617.2 (δ) lineage shares the same mutation in position 452, but it has another mutation in position 478 where the neutral residue Thr changes to the positively charged Lys. The presence of two positively charged residues in the variant B.1.617.2 (δ) increases the positive electrostatic potential surface (Figure 2). 4 \| DISCUSSION Given that SARS‐CoV‐2 variants are characterized by evolutionary and genetic changes accumulated in the genome, the use of new and improved phylodynamic techniques for the study of how epidemio- logical, immunological, and evolutionary processes contribute to shaping viral phylogenies is extremely important and useful. Here, using a well‐structured software workflow, we provide evidence of a strong system able to carry out a quick, systematic, and reproducible screening of the SARS‐CoV‐2 genome isolates. Protein mutations were identified by scanning high‐quality SARS‐CoV‐2 genomes var- iants downloaded from the GISAID databank.16 We reasoned that the most likely health‐threatening properties of SARS‐CoV‐2 VOC rely on fine biochemical and biophysical changes that eventually impact the RBD interaction with the ACE2 receptor present on the host's cell surface. We thus characterized B.1.617+ SARS‐CoV‐2 VUI using in‐silico methods capable of predicting the effect of mutations on S‐RBD protein stability and its electrostatic potential. The 3.2 Starting with position 501 within a loop in the RDB interacting with the ACE2 receptor, we note that the mutation N501Y does not show any clear structural effect as the negative ΔΔG values are very close to 0.0 kcal/mol in DynaMut. At variance with DynaMut, DUET The entire set of mutations in positions 417, 452, 478, 484, and 501 are in the spike RBD at the interface with the ACE2 N‐terminal helix (Figure 1). However, we note that the mutant positions 452, 478, FIGURE 1 Comparison between the wild‐type and variant spike receptor‐binding domains (RBDs). Protein surface is colored according to the electrostatic potential. Color scale ranges from −5 kT/e (red) to +5 kT/e (blue) as reported by the bar under the wild‐type RBD. Position of the mutant sites is indicated by a circle and an attached label. Red arrows mark the area of increased positive potential in the RBD Indian variants FIGURE 1 Comparison between the wild‐type and variant spike receptor‐binding domains (RBDs). Protein surface is colored according to the electrostatic potential. Color scale ranges from −5 kT/e (red) to +5 kT/e (blue) as reported by the bar under the wild‐type RBD. Position of the mutant sites is indicated by a circle and an attached label. Red arrows mark the area of increased positive potential in the RBD Indian variants FIGURE 1 Comparison between the wild‐type and variant spike receptor‐binding domains (RBDs). Protein surface is colored according to the electrostatic potential. Color scale ranges from −5 kT/e (red) to +5 kT/e (blue) as reported by the bar under the wild‐type RBD. Position of the mutant sites is indicated by a circle and an attached label. Red arrows mark the area of increased positive potential in the RBD Indian variants 4 PASCARELLA ET AL. 4 3.3 \| Electrostatic potential We note that a major, global effect of the mutations characterizing the Indian variants is represented by the alteration of the RBD surface electrostatic potential. In particular, in the lineage B.1.617.1 (κ) the uncharged and hydrophobic residue Leu452 changes to the positively charged residue Arg, and the negatively charged residue FIGURE 2 Ribbon model of the interface between ACE2 (deep teal) and receptor‐ binding domain (RBD) (orange). Sidechains of relevant residues are displayed as stick models and labeled. The two mutations at the RBD sites 417 and 484 have been simultaneously displayed FIGURE 2 Ribbon model of the interface between ACE2 (deep teal) and receptor‐ binding domain (RBD) (orange). Sidechains of relevant residues are displayed as stick models and labeled. The two mutations at the RBD sites 417 and 484 have been simultaneously displayed PASCARELLA ET AL. 5 B.1.617+ mutations have been investigated by comparing most of the already known mutations of previously reported VOCs.17 To further justify our assumption, we note that the intensely investigated D614G substitution of the spike protein, early reported in Italian isolates,18–20 and subsequently attributed with increased virus transmissibility21 was found to enhance the protein torsional ability and potentially affecting its stability.21 Regarding the commonly denominated Indian variants, these constitute the new SARS‐CoV‐2 lineage B.1.617, which is actually composed of a family of three subvariants, namely B.1.617.1 (κ), B.1.617.2 (δ), and B.1.617.3. This lineage, which emerged in India in October 2020, has since spread to other countries, particularly in the United Kingdom, in settings with a high density of resident immigrants from India. Data from Public Health England registry shows that the subvariant B.1.617.2 (δ) has become epidemiologically prevalent.22 Due to its subvariants com- position, the variant has been designed as B.1.617+ by the WHO.23 mutations and how the interactions between mutations and local cellular microenvironment influence the clinical outcome and the transmission dynamics of this virus. ORCID Stefano Pascarella http://orcid.org/0000-0002-6822-4022 Massimo Ciccozzi http://orcid.org/0000-0003-3866-9239 Davide Zella http://orcid.org/0000-0001-5576-5770 Martina Bianchi http://orcid.org/0000-0002-2547-5999 Francesca Benedetti http://orcid.org/0000-0001-8720-090X Domenico Benvenuto http://orcid.org/0000-0003-3833-2927 Silvia Angeletti http://orcid.org/0000-0002-7393-8732 Marta Giovanetti http://orcid.org/0000-0002-5849-7326 Antonio Cassone http://orcid.org/0000-0002-0434-0761 Our data indicate that most of the mutants are predicted to destabilize the RBD structure, except for E484K and T478K. It is conceivable that destabilization alters binding affinity to ACE2 and to neutralizing antibodies. As noticed above, the influence of the mutations on the spike surface properties is evident in the B.1.617+ lineage, particularly in the B.1.617.2 (δ) lineage subvariant, where the positive electrostatic surface potential is markedly increased. This may favor RBD interaction with the negatively charged ACE2,26 which, in turn, would then increase affinity for the ACE2 receptor. All of these changes have the potential to eventually modify infectivity, pathogenicity, and virus spread. Regarding differential binding to neutralizing antibodies, previous studies suggested that VOCs RBD changes in the electrostatic potential surface could induce SARS‐CoV‐2 antibody evasion, and even single amino acid changes that marginally affect ACE2 affinity could greatly influence nAbs affinity.27 Several factors have been demonstrated to affect the impact of VOCs. For example, it has been observed an increased effect at pH associated with nasal secretions (from 5.5 to 6.5).28 For this reason, additional experiments both in vitro and in vivo are needed to establish the biological significance of SARS‐CoV‐2 ACKNOWLEDGMENTS The authors are grateful to Alice Vinciguerra for her precious assistance. We also would like to thank all the authors who have kindly deposited and shared genome data on GISAID. Stefano Pascarella has been partially supported by the Sapienza grant RP120172B49BE24. Open access funding enabled and organized by Projekt DEAL. AUTHOR CONTRIBUTIONS Stefano Pascarella and Martina Bianchi performed the in‐silico ana- lysis and proposed data interpretation. Antonio Cassone and Mas- simo Ciccozzi proposed addressing the issue of this study, wrote the initial draft of the manuscript, and contributed to writing the final version of the manuscript. Stefano Pascarella, Martina Bianchi, An- tonio Cassone, Massimo Ciccozzi, Davide Zella, Francesca Benedetti, Francesco Broccolo, Roberto Cauda, Arnaldo Caruso, Silvia Angeletti, Marta Giovanetti, and Domenico Benvenuto discussed the data, their interpretation, and contributed to writing the final version of the manuscript. All Authors have seen and approved the initial draft and the revised, final text of the manuscript. A dichotomic behavior is observed with variant B.1.617.2 (δ). In contrast, it lacks mutation E484Q which is present in the other two lineages and was initially suspected to confer a certain degree of resistance to antibody neutralization. In contrast, this subvariant has a mutation at site 478 where a lysine replaces the proline. Of note, variant B.1.617.2 (δ) is indeed characterized by a major shift toward increased positive electrostatic potential because of three amino acid changes from negative or neutral to clearly positive charge, as shown in Section 3. DATA AVAILABILITY STATEMENT Subvariants B.1.617.1 (κ) and B.1.617.3 have both the double mutations E484Q and the L452R. Although initially believed to en- hance the antibody‐escape potential, it has been shown that the B.1.617.1 (κ) subvariant is pretty neutralized by the majority of sera from convalescent individuals and all sera from vaccinated sub- jects.24 Nonetheless, this variant has been shown to be more pa- thogenic than the B.1.1.7 (α) variant in an experimental model of SARS‐CoV‐2 infection in hamsters.25 The data that support the findings of this study are openly available in bioRxiv at https://submit.biorxiv.org/. 6 \| 6 \| 6 PASCARELLA ET AL. 19. Korber B, Fischer WM, Gnanakaran S, et al. Tracking changes in SARS‐CoV‐2 spike: evidence that D614G increases infectivity of the COVID‐19 virus. Cell. 2020;182:812‐827. 6. Voysey M, Clemens S, Madhi SA, et al. 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https://openalex.org/W4389731129	http://j.uniyar.ac.ru/index.php/vyrgu/article/download/1462/1239	Russian	null	Historical trends in the study of the phenomenon of wisdom in psychological science	Vestnik Âroslavskogo gosudarstvennogo universiteta imeni P.G. Demidova. Seriâ, Gumanitarnye nauki/Vestnik Âroslavskogo gosudarstvennogo universiteta im. P. G. Demidova. Seriâ, Gumanitarnye nauki	2,023	cc-by	4,152	Historical trends in the study of the phenomenon of wisdom in psychological science N. N. Mekhtikhanova1 1P. G. Demidov Yaroslavl state University, 14 Sovetskaya str., Yaroslsvl 150003, Russian Federation DOI: 10.18255/1996-5648-2023-4-620-630 Research article Full text in Russian © Yaroslavl State University, 2023 This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: 10.18255/1996-5648-2023-4-620-630 The article briefly describes the traditions of the study of wisdom in the philosophical period of the development of psychology. Wisdom was associated with moral virtue and a good solution to life’s problems. This served as the basis for the modern understanding of the phenomenon. The chronology of the study of wisdom in foreign and domestic psychology is presented. It is shown that there are two main approaches in research – implicit and explicit. The reasons for the long-term neglect of the phenomenon in psychology and the revival of interest in modern science are analyzed. Keywords: wisdom; philosophy; theoretical and empirical studies of wisdom; implicit and explicit approaches; difficulties in the study of wisdom; reasons for the revival of interest in wisdom the revival of interest in wisdom FORMATION ABOUT AUTHORS Mekhtikhanova, Natalya N. E-mail: natnik1@list.ru Cand. Sc. (Psychology), Associate Professor Vestnik YarGU. Seriya Gumanitarnye nauki. 2023. Vol. 17, No 4 journal homepage: http://j.uniyar.ac.ru/index.php/vyrgu Vestnik YarGU. Seriya Gumanitarnye nauki. 2023. Vol. 17, No 4 journal homepage: http://j.uniyar.ac.ru/index.php/vyrgu PSYCHOLOGY Исторические тенденции в изучении феномена «мудрость» в психологической науке 10.18255/1996-5648-2023-4-620-630 Научная статья 159.9.072.59 Полный текст на русском языке Кратко описываются традиции изучения мудрости в философский пе риод развития психологии. Мудрость связывалась с нравственной доброде телью и хорошим решением жизненных проблем. Это послужило основой для современного понимания феномена. Излагается хронология изучения мудрости в зарубежной и отечественной психологии. Показано, что суще ствуют два основных подхода в исследованиях – имплицитный и эксплицит ный. Анализируются причины долгого игнорирования феномена в психоло гии и возрождения интереса в современной науке. Ключевые слова: мудрость; история развития; теоретические и эмпирические исследования мудрости; имплицитный и эксплицитный подходы; трудности в исследованиях мудрости; причины возрождения интереса к мудрости INFORMATION ABOUT AUTHORS FORMATION ABOUT AUTHORS Mekhtikhanova, Natalya N. E-mail: natnik1@list.ru Cand. Sc. (Psychology), Associate Professor 620 Вестник ЯрГУ. Серия Гуманитарные науки. 2023. Том 17, № 4 веб-сайт: http://j.uniyar.ac.ru/index.php/vyrgu ПСИХОЛОГИЯ ИНФОРМАЦИЯ ОБ АВТОРАХ ИНФОРМАЦИЯ ОБ АВТОРАХ Мехтиханова, Наталья Николаевна E-mail: natnik1@list.ru Кандидат психологических наук, доце Мехтиханова, Наталья Николаевна E-mail: natnik1@list.ru Кандидат психологических наук, доцент © ЯрГУ, 2023 Почти полвека назад психологи В. Клейтон и Дж. Биррен писали, что в технологических обществах гораздо больше внимания уделяется когнитивным навыкам, которые нужны для продуктивной деятельно сти, чем личной мудрости, необходимой для хорошей жизни [1]. За про шедшие годы материальная жизнь, несомненно, изменилась к лучшему, но стали ли люди счастливее? Изучение мудрости с древности имело цель помочь людям понять свою природу, причины страданий и благополучия, но даже эта высоко гуманная цель не облегчила разработку научного под хода к мудрости. Путь, который прошла проблема мудрости в психологии, оказался очень непростым. По сей день отсутствует единство в понима р , Статья открытого доступа под лицензией CC BY (https://creativecommons.org/licenses/by/4.0/) 621 Н. Н. Мехтиханова нии, определении и объяснении феномена мудрости. Отчасти это можно объяснить противоречивой природой мудрости. Отметим некоторые са мые важные парадоксы [2]. нии, определении и объяснении феномена мудрости. Отчасти это можно объяснить противоречивой природой мудрости. Отметим некоторые са мые важные парадоксы [2]. Мудрость субъективна и объективна. В объективных документах, где описываются мудрые люди и их действия, отражается субъективный опыт человека. Мудрость предполагает как знание, так и неуверенность; основана на знаниях, но формируется в неопределенности. Мудрые люди знают, что они не знают многое, и действуют соответствующим образом: или ищут новую информацию, или принимают, что некоторые результаты по своей сути неопределенны и непостижимы. Мудрость предполагает как потери, так и выгоду. Иногда мы обре таем мудрость через неудачу, через утрату иллюзий, привязанностей. И эти потери могут быть источником если не радости, то хотя бы личност ного роста. Мудрость требует перемен через принятие. Принимая обстоятель ства такими, как они есть, мудрость позволяет возникнуть новым возмож ностям и перспективам. Мудрость своевременна и вечна. Мудрость предполагает саморазви тие через самоотверженность. Мудрость требует и личной отстраненно сти, и в то же время вовлеченности в заботу о коллективном благополучии. Мудрость предполагает как действие, так и активное разумное бездей ствие. Эмоции имеют центральное значение для мудрости, но эмоцио нальная отрешенность обязательна. Мудрый поступок в одном контек сте может быть чистой глупостью в другом [3]. В российской психологической науке к этим парадоксам можно доба вить ещё один: все хотят знать, что такое мудрость, но считают её слиш ком загадочной и неуловимой, опасаются начать разработку проблемы и явно недостаточно изучают её. Наконец, нам нужна мудрость, чтобы понять мудрость [1]. ИНФОРМАЦИЯ ОБ АВТОРАХ Многие исследователи проблемы мудрости пришли к её изучению в силу определенных личных обстоятельств. Так, В. Клейтон, была пора жена различиями между своими близкими при решении сложных жиз ненных проблем [1]. Кто-то был удивлен тайной, окружающей фено мен мудрости. Для кого-то открытие мудрости началось с мудрых книг, а кто-то получил своевременный и конструктивный мудрый совет. Мудрость является неотъемлемой частью наших имплицитных пред ставлений о людях, а сам термин часто используется в обыденном обще нии. Народная мудрость нашла отражение в устном творчестве, в сказках, песнях, поговорках, благодаря чему передавалась из поколения в поколе ние. Мудрецами считались философы, уважаемые старцы, люди, имею щие богатый жизненный опыт и всесторонние знания. 622 Исторические тенденции в изучении феномена «мудрость» … Для Святого Фомы было важно различать веру и разум, что имело следствием дифференциацию науки (в те времена – философии) и религии. Поэтому «добродетельное поведение или практическая мудрость (Фронезис) стала в большей мере областью церкви, в то время как изучение философской мудрости (Со фия) наряду с изучением научного знания (Эпистема) стала областью на учного знания. Это разделение можно считать одной из причин долгого невнимания психологической науки к феномену мудрости» [9, с. 251]. стойной целью для каждого человека и приближает его к Богу. Эти идеи, в частности, прослеживаются в работах Святого Августина, Фомы Ак винского и других философов и богословов [4]. Для Святого Фомы было важно различать веру и разум, что имело следствием дифференциацию науки (в те времена – философии) и религии. Поэтому «добродетельное поведение или практическая мудрость (Фронезис) стала в большей мере областью церкви, в то время как изучение философской мудрости (Со фия) наряду с изучением научного знания (Эпистема) стала областью на учного знания. Это разделение можно считать одной из причин долгого невнимания психологической науки к феномену мудрости» [9, с. 251]. Развитие европейской культуры и науки периодов Возрождения и Но вого времени не стало принципиальным прорывом в изучении мудрости. Секуляризация знания подразумевала научный метод, логику, матема тику. Это противоречило глубинным основаниям мудрости – созерцанию, интуиции, откровению. Мудрость по-прежнему считалась недоступной для научного изучения, её достижение рассматривалось как божий дар, что не позволяло ввести её познание в круг научных задач. Тем не менее некоторые философы развивали идеи Аристотеля о мно гообразии видов мудрости, её роли в жизни людей. Так, у Декарта му дрость варьировалась от земной до божественной. Мудрость имеет раз ные уровни и может развиваться от здравого смысла до вдохновенного суждения и понимания. Декарт рассматривал её как гармоничное допол нение к научному знанию: знание включало накопление фактов, тогда как мудрость организовывала и интерпретировала эти факты [4]. Вместе с тем возможность понимания и развития мудрости продол жала оцениваться философами очень по-разному. И. Кант, например, пи сал о необходимости любви к мудрости, ее почитанию, но недосягаемости в обычной жизни. Как мы уже отмечали, «долгое время право на суждения о мудрости принадлежало философии и религии, а психология скромно ожидала своей очереди в познании этого феномена. Даже когда в XIX в. произо шло революционное отделение психологии от философии, а романтизм и трансцендентализм возродили интерес к мудрости в гуманитарных науках, обращение к исследованию мудрости было не выгодно для пси хологической науки. Как справедливо замечает Робинсон, расцветающая самостоятельная психология искала признания в области естественных наук. Исторические тенденции в изучении феномена «мудрость» … Одними из первых книг о мирской мудрости, как принято считать, являются книги визиря фараона Исси Птаха Хотепа, написанные где- то между 2870 и 2675 годами до н. э. Книги, посвященные его сыну, со держали инструкции, описывающие правильное поведение, принципы достойной жизни, и, вероятно, использовались для изучения философии в школах [4]. В дальнейшем феномен мудрости стал объектом для обсуждения в различных религиозных учениях: в Библии, Коране, трудах Конфуция, буддийских трактатах и др. Эти работы в значительной мере повлияли на понимание мудрости в философии [4–8]. В различные периоды истории обсуждение проблемы мудрости ве лось, главным образом, в философских и религиозных учениях. Первона чально, до Сократа, философы наделяли мудростью только божественных существ, а для людей она была недостижимым и непостижимым идеалом, к которому необходимо стремиться. В произведениях Платона мудростью наделяется живой человек – Сократ, а это значит, что она является элементом человеческой жизни. Как известно, Сократ, признанный мудрейшим из людей, предпочитал знание о добродетели знанию о физической вселенной. Платон, ученик Сократа, писал, что мудрость понимается им как знание о наилучших действиях по отношению к себе и другим, как знание того, что полез но для каждого. В работах Платона и Аристотеля мудрость предстаёт как идеальная интеграция знания и действия, ума и добродетели. Му дрость неодинакова по своим целям, происхождению, структуре. Прежде всего, это философская мудрость – София – знание ради самого знания. Аристотель ввел важное различие между теоретической мудростью – «episteme» и тем, что он назвал «phronesis» – практической мудростью. Он утверждал, что «теоретическая мудрость» связана с объяснительным знанием в «непрактичной» области (например, физика или метафизика), тогда как знание о том, как хорошо жить, как выбирать этические средства для достижения правильных целей в повседневной жизни, в конкретных ситуациях, является «практической мудростью». Практическая мудрость по Аристотелю предполагает, что человек должен быть морально добро детельным, стремиться к процветанию как личному, так и общечелове ческому. Мудрость является врожденной способностью, но её можно раз вить, постоянно практикуясь [1, 4]. Эти и другие идеи древних ученых послужили основой для форму лирования основных проблем психологии мудрости, которые продолжа ют разрабатываться в современных исследованиях [8]. Период средневековья в европейской христианской традиции озна меновался обращением вновь к идеальной, недостижимой мудрости, ка ковой является божественная мудрость. Стремление к ней является до 623 Н. Н. Мехтиханова стойной целью для каждого человека и приближает его к Богу. Эти идеи, в частности, прослеживаются в работах Святого Августина, Фомы Ак винского и других философов и богословов [4]. Исторические тенденции в изучении феномена «мудрость» … Мудрость как объект исследования казалась чем-то очень далеким, исключительным, непостижимым: она была, как считалось, недоступна эмпирическим замерам, мудрецы, как потенциальные испытуемые встре чались редко. Не в должной мере оценивалась и роль мудрости, ее функ ции в обыденной жизни каждого человека и общества» [9, с. 251]. 624 Исторические тенденции в изучении феномена «мудрость» … Исторические тенденции в изучении феномена «мудрость» … И только почти пять тысячелетий спустя после первых упоми наний мудрости психологи стали её изучать. Первоначально пробле ма мудрости была частью более широких теорий. Это, главным образом, теории развития личности, и мудрость считалась идеальной, конеч ной точкой жизненного пути. С. Холл в фундаментальном труде «Ста рение» рассуждал о слабостях и возможностях старения. В этот период человеческой жизни в силу биологических законов происходит сниже ние интеллектуальных и других функций, но мудрость компенсирует это, что позволяет человеку успешно и благополучно существовать. Кро ме того, мудрость в зрелом возрасте помогает извлечь моральные уроки из прошлой жизни, формирует спокойствие и беспристрастность [3]. В концепции личности К. Г. Юнга описывается «архетип мудрого ста рого человека». Мудрец помогает в решении сложных жизненных про блем, хранит и развивает культуру и традиции [10]. Мудрость считалась фактором и результатом конструктивного реше ния последнего психосоциального конфликта, или эго-целостности в кон цепции Э. Эриксона. Это особый, высший уровень развития человека, когда он понимает смысл жизни, свое положение в мире и т. п. Самы ми главными детерминантами появления мудрости являются саморазви тие и самотрансценденция. Благодаря им человек признает, что, несмотря на возрастные потери, можно открыть много нового в жизни [7]. «Вместе с тем, до 1980-х гг. все обсуждения феномена мудрости носи ли теоретический характер. Важнейшим шагом на пути психологических исследований мудрости стали работы Вивиан Клейтон. По всеобщему признанию, Клейтон – психолог, положивший начало всем последующим эмпирическим исследованиям мудрости. Значимость ее работ высоко оценили Р. Дж. Стернберг, П. Балтес, Д. Смит и др. По их мнению, утверж дение В. Клейтон, что мудрость может быть законной темой для эмпири ческого изучения, было большим прорывом в науке [9, с. 252]. Будучи очень наблюдательной, В. Клейтон заметила множество осо бенностей поведения некоторых своих родственников: они были эмо ционально устойчивы, скромны, добры, принимали решения, полезные для всех. Все эти характеристики она обозначила как мудрость, которая включает три составляющих: когнитивную, аффективную и рефлексив ную. Наличие первой из них позволяет считать мудрость другим образо ванием, нежели интеллект. Мудрость связана в большей мере с личностью, чем с интеллектом и с познавательными способностями. В. Клейтон так же считала, что возраст и жизненный опыт не являются залогом обрете ния мудрости [7]. Первоначально публикации по психологии мудрости были единичны ми, но далее произошел лавинообразный рост исследований. Мудрость стала полноправным объектом изучения в нашей науке. В 1990 году вы 625 Н. Н. Мехтиханова Н. Н. Мехтиханова ходит книга под редакцией Р. Стернберга «Мудрость: ее природа, проис хождение и развитие». Она стала первым обобщающим трудом многих эмпирических и тео ретических исследований, выполненных на тот момент времени. Преди словие к сборнику, написанному главным редактором, содержит описание общих подходов к изучению мудрости: а) философского, б) психологиче ского, который, в свою очередь делится на имплицитный и эксплицитный [1]. «Имплицитные» исследования изучают неявные теории мудрости обычного человека и основаны на непрофессиональном, житейском пони мании термина. Эти теории содержат верования, представления людей о мудрости и особенностях мудрого человека. Житейские представления вносят определенный вклад в научное изучение мудрости. Вторая группа – эксплицитные исследования, в них ученые пыта ются применять традиционные эмпирические методы. Главная цель этих работ – сформировать теоретическую модель мудрости. Явные те ории мудрости являются производными от экспертного мнения и знаний. Они часто основаны на теоретических моделях развития, например, тео рии психосоциального развития Эриксона, теориях когнитивного разви тия. Акцент в явных теориях мудрости ставится на познавательных и по веденческих выражениях мудрости [9, с. 254]. Примером эксплицитных теорий могут служить разработки П. Бал теса и его коллег, выполненные в Институте Макса Планка в Берлине. Теория основывается на когнитивистском подходе, который доминиро вал в психологии 1990-х гг. Мудрость понималась как экспертная система знаний и суждений, выдающаяся способность понимания фундаменталь ных проблем, касающихся смысла и течения [7]. Теория группы Балтеса подверглась критике, но многих ученых это подтолкнуло к созданию собственных моделей мудрости. Появились мно гочисленные обзоры исследований, выполненных в зарубежной психоло гии. Так, Р. Троубридж в 2005 году в результате анализа работ, выполнен ных за последние 25 лет, сделал следующие выводы: Теория группы Балтеса подверглась критике, но многих ученых это подтолкнуло к созданию собственных моделей мудрости. Появились мно гочисленные обзоры исследований, выполненных в зарубежной психоло гии. Так, Р. Троубридж в 2005 году в результате анализа работ, выполнен ных за последние 25 лет, сделал следующие выводы: «1. Мудрость была вновь введена в современную науку через создание эмпирических исследований. «1. Мудрость была вновь введена в современную науку через создание эмпирических исследований. 2. Мудрость операционализируется многими различными способами. Это уникальная конструкция, связанная с эмоциональным и когнитив ным интеллектом или любой другой личностной чертой, но отличающаяся от них. 3. Хотя многие исследования были направлены на определение му дрости, этот вопрос так и не решен. 3. Хотя многие исследования были направлены на определение му дрости, этот вопрос так и не решен. 4. Мудрость – это область, в которой пожилые люди могут преуспеть. 4. Н. Н. Мехтиханова Мудрость – это область, в которой пожилые люди могут преуспеть. 5. Мудрость как опыт и как эвристическое отношение к смыслу и по ведению жизни богата возможностями. 4. Мудрость – это область, в которой пожилые люди могут преуспеть. 5. Мудрость как опыт и как эвристическое отношение к смыслу и по ведению жизни богата возможностями. 5. Мудрость как опыт и как эвристическое отношение к смыслу и по ведению жизни богата возможностями. 626 626 Исторические тенденции в изучении феномена «мудрость» … 6. Самым большим прогностическим фактором для мудрости, как было установлено, является профессия в такой области, как клиническая пси хология и пастырская деятельность. 6. Самым большим прогностическим фактором для мудрости, как было установлено, является профессия в такой области, как клиническая пси хология и пастырская деятельность. 7. Были обнаружены положительные корреляции между мудростью и удовлетворенностью жизнью, а также особыми личностными чертами, такими как открытость, социальный интеллект и моральное мышление. 7. Были обнаружены положительные корреляции между мудростью и удовлетворенностью жизнью, а также особыми личностными чертами, такими как открытость, социальный интеллект и моральное мышление. 8. Исследователи обнаружили, что мудрость можно развить, кон сультируясь с другими или давая человеку время обдумать ситуацию» [9, с. 255]. 8. Исследователи обнаружили, что мудрость можно развить, кон сультируясь с другими или давая человеку время обдумать ситуацию» [9, с. 255]. В 2019 г. в г. Торонто (Канада) были приглашены исследователи му дрости из многих стран с целью оценить доминирующие методологиче ские, теоретические и эмпирические позиции и выработать общую пози цию по проблеме мудрости. Итогом работы Рабочей группы по мудрости стала общая модель мудрости [11–12]. За время, прошедшее с момента встречи ученых, количество публикаций по проблеме мудрости в зару бежной психологии продолжает неуклонно расти. В 2019 г. в г. Торонто (Канада) были приглашены исследователи му дрости из многих стран с целью оценить доминирующие методологиче ские, теоретические и эмпирические позиции и выработать общую пози цию по проблеме мудрости. Итогом работы Рабочей группы по мудрости стала общая модель мудрости [11–12]. За время, прошедшее с момента встречи ученых, количество публикаций по проблеме мудрости в зару бежной психологии продолжает неуклонно расти. Отечественная психология сравнительно недавно обратилась к про блеме мудрости. Прежде всего ученые описывали, анализировали, дела ли выводы об исследованиях, выполненных за рубежом [5–6, 10, 13–20]. В целом оценки этих работ являются достаточно высокими. Тем не менее определенный скепсис по отношению к возможности изучать мудрость, особенно эмпирически, имеется. Н. Н. Мехтиханова Российские ученые настаивают на чрез мерной сложности эмпирического изучения мудрости, говорят об ее ис ключительности, малой распространенности, предупреждают о подмене феномена мудрости некими мудроподобными решениями [9]. Несомненно, что такие проблемы существуют, но действие в любом случае лучше бездействия, и не надо искать «абсолютных мудрецов», как справедливо отмечает В.В. Нуркова и ее коллеги [15]. В частности, они ставят задачу изучения мудрых решений и советов в контексте пси хологии развития, используя методы исследования автобиографической памяти. Исследования Н. Н. Мехтихановой, С. Э. Дровосекова выполне ны с помощью психометрически проверенных диагностических методик [13, 19]. Работы А. С. Огнева, Е. А. Никитиной посвящены изучению им плицитных представлений о мудрости и также основаны на эмпириче ских методах [14, 16]. Таким образом, можно констатировать недостаточное внимание рос сийских ученых к исследованию проблем психологии мудрости. Причин для такого положения дел можно найти немало. Как нами уже было отмечено, прежде всего «…отсутствие надежных методов яв ляется одной из причин затруднений в изучении мудрости в российской науке, но, как показал анализ и зарубежных, и отечественных работ, 627 Н. Н. Мехтиханова не только это, а еще целый ряд обстоятельств обусловили долгое забвение проблемы мудрости в психологической науке. не только это, а еще целый ряд обстоятельств обусловили долгое забвение проблемы мудрости в психологической науке. Среди причин длительного отсутствия интереса у психологов к проблеме мудрости также следует отметить многогранность, сложность и даже некую иллюзорность, неуловимость самого конструкта. Многие ученые, как и обычные люди, видели и видят в мудрости человеческий идеал, который практически не достижим, не объективируем, а значит, и не изучаем. Также сама психология, стремясь стать самостоятельной наукой, мак симально дистанцировалась от философии и ее понятий, а тема мудрости на протяжении тысячелетий как раз была в центре внимания философов и религиозных деятелей. Более того, западная культура была склонна приравнивать любое знание к техническому или научному знанию и за крывать глаза на другие виды знания, такие как мудрость» [9, с. 255]. Как показал проведенный анализ работ по истории изучения мудро сти, обращение ученых к этой проблеме в конце ХХ в. было не случайным, а обусловленным рядом причин. По мнению Д. А. Леонтьева, «идея мудро сти утратила актуальность, обесценилась — общественный запрос на му дрость стал размываться и ослабевать. Это во многом связано с наступле нием «массовой культуры» на просто культуру, с омассовлением» [6, с. 20]. Н. Н. Мехтиханова В ХХ веке произошло осознание относительности всех ценностных си стем, но понимание в обществе того, что социальная жизнь, коммуника ция оказываются невозможны в отсутствие разделяемых всеми основа ний, побудило обратиться к мудрости. Кроме того, возникло «осознание необходимости преодолеть интеллектуальную фрагментарность, восста новить целостность человека и стимулировать рост интереса к такому хо листическому и междисциплинарному понятию, как “мудрость”» [6, с. 21]. В. В. Нуркова с соавторами отмечают, что «рост катастроф, рисков, не определенности в кризисном обществе возрождает гуманитарный идеал эффективного мышления, результаты которого должны соответствовать общечеловеческим ценностям и жизненному опыту субъекта. Надежды возлагаются на мудрость — «благоразумие, соединенное с добродетелью» [16, с. 68–69]. Кроме того, и сама логика развития науки заставляет исследовате лей обратиться к феномену мудрости «в связи с изменившейся структу рой развития психологии, а именно с выдвижением в центр исследования когнитивных процессов, постановкой новой проблемы социального интел лекта, а также повышением внимания к поздним периодам жизни чело века» 10, с. 17]. Таким образом, в современную эпоху мудрость как вид знания, кото рый гибко приспосабливается к сложным ситуациям, оказалась в центре внимания. В течение примерно пятидесяти лет эмпирических исследова 628 Исторические тенденции в изучении феномена «мудрость» … ний мудрости ученые создали многочисленные концепции мудрости, осно ванные на «тщательных обзорах литературы». Тем не менее каждая группа исследователей читала литературу по-своему и в конечном итоге разра ботала интересные, но часто взаимно противоречащие концепции. 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https://openalex.org/W2090933140	https://europepmc.org/articles/pmc3733502?pdf=render	English	null	Joint action on monitoring injuries in Europe (JAMIE)	Archives of public health	2,012	cc-by	4,441	Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 ARCHIVES OF PUBLIC HEALTH Open Access RESEARCH © 2012 Rogmans; 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. Correspondence: w.rogmans@eurosafe.eu.com European Association for Injury Prevention and Safety Promotion (EuroSafe), Amsterdam, The Netherlands Joint action on monitoring injuries in Europe (JAMIE) W. H. J. Rogmans* Abstract Background: Injuries due to accidents or violence constitute a major public health problem globally and also within the 27 member states of the European Union (EU-MSs). In spite of the magnitude and the severity of the problem, injury surveillance systems are not yet sufficiently well developed to accurately quantify the burden of injuries on individuals, health services and society in the EU-region. Much of the injury information generated up until now is not comparable between countries, and not between registers, due to the lack of harmonised methodology and classification. The hospital sector provides the best setting for collecting information as this information relates to the most severe cases (while less severe cases are treated by family doctors of school nurses for instance) and information can be obtained easily on a large number of cases at low cost (while surveys are expensive and suffering serious deficiencies as regards the specificity of data obtained). The WHO-International Classification of Diseases and its derivative classification on external causes of injuries provide the proper tools for standardised data collection on injuries treated within the health sector. Project Objectives: JAMIE project aims at having by 2015 a common emergency departmental-based surveillance system for injury prevention in operation in all MS. Such a system should report on external causes of injuries due to accidents and violence as part of the Community Statistics on Public Health. The project will build on previous work on injury data exchange initiated by the European Commission (EC) and a number of EU-member states, which resulted to the so called Injury Data Base hosted by the EC. In order to make injury data collection affordable for countries to collect and to have a greater number of countries joining the data exchange efforts, JAMIE envisages to have a relatively limited set data elements being collected in a representative sample of emergency departments in countries, while collecting in a few departments deeper information on the circumstances of the injury event. Background interacting with the body in amounts or rates that exceed the threshold of human tolerance. In some cases (e.g. drowning and frostbites) injuries result from sudden lack of essential agents such as oxygen or heat" [2]. Injuries due to accidents or violence constitute a major public health problem globally and also within the 27 member states of the European Union (EU-MSs). Within the EU-region, each year injuries result in an estimated 256,000 deaths, 7,200,000 hospital admissions, a further 34,800,000 emergency department (ED) attendances and 18,600,000 other medical treatments, totalling 60,600,000 medical treatments [1]. In spite of the magnitude and the severity of the prob- lem, injury surveillance systems are not yet sufficiently well developed to accurately quantify the burden of in- juries on individuals, health services and society in the EU-region. Injuries are commonly defined as being "caused by acute exposure to physical agents such as mechanical en- ergy, heat, electricity, chemicals, and ionizing radiation What information is available tends to focus on fatal injuries. So also most of the targets of EU- and national policies with respect to road traffic safety, safety at work, consumer safety, violence and suicide prevention have been primarily focused on the reduction of deaths. How- ever, deaths are only one aspect of the total injury Page 2 of 5 Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 states to develop national injury surveillance and report- ing systems. The Council also invited the Commission to establish a Community-wide injury surveillance sys- tem to make the information contained in the database easily accessible to all stakeholders. problem; for every person killed, many more are ser- iously and permanently disabled and many more again suffer minor, short-term disabilities. Not only the costs of injury mortality but also the costs of morbidity are immense, not only in terms of lost economic opportun- ity and demands on national health budgets, but also in terms of personal suffering. Over the past few years, several projects have been initiated by the Commission to develop such an ex- change of injury data at the EU level based on data col- lected in accident and emergency departments at general hospitals. Background In 2010, thirteen EU-MSs were rou- tinely collecting injury data in a sample of hospitals and delivering these data to the Commission, in line with the Injury Data Base (IDB) methodology [5]. This method- ology allowed countries to collect accident and injury data from a representative sample of emergency depart- ments in the participating countries and to use a stan- dardised classification for coding the circumstances of the injury-event and its outcome (as a derivative classifi- cation of the WHO-International Classification of Exter- nal Causes of Injuries, ICECI [6]). It is now increasingly acknowledged that deaths are only one measure of the magnitude of the injury prob- lem. In fact, in many EU Member States deaths in road traffic or for instance at work, have been declining over the last several decades due in part to improvements in medical care (prompt emergency response, early diagno- sis, and treatment capabilities) as well as to advances in road and vehicle design and in technology. In contrast to this development, non-fatal injuries are increasing in importance in terms of both societal and economic costs as well as loss of productivity. Consequently, there is a growing need for separate targets related to the reduc- tion of non-fatal injuries, in particular those leading to permanent impairments. Such indicators are gradually being introduced at the EU level for target setting and for measuring progress in policies for road safety and for health and safety at work. IDB-system complements existing data sources such as the routine causes of death statistics, hospital dis- charge registers and data sources specific to injury areas, including road accidents and work related accidents. Currently 13 countries are still collecting injury data in line with this methodology, although some only for a selected population, e.g. by collecting information in pediatric hospitals only or by collecting only injuries due to home and leisure accidents. Much of the injury information generated up until now is not comparable between countries, and not be- tween registers, due to the lack of harmonised method- ology and classification. Injury surveillance in the EU – and in most MSs – can be characterized as operating on an incomplete puzzle of data sources that only provides a notion of the complete picture but lacks important details [3]. A new three year injury-data collection initiative d f h d In order to encourage continuation of these data collec- tion efforts and the inclusion of the remaining EU-MSs in EU-wide injury data exchange, the European Com- mission, DG for Health and Consumers (DG Sanco) is currently funding a public health Joint Action on injuries known as JAMIE (Joint Action on Monitoring Injuries in Europe) from 2011–2013. The project is being endorsed by the Ministries of Health in 22 EU-MSs (see list in Appendix I). Each of these Ministries have desig- nated a internal unit or a national competent organisa- tion to contribute to the JAMIE-project and to test the feasibility of introducing an pragmatic and sustainable injury surveillance in their country. It is obvious that the hospital sector provides the best setting for collecting information as this information relates to the most severe cases (while less severe cases are treated by family doctors of school nurses for in- stance) and information can be obtained easily on a large number of cases at low cost (while surveys are expensive and suffering serious deficiencies as regards the specifi- city of data obtained). Technological developments in medical administration and data linkage, also offers new opportunities for recording information that is also relevant for injury prevention. The JAMIE project aims at having by 2015 a com- mon hospital-based surveillance system for injury pre- vention in operation in all MS. Such a system should report on external causes of injuries due to accidents and violence as part of the Community Statistics on Public Health. Background However these challenges can be met by using health based data that provide the ‘cement’ to glue the jigsaw pieces of understanding the injury field to- gether and will serve as common denominator for all policy sectors and MSs. EU-policy response In response to the growing but incomplete evidence on the scale of the injury problem the EU Council issued in 2007 a Recommendation on the Prevention of Injuries and the Promotion of Safety [4] that urged all member Up to mid 2014 a series of actions are envisioned that will lay the groundwork for a genuine EU-wide injury in- formation system through the following steps: Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Page 3 of 5 previously implemented as the Injury Data Base or IDB); and 2. a new Minimum Data Set (MDS). – within twelve months criteria for IDB data quality, such as representativeness and comparability (taking into account the differences in the organisation of emergency services in countries), will be clearly defined, in line with the respective requirements of the European Statistical System (ESS); and – within twelve months criteria for IDB data quality, such as representativeness and comparability (taking into account the differences in the organisation of emergency services in countries), will be clearly defined, in line with the respective requirements of the European Statistical System (ESS); and – within twelve months criteria for IDB data quality, such as representativeness and comparability (taking into account the differences in the organisation of emergency services in countries), will be clearly defined, in line with the respective requirements of the European Statistical System (ESS); and – over the years 2012–2014 an increasing number of countries will be assisted in collecting and injury data in accordance with these quality criteria for uploading in the EU central Injury Database (IDB), hosted by the European Commission, DG Health and Consumers. – within twelve months criteria for IDB data quality, such as representativeness and comparability (taking into account the differences in the organisation of emergency services in countries), will be clearly defined, in line with the respective requirements of the European Statistical System (ESS); and – over the years 2012–2014 an increasing number of countries will be assisted in collecting and injury data in accordance with these quality criteria for uploading in the EU central Injury Database (IDB), hosted by the European Commission, DG Health and Consumers. Decision about the content of these datasets has been based on a review of the existing literature and practices around the world and discussion between experts on the feasibility of collecting such data whilst ensuring consistency as far as possible with existing classification systems. The JAMIE-approach The proposed Full Data Set (FDS) is in line with the original IDB-classification as it has been implemented over the past few years in 13 countries [1]. The categor- ies of external cause variables included in the proposed FDS (see Appendix II) reflect the responsibility of the major agencies and bodies involved in prevention in many countries, including the prevention of injuries in specific domains such as road traffic, consumer products Whilst from an EU perspective the main focus of JAMIE is to develop a system to enable the incidence of home and leisure injuries to be monitored by an increasing number of countries, it is clear that there are many other needs for injury data to support policy development, ap- praisal, prevention and research in relation to injuries from defective products, or resulting from violence or road traffic accidents, to name but a few. With not too much effort and within the existing resources provided through JAMIE it would be possible to provide tools to answers most of these questions at individual member state or EU level. Big samples of MDS are needed for ac- curate estimates for incidences (not only for home and leisure injuries). Additional in-depths information on ex- ternal causes, circumstances, locations, activities, and products are needed for developing preventive measures, guiding and evaluating prevention programmes. Table 1 The single screen minimum data set: external factors of injury Intent: Selected mechanisms: –Accidental injury –Fall –Deliberate (intentional) self harm –Cut/pierce –Assault related injury –Road traffic –Unknown intent –Poisoning –Burn/scald –Other –Unknown Location (setting): Selected activities: –Workplace –Sport –Road (incl. pavement) –Work –Educational establishment –Other –Leisure area (incl. sport/fitness, shops, pubs, clubs and recreation) –Unknown –Home (includes garden) –Other (includes health facilities) –Unknown Table 1 The single screen minimum data set: external factors of injury Intent: Selected mechanisms: –Accidental injury –Fall –Deliberate (intentional) self harm –Cut/pierce –Assault related injury –Road traffic –Unknown intent –Poisoning –Burn/scald –Other –Unknown Location (setting): Selected activities: –Workplace –Sport –Road (incl. pavement) –Work –Educational establishment –Other –Leisure area (incl. EU-policy response The proposed MDS-Injury is now presented in Table 1 as a single screen reporting tool. This data set serves as complementary items to the data elements related to the 'nature of injury' and 'body part affected' that are already collected in all emergency departments in a routinely manner. – over the years 2012–2014 an increasing number of countries will be assisted in collecting and injury data in accordance with these quality criteria for uploading in the EU central Injury Database (IDB), hosted by the European Commission, DG Health and Consumers. By the end of the action (mid 2014), in at least 26 countries National IDB Data Administration Centre (’NDA’) shall be designated by the competent national or regional authority and be in full operation, and at least 22 countries shall collect IDB data in a sustainable man- ner. Four more countries are expected to have imple- mentation plans in place endorsed by the competent authorities. The MDS is designed to be implemented in many differ- ent ways, including the creation of de novo computer sys- tems, the adaptation of existing systems, or using check boxes in existing or new paper based clinical records. The simple MDS for Europe reflects the need to meet many different agendas in relation to data collection, such as supporting the development of high level EU and MS injury indicators, being feasible to implement in MSs with wide variation in existing practice, and maxi- mising the potential to support prevention and research. The JAMIE-approach sport/fitness, shops, pubs, clubs and recreation) –Unknown –Home (includes garden) –Other (includes health facilities) –Unknown Table 1 The single screen minimum data set: external factors of injury Table 1 The single screen minimum data set: external factors of injury That is why JAMIE allows EU-MSs to supply ED in- jury data with two levels of depth on injury determi- nants, the minimum and full level datasets. The combination of much larger amounts of cases at a lower level of detail as to the injury circumstances, sufficient for developing the accurate estimates of population inci- dence, with data at high levels of detail from a relatively small number of hospitals provides information for a wide range of policy makers and health, transportation and consumer protection authorities. Location (setting): –Workplace –Road (incl. pavement) –Educational establishment –Leisure area (incl. sport/fitness, shops, pubs, clubs and recreation) –Home (includes garden) –Other (includes health facilities) –Unknown The proposed two level system involves the implementa- tion of emergency department datasets at different levels of sophistication: 1. the Full injury surveillance Data Set (FDS, Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Page 4 of 5 Page 4 of 5 and services, or in work environments. In creating such a dataset we have been guided by the need to be able to capture the required variables efficiently and from a var- iety of staff in emergency departments including recep- tion staff and clinicians. In response to the latter requirement we have chosen terminology for categories which are widely understood both by the general public and clinical staff, e.g. put ‘accidental injury’ instead of ‘unintentional injury’ on the form. Hungary, National Institute for Health Development Sweden, National Board of Health and Welfare Ireland, National Suicide Research Foundation Slovenia, National Institute of Public Health Iceland, The Directorate of Health Spain, Servicio Navarra de Salud Italy, Instituto Superiore di Sanità United Kingdom, Swansea University Hungary, National Institute for Health Development United Kingdom, Swansea University Italy, Instituto Superiore di Sanità Collaboration has been established with a number of other countries, including Luxembourg, Poland and Croatia. Appendix I Wh ppe d Who we are To date, the following countries joined the project as associated partners in JAMIE: Health policy use h f Given the range of data being collected as part of the MDS proposed in the JAMIE project, including information on the age/gender of the injured individual, the nature of the injury sustained, the mechanism of the injury and the activ- ity/location/intent associated with the injury, the opportun- ity exists for each member state to use this information to calculate the number of DALYs and the size of the direct medical costs applicable to their own country. Such infor- mation is extremely valuable for undertaking economic analyses to assess the effectiveness or cost-benefit of injury prevention strategies in the EU-region. The JAMIE-project has been initiated, with the en- dorsement of governments in the EU-Member States, by a consortium of centres of excellence in injury surveil- lance based in the EU region: the Austrian Road Safety Board (KfV), Vienna, Austria; the European Association for Injury Prevention and Safety Promotion (EuroSafe), Amsterdam, the Netherlands; the National Institute for Health Development (NIHD), Budapest, Hungary; Due to the complexities of these calculations the project will provide instructions relating to how DALYs and dir- ect medical costs can be measured, utilising the know- ledge gained and findings resulting from the GBDI study [7] and the UK Burden of Injury (UK BOI) study [8]. the Swansea University School of Medicine, Health Information Research Unit (SU), Swansea, Wales, UK; and the Brandenburg University of Technology, Information Systems Unit, Cottbus, Brandenburg, Germany. The complexity of the project as well as the diversity of stakeholders involved calls for a comprehensive communi- cation plan in order to ensure focus in all activities and among all involved partners. The project group will work closely together with the competent authorities in MSs and those involved in the development of health sector based in- jury data exchange, including the European Statistical Office and World Health Organization ICD Revision Group. The European Association for Injury Prevention and Safety Promotion (EuroSafe) provides leadership to the project. Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Page 5 of 5 Rogmans Archives of Public Health 2012, 70:19 http://www.archpublichealth.com/content/70/1/19 Page 5 of 5 Page 5 of 5 Transport injury event – Any incident involving a trans- port device and resulting in injury Place of occurrence – Where the injured person was when the injury event started Mechanism of injury – The way in which the injury was sustained (i.e. how the person was hurt) Activity when injured – The type of activity the injured person was engaged in when the injury occurred Object/substance producing injury – Matter, material or thing being involved in the injury event Type of injury – Type of injury sustained Part of the body injured – Region or part of the body where the injury is located Narrative – Description of the event leading to the (sus- pected) injury Transport injury event – Any incident involving a trans- port device and resulting in injury Transport injury event – Any incident involving a trans- port device and resulting in injury Received: 9 July 2012 Accepted: 20 August 2012 Published: 28 August 2012 Received: 9 July 2012 Accepted: 20 August 2012 Published: 28 August 2012 Place of occurrence – Where the injured person was when the injury event started Violence module Victim/perpetrator relationship – The relationship of the person committing the violent act to the injured per- son. doi:10.1186/0778-7367-70-19 Cite this article as: Rogmans: Joint action on monitoring injuries in Europe (JAMIE). Archives of Public Health 2012 70:19. Sex of perpetrator – The sex of the person who inflicted the injury. Age group of perpetrator – The age group of the person who inflicted the injury. Context of assault – The circumstances surrounding the violent injury event. Submit your next manuscript to BioMed Central and take full advantage of: Counterpart – The other vehicle, object, person, or ani- mal (if any) with which the injured person, or the vehicle in which the injured person was travelling, collided. • Convenient online submission • Thorough peer review Intentional Self-harm module Proximal risk factor – The most recent crises that led to the self-harm incident. Previous intentional self-harm – Whether or not the injured person attempted intentional self-harm before. Admission module 7. Murray CJL, Lopez AD: The Global Burden of Disease. Geneva: World Health, Organization, Harvard School of Public Health, World Bank; 1996. Number of days in hospital – The number of days the injured person is admitted in the recording hospital. 8. Lyons RA, Kendrick D, Towner EM, Christie N, Macey S, et al: Measuring the Population Burden of Injuries—Implications for Global and National Estimates: A Multi-centre Prospective UK Longitudinal Study. PLoS Med 2011, 8(12): e1001140. doi:10.1371/journal.pmed.1001140. Core IDB FDS data elements Core IDB FDS data elements Recording country – Country that provides the data Unique national record number – Number of the emer- gency department case or record Age of patient – Person’s age at the time of the injury Sex of patient – Person’s sex at the time of the injury Country of permanent residence – Person’s country of residence at the time of the injury Date of injury – The date the injury was sustained Time of injury – The time the injury was sustained Date of attendance – The date the injured person attended the Emergency Department Time of attendance – The time the injured person attended the Emergency Department Treatment and follow-up – Status of treatment after at- tendance at the Emergency Department Intent – The role of human purpose in the injury event To date, the following countries joined the project as associated partners in JAMIE: Austria, Kuratorium für Verkehrssicherheit Lithuania, Institute of Hygiene Cyprus, Ministry of Health Latvia, National Health Services Czech Republic, The University Hospital Brno Malta, Directorate General Strategy & Sustainability Denmark, Syddansk Universitet Netherlands, Consumer Safety Institute Estonia, Ministry of Social Affairs of Estonia Norway, Norwegian Safety Forum Germany, Landesgesundheitsamt Brandenburg Portugal, Instituto Nacional de Saúde-Dr. Ricardo Jorge IP Greece, National School of Public Health Romania, Babeş-Bolyai University Cluj-Napoca Austria, Kuratorium für Verkehrssicherheit Lithuania, Institute of Hygiene Cyprus, Ministry of Health Latvia, National Health Services Czech Republic, The University Hospital Brno Malta, Directorate General Strategy & Sustainability Denmark, Syddansk Universitet Netherlands, Consumer Safety Institute Estonia, Ministry of Social Affairs of Estonia Norway, Norwegian Safety Forum Germany, Landesgesundheitsamt Brandenburg Portugal, Instituto Nacional de Saúde-Dr. Ricardo Jorge IP Greece, National School of Public Health Romania, Babeş-Bolyai University Cluj-Napoca Transport module Mode of transport – The means by which the injured person was travelling from one place to another. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • 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 Role of the injured person – How the injured person was involved with the specified mode of transport at the time of the injury event. Submit your next manuscript to BioMed Central and take full advantage of: References 1. Kisser R, et al: Injuries in the European Union, Statistics Summary 2005–2007 Kuratorium für Verkehrssicherheit, 2009. Vienna: EuroSafe; 2009. website: http://www.eurosafe.eu.com/csi/eurosafe2006.nsf/wwwVwContent/ l3reportsonidb-1.htm. 1. Kisser R, et al: Injuries in the European Union, Statistics Summary 2005–2007, Kuratorium für Verkehrssicherheit, 2009. Vienna: EuroSafe; 2009. website: http://www.eurosafe.eu.com/csi/eurosafe2006.nsf/wwwVwContent/ l3reportsonidb-1.htm. Mechanism of injury – The way in which the injury was sustained (i.e. how the person was hurt) 2. Baker SP, O'Neill B, Ginsberg MJ, Guohua L: The injury fact book. Oxford Press: New York; 1992. 2. Baker SP, O'Neill B, Ginsberg MJ, Guohua L: The injury fact book. Oxford Press: New York; 1992. 3. Kisser R, et al: Injury data needs and opportunities in Europe. Int. J. Injury Control and Safety Promotion 2009, 16(2):103–112. p 106. 3. Kisser R, et al: Injury data needs and opportunities in Europe. I Kisser R, et al: Injury data needs and opportunities in Europe. Int. J. Injury Control and Safety Promotion 2009, 16(2):103–112. p 106. Object/substance producing injury – Matter, material or thing being involved in the injury event Control and Safety Promotion 2009, 16(2):103–112. p 106. 4. Council Recommendation on the prevention: Council Recommendation on the prevention of injury and the promotion of safety. Official Journal of the European Union 2007/C164/01 of July 18, 2007. (CELEX-Nr. 32007H0718). website: http://www.eurosafe.eu.com/csi/eurosafe2006.nsf/wwwVwContent/ l4councilrecommendation.htm. Part of the body injured – Region or part of the body where the injury is located 5. European Commission: Health in Europe- Information and data interface. https://webgate.ec.europa.eu/sanco/heidi/index.php/IDB. Narrative – Description of the event leading to the (sus- pected) injury Narrative – Description of the event leading to the (sus- pected) injury 6. CECI Coordination and Maintenance Group: International Classification of External Causes of Injuries (ICECI) version 1.2. Adelaide: Consumer Safety Institute, Amsterdam and AIHW National Injury Surveillance Unit; 2004. website: http://www.who.int/classifications/icd/adaptations/iceci/en/index. html. Sports module Type of sport/exercise activity – The type of sport or ex- ercise activity in which the injured person was engaged at the time of the injury.
https://openalex.org/W1994948141	https://www.nature.com/articles/srep09974.pdf	English	null	Effects of substrate conductivity on cell morphogenesis and proliferation using tailored, atomic layer deposition-grown ZnO thin films	Scientific reports	2,015	cc-by	10,034	OPEN SUBJECT AREAS: FOCAL ADHESION MECHANOTRANSDUCTION SYNTHESIS AND PROCESSING Won Jin Choi1, Jongjin Jung2, Sujin Lee3, Yoon Jang Chung1, Cheol-Soo Yang1, Young Kuk Lee1, You-Seop Lee4, Joung Kyu Park2, Hyuk Wan Ko3 & Jeong-O Lee1 Received 28 October 2014 Accepted 24 March 2015 Published 21 April 2015 1Advanced Materials Division, Korea Research Institute of Chemical Technology (KRICT), Daejeon, 305-343, South Korea, 2Research Center for Convergence Nanotechnology, Korea Research Institute of Chemical Technology (KRICT), Daejeon, 305-343, South Korea, 3College of Pharmacy, Dongguk University, Gyeonggido, 410-820, South Korea, 4Eco-Solution Team, DMC R&D Center, Samsung Electronics, Suwon, 443-742, South Korea. We demonstrate that ZnO films grown by atomic layer deposition (ALD) can be employed as a substrate to explore the effects of electrical conductivity on cell adhesion, proliferation, and morphogenesis. ZnO substrates with precisely tunable electrical conductivity were fabricated on glass substrates using ALD deposition. The electrical conductivity of the film increased linearly with increasing duration of the ZnO deposition cycle (thickness), whereas other physical characteristics, such as surface energy and roughness, tended to saturate at a certain value. Differences in conductivity dramatically affected the behavior of SF295 glioblastoma cells grown on ZnO films, with high conductivity (thick) ZnO films causing growth arrest and producing SF295 cell morphologies distinct from those cultured on insulating substrates. Based on simple electrostatic calculations, we propose that cells grown on highly conductive substrates may strongly adhere to the substrate without focal-adhesion complex formation, owing to the enhanced electrostatic interaction between cells and the substrate. Thus, the inactivation of focal adhesions leads to cell proliferation arrest. Taken together, the work presented here confirms that substrates with high conductivity disturb the cell-substrate interaction, producing cascading effects on cellular morphogenesis and disrupting proliferation, and suggests that ALD-grown ZnO offers a single-variable method for uniquely tailoring conductivity. S S tudies of various organic/inorganic structures and materials as cellular substrates are a current research priority, reflecting the fundamental importance of understanding cellular interfaces and their applications, which range from wound healing and bone and nerve regeneration to prosthetics and artificial tissues and organs. Cells are extremely sensitive to nano- or micron-sized natural/artificial surface topographies and chemistries, which may permanently change cell fate1–7. Depending on the cell type or application, different materials/topographies are required as cell substrates. For example, neuronal cells prefer conductive substrates, such as carbon nanotubes8, whereas bone tissue regeneration requires mechanically robust substrates9, and vascular implants favor fibrous supports10,11. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.-O.L. (jolee@krict.re. kr); J.K.P. (parkjk@ krict.re.kr) or H.W.K. (kohw@dongguk.edu) OPEN The conductivity of ZnO films is generally governed by film thickness in the nanometer range, allowing the electrical properties of the film to be carefully tuned without altering other characteristics. Such tunable conductivity is unique to ALD-processed ZnO thin films, distinguishing this approach from other fabrication methods, such as sputtering, chemical vapor deposition, hydrothermal and sol-gel-derived ZnO. promote astrocyte cell growth; the same metallic Au surfaces exerted the opposite effect. Although Au and Pt are metallic substrates, Si a semiconducting one, and SiO2 could be classified as an insulating substrate. Thus, the cell growth effects cannot be exclusively attrib- uted to differences in electrical conductivity because these substrates possess chemically and physically diverse properties. These studies highlight the importance of being able to vary a single physical parameter while holding all other physicochemical parameters constant to develop a clear understanding of the effect of electrically conducting substrates on cell behavior. In this work, we investigated ZnO films grown by atomic layer deposition (ALD) as cell-interfacing substrates with variable electrical conductivity. Depending on their thickness, ALD-grown ZnO films displayed a wide range of electrical properties, encom- passing insulating, semiconducting and metallic properties, whereas their chemical and topological properties remained constant. SF295 glioblastoma cells grown on ZnO films with different conductivities exhibited marked differences in cell morphogenesis and proliferation that depended on the conductivity of the film. Figure 1 (b–f) shows the properties of ALD ZnO thin films as a function of cycle number (50–500 cycles). A plot of the electrical characteristics of the ZnO thin films (Figure 1b), measured by the Hall effect (Figure 1c), shows that the very thin films (,7 nm; 50 cycles) are similar to insulators, exhibiting no measurable conduction. At intermediate thicknesses of ,18 nm (100 cycles) and ,25 nm (150 cycles), the films show semiconducting behavior with moderate conductivity (0.4–78 S/cm) and typical field effect transistor characteristics (Supplementary Figure S1). When the film thickness exceeds 34 nm (.200 cycles), the conductivity values are large enough to allow implementation as a metallic electrode (.100 S/cm). The conductivity values (Figure 1b) and charge carrier concentration values extracted/derived from them increased linearly with increases in the number of ALD cycles. Although the effect of substrate conductivity has been studied previously12, the present work employed the widest conductivity range, as well as much higher conductivity, highlighting the appropriateness of the current study. OPEN Despite these general trends, a fundamental understanding of the mechanisms underlying such tendencies has remained elusive owing to the simultaneous contributions of multiple cell substrate parameters. p p Electrically conductive substrates have recently been used as cell-stimulating interfaces, and the effects of electrical conductivity on cell behavior have been extensively investigated12–15. For example, Thrivikraman and colleagues investigated the cell behavior with hydroxyapatite (HA) and calcium titanate (CA) and concluded that cell proliferation was enhanced on more highly conducting CA12. Jun et al. showed that electrically conductive composite fibers of poly(L-lactide-co-e-caprolactone) blended with polyaniline stimulate the differentiation of myoblast cells13. Baxter and colleagues showed that electrically active (polarized) hydroxyapatite exerts positive effects on bone cell growth14 and suggested that the adsorption of proteins and ions on the polarized substrate might be a possible mechanism. However, conductivity of the substrates investigated was too low (,1029/Ohm?cm for CA) to draw meaningful conclusions. Maydanov et al. investigated the role of an electrically conductive cell substrate by growing astrocytes on Au, Pt, Si, or SiO2 substrates15. Pt substrates were found to SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 1 www.nature.com/scientificreports www.nature.com/scientificreports promote astrocyte cell growth; the same metallic Au surfaces exerted the opposite effect. Although Au and Pt are metallic substrates, Si a semiconducting one, and SiO2 could be classified as an insulating substrate. Thus, the cell growth effects cannot be exclusively attrib- uted to differences in electrical conductivity because these substrates possess chemically and physically diverse properties. These studies highlight the importance of being able to vary a single physical parameter while holding all other physicochemical parameters constant to develop a clear understanding of the effect of electrically conducting substrates on cell behavior. The ZnO thin films used here were grown on glass substrates using the ALD process shown in Figure 1a. A single cycle of ALD is composed of a pulse of diethyl zinc (DEZ) followed by a purge process, resulting in the formation of a layer of Zn-terminated bonds on the surface of the glass substrate. This cycle is then followed by a subsequent pulse of H2O to attach O atoms to these chains to form a layer (,0.2 nm) of ZnO23,24. The self-limited nature of ALD enables atomic-scale control of the thickness of ZnO films while maintaining other factors, such as surface roughness and chemical composition. OPEN The correlation between the number of ALD cycles and film Results Preparation and characterizations of ZnO films. ZnO is a wide bandgap (3.37 eV at room temperature) group II-VI semiconductor material that is used in numerous fields of materials research16. Its optical clarity and relatively metallic properties allow it to be implemented as a transparent, conductive, oxide material for electrodes in smart windows and touch screens. In the semiconductor industry, ZnO is widely used as the active channel material in thin film transistors owing to its large on/off ratio and moderate field effective mobility, possibly even challenging classic Si-based devices in some applications17–21. ZnO is also commonly found as an optoelectronic film in various optical applications22, and its piezoelectric properties have opened a broad avenue of research in energy devices. Figure 1 \| Characteristics of ALD-grown ZnO thin films. (a) A schematic depiction of the ALD process used to prepare ZnO thin films with different nanometer-depth thicknesses. (b) Conductivity versus ALD cycle number in ZnO thin films. ZnO films prepared with 50 and 100 ZnO-layering cycles are classified as insulators, those prepared with 150 cycles are classified as semi-conductors, and those prepared using more than 200 cycles are categorized as metal. (c) A scheme showing Hall measurements of ZnO thin films deposited on glass. (d) ZnO film thickness versus the number of ALD cycles. (e) The degree of hydrophobicity of ZnO thin films determined by measuring the contact angles. As the ZnO film becomes thicker, the contact angle increases from 40u to 64u. The contact angle becomes saturated after 200 cycles of ALD. (f) Plot of surface roughness, analyzed by AFM, versus the number of ALD cycles. The surface roughness curve exhibits a hyperbolic relationship with the cycle number (from 0.15 nm to 0.7 nm) and saturates at 150 cycles. SCIENTIFIC REPORTS \| 5 9974 \| DOI 10 1038/ 09974 2 Figure 1 \| Characteristics of ALD-grown ZnO thin films. (a) A schematic depiction of the ALD process used to prepare ZnO thin films with different nanometer-depth thicknesses. (b) Conductivity versus ALD cycle number in ZnO thin films. ZnO films prepared with 50 and 100 ZnO-layering cycles are classified as insulators, those prepared with 150 cycles are classified as semi-conductors, and those prepared using more than 200 cycles are categorized as metal. (c) A scheme showing Hall measurements of ZnO thin films deposited on glass. (d) ZnO film thickness versus the number of ALD cycles. Results In the presence of serum, SF295 cells spread out properly but exhibited different patterns of mem- brane protrusion that depended on the thickness of the ZnO film. As shown in Figure 2a and Supplementary Figure S5, cells grown on highly conductive ZnO films adopted a more elongated shape and were well spread out. As the number of ZnO cycles increased, the cell elongation factor values, measured as the long axis/short axis ratio, also increased. Supplementary Figure S6 shows actin filament structures in SF295 cells, revealing morphological changes in cells Figure 1 (e and f) shows the hydrophobicity of the films determined from contact angle measurements and their roughness, measured by AFM. Both profiles clearly show that the surface prop- erties, except charge carrier concentration, remained constant above ,200 cycles of ZnO thin film layering, implying that any changes in cell behavior beyond this point are unrelated to differences in the surface roughness or contact angle. Typical topographic images and wetting-angle measurements corresponding to a range of ALD cycles (50–500) are shown in Supplementary Figures S2 and S3. SF295 cell growth on ZnO thin films. To examine the effects of variable conductivity on cellular responses, the SF295 cell line was used as a model system. SF295 is an established high-grade brain tumor cell line derived from human malignant glioma25. Brain tumors are highly aggressive, yet the treatment therapy for brain tumors remains limited. Tumor treating fields (TTF) therapy was recently approved by the U.S. Food and Drug Administration (FDA) as a treatment method for this devastating malignant glioma26,27. TTF Figure 2 \| Cell viability and proliferation on ZnO films. (a) Differential-interference contrast (DIC) images of SF295 cells on ZnO thin films with different charge carrier densities (50–500 cycles of ZnO layering). SF295 cells on substrates with a higher carrier density exhibited more elongated and narrowed ends. Scale bar 5 20 mm. (b) Histogram showing the optical density of SF295 cells grown on ZnO substrates expressed as absorbance units (a.u.) and determined using MTS assays. Assays were performed 7 days after seeding cells on each ZnO substrate. Data are presented as the means 6 S.E.M. (n53). Significance was tested using one-way analysis of variance (ANOVA) with post hoc Fisher’s least significant difference (LSD) analysis. , P , 0.05; NS, not significant. Results (c) Possible contribution of the toxicity of released Zn21 ions to the ALD cycle number-dependent effects of ZnO thin films on proliferation (shown in 2b). Left: Illustration of the experimental setup. ZnO thin films were incubated (or soaked in cell culture medium) for 24 hours in a CO2 incubator, after which the conditioned medium was collected. SF295 cells grown on glass slides were then incubated with the collected ZnO thin film-conditioned medium for 24 hours, and their viability was assayed by MTS assay. Right: Results of cell viability assays. The decrease in cell viability did not exceed 10% compared with controls incubated in conditioned medium from the glass substrate. Zn21 ion release from ZnO thin films was further examined by measuring the concentration of Zn21 ions using ICP-AES (see Supplementary Figure S7). Data are presented as the means 6 S.D. (n 5 3). Significance was tested using one-way ANOVA with post hoc Fisher’s LSD analysis. *, P , 0.001; NS, not significant. (d) Cell counting assay to quantify the proliferation of SF295 cells cultured on each ZnO substrate from 24 to 140 hours. Cell growth curves indicate that SF295 cells grown on glass or 50- to 200-cycle ZnO films proliferated exponentially, whereas cells grown on 250- and 500-cycle ZnO films showed linear growth. Figure 2 \| Cell viability and proliferation on ZnO films. (a) Differential-interference contrast (DIC) images of SF295 cells on ZnO thin films with different charge carrier densities (50–500 cycles of ZnO layering). SF295 cells on substrates with a higher carrier density exhibited more elongated and narrowed ends. Scale bar 5 20 mm. (b) Histogram showing the optical density of SF295 cells grown on ZnO substrates expressed as absorbance units (a.u.) and determined using MTS assays. Assays were performed 7 days after seeding cells on each ZnO substrate. Data are presented as the means 6 S.E.M. (n53). Significance was tested using one-way analysis of variance (ANOVA) with post hoc Fisher’s least significant difference (LSD) analysis. , P , 0.05; NS, not significant. (c) Possible contribution of the toxicity of released Zn21 ions to the ALD cycle number-dependent effects of ZnO thin films on proliferation (shown in 2b). Left: Illustration of the experimental setup. ZnO thin films were incubated (or soaked in cell culture medium) for 24 hours in a CO2 incubator, after which the conditioned medium was collected. Results (e) The degree of hydrophobicity of ZnO thin films determined by measuring the contact angles. As the ZnO film becomes thicker, the contact angle increases from 40u to 64u. The contact angle becomes saturated after 200 cycles of ALD. (f) Plot of surface roughness, analyzed by AFM, versus the number of ALD cycles. The surface roughness curve exhibits a hyperbolic relationship with the cycle number (from 0.15 nm to 0.7 nm) and saturates at 150 cycles. Figure 1 \| Characteristics of ALD-grown ZnO thin films. (a) A schematic depiction of the ALD process used to prepare ZnO thin films with different nanometer-depth thicknesses. (b) Conductivity versus ALD cycle number in ZnO thin films. ZnO films prepared with 50 and 100 ZnO-layering cycles are classified as insulators, those prepared with 150 cycles are classified as semi-conductors, and those prepared using more than 200 cycles are categorized as metal. (c) A scheme showing Hall measurements of ZnO thin films deposited on glass. (d) ZnO film thickness versus the number of ALD cycles. (e) The degree of hydrophobicity of ZnO thin films determined by measuring the contact angles. As the ZnO film becomes thicker, the contact angle increases from 40u to 64u. The contact angle becomes saturated after 200 cycles of ALD. (f) Plot of surface roughness, analyzed by AFM, versus the number of ALD cycles. The surface roughness curve exhibits a hyperbolic relationship with the cycle number (from 0.15 nm to 0.7 nm) and saturates at 150 cycles. SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 2 www.nature.com/scientificreports thickness is shown in Figure 1d. The thickness of the film was confirmed by atomic force microscopy (AFM). therapy uses low intensity alternating electric fields and currents, and the molecular basis of TTF presumably lies in the disruption of the mitotic process of proliferating cancer cells by alternating electric fields. In this regard, the interaction of glioma cells and the electric field (current) need to be explored at a basic level; thus, we explored cell motility and proliferation on substrates with varying electrical conductivity. SF295 cells were cultured on ZnO thin films with or without serum (see Materials and Methods). In serum-free medium, SF295 cells attached normally to ZnO substrates but failed to form proper membrane protrusions, such as filopodia and lamellipodia. In addition, the cells could not spread out and instead displayed round shapes (Supplementary Figure S4). Results SF295 cells grown on glass slides were then incubated with the collected ZnO thin film-conditioned medium for 24 hours, and their viability was assayed by MTS assay. Right: Results of cell viability assays. The decrease in cell viability did not exceed 10% compared with controls incubated in conditioned medium from the glass substrate. Zn21 ion release from ZnO thin films was further examined by measuring the concentration of Zn21 ions using ICP-AES (see Supplementary Figure S7). Data are presented as the means 6 S.D. (n 5 3). Significance was tested using one-way ANOVA with post hoc Fisher’s LSD analysis. **, P , 0.001; NS, not significant. (d) Cell counting assay to quantify the proliferation of SF295 cells cultured on each ZnO substrate from 24 to 140 hours. Cell growth curves indicate that SF295 cells grown on glass or 50- to 200-cycle ZnO films proliferated exponentially, whereas cells grown on 250- and 500-cycle ZnO films showed linear growth. eration on ZnO films. (a) Differential-interference contrast (DIC) images of SF295 cells on ZnO thin films with SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 3 www.nature.com/scientificreports grown on ZnO films with different conductivities. Cells were stained using the F-actin-specific binding peptide, phalloidin, conjugated with an Alexa Fluor 488 fluorescent probe. On highly conductive ZnO thin films (.250 cycles), the cells formed actin stress fibers aligned along the long axis of the cells. A previous study showed that cells tend to align themselves along extracellular cues, such as mechanical, chemical and electrical stimulation28. In a recent review by Li et al., various examples of in vitro cell alignment techniques were summarized28. However, no external stimulation sources, such as electric field (current)29, unidirectional mechanical stress (stretch30, flow31, compression32 or topographic patterning33) or line-shaped chemical stimulation34, were applied to the system in this work; the directions of cell alignment were randomly oriented as shown in Figure 2 (a). released from ZnO thin films did not significantly influence cell viability. MTS assays not only provide information about cell viability, they also provide a measure of cell proliferation. Thus, a lower O.D. value in the MTS assay might indicate a reduced level of cell proliferation on ZnO thin films instead of poor cell viability. Results To measure the degree of cell pro- liferation in SF295 cells, we used a labeling technique employing the non-radioactive thymidine analog, 5-ethynyl-2’-deoxyuridine (EdU), which is readily incorporated into the double-stranded DNA of proliferating cells. A single 1-hour pulse treatment of cells with EdU was followed by a 24-hour incubation, after which nascent DNA staining was analyzed using a copper-catalyzed reaction. This analysis revealed a significant decrease in the percentage of EdU- positive cells in ZnO films with a higher charge carrier density (Figure 3a and b). Cells grown on 500-cycle ZnO films showed an approximately 50% decrease in the EdU-positive fraction compared with control cells cultured on a glass plate. These results indicate that the electrical conductivity of the underlying substrate has an impact on the cell division rate. Then, what aspects of the electrically con- ductive substrate hinder cell proliferation? We found a clue from previous experimental work that showed that cell proliferation was reduced on adhesive substrates42. y g Previous studies have suggested two primary possibilities to account for the cytotoxic properties of ZnO nanomaterials on cul- tured cells: i) the dissolution and release of toxic cations35,36 and ii) the production of reactive oxygen species (ROS)37,38. In the first case, a likely mechanism begins with the endocytosis of ZnO nanorods. In this scenario, the acidic environment generated inside endosomes leads to the ionization of ZnO into O22 and free Zn21 ions; the latter then causes cytotoxic effects on cells. These events could occur if the ZnO particle size is small enough to be confined within the cytoplasm of the cell. ROS-mediated cytotoxicity could occur if sam- ple-handling procedures are not sufficiently stringent. For example, the exposure of ZnO films to UV light might result in generation of ROS that is due to oxygen defects in the films. The underlying molecular mechanism of ROS-dependent ZnO nanoparticle- induced cell death might be attributable to mitochondrial damage and the subsequent disruption of cellular respiratory functions39. However, such a scenario cannot be directly applied to our system that used a relatively smooth, continuous film-type ZnO as a stimulant. Adhesion force measurements of SF295 cells grown on ZnO thin films. The adhesion forces of cells grown on ZnO films with varying conductivity were analyzed as follows. First, cells growing on different substrates (varying conductivity) were treated with trypsin for 150 seconds and then centrifuged to remove weakly bound cells. Results To clarify the effects of ZnO on cell fate, we quantified viable cells by fixing and counting cells at four different time points (from 24 to 140 hours) after cul- turing on ZnO thin films (Figure 2d). As shown in Supplementary Table 1 and Supplementary Figure S8, the proliferation rate of SF295 cells varied according to the conductivity of the ZnO films. SF295 cells grown on 250- and 500-cycle ZnO thin films exhibited a linear increase in proliferation, whereas cells grown on all other ZnO thin films grew exponentially. Because all other physical parameters (wetting angle, surface roughness) become saturated after approxi- mately 150–200 cycles, these findings suggest that the three different electrical conductivity states of ZnO thin films—metallic, semicon- ducting and insulating—that depend on the charge carrier density, caused SF295 glioblastoma cells to proliferate at different rates. Therefore, we conclude that the electrical conductivity of the sub- strate is the dominating factor for cell proliferation above 200 cycles of ZnO (metallic conductivity range where other parameters, such as wettability and surface roughness of ZnO, saturate). However, other physical parameters may also aid cell proliferation when the conduc- tivity ranges of the ZnO films lie in the insulating or semiconducting regions (below 200 cycles). We also observed a decrease in the total number of cells with increases in conductivity, as shown in Figure 2a. To assess the viability of SF295 cells grown on ZnO thin films with different thick- nesses (50–500 cycles), we performed MTS (3-[4,5-dimethylthiazol- 2-yl]-2,5 diphenyl tetrazolium bromide) assays after culturing cells for 7 days. As shown in Figure 2b, the measured optical density (O.D.) decreased dramatically with increasing ZnO thickness; on 500-cycle ZnO thin films, the cell viability decreased to less than 50% of that in controls (cells cultured on glass slides). Although the physicochemical features of the cell substratum are critically important in determining cell adhesion and viability, our results indicate that decreases in total cell numbers might be attributable to the charge carrier concentration rather than other surface prop- erties. The surface roughness and hydrophobicity of ZnO thin films increased linearly over a limited thickness window corresponding to 0 to 150 cycles, but both parameters saturated at approximately 150 cycles and remained constant (see Figure 1e and f). We further examined the effects of changes in electrical conduc- tivity on SF295 cell proliferation. Results The details of the adhesion force measurements are described in the Materials and Methods section. As shown in Figure 4a, the number of cells that remained attached to the substrate dramatically increased with increasing ZnO thickness. Figure 4b and Supplementary Figure S9, which compare cells grown on 50-cycle ZnO and 250-cycle ZnO after trypsin treatment for 150 seconds, clearly show that a larger number of cells grown on thicker ZnO films exhibit elongated cell shapes, indicating resistance to trypsin-EDTA or stronger adhesion. y yp To determine whether the Zn21 ions released from thicker ZnO films mediate the cytotoxic effects on SF295 cells40,41, we performed the following control experiment. First, ZnO films of different thickness were incubated for 24 hours in growth medium, and the conditioned media containing released Zn21 ions were collected. SF295 cells on a glass substrate were then cultured in the collected media for 48 hours, after which the percentage of viable cells was determined using MTS assays (Figure 2c). The amount of Zn21 ions released was also determined by measuring the Zn21 concentration in the conditioned medium using inductively coupled plasma atomic emission spectroscopy (ICP-AES). As shown in Supplementary Figure S7, the concentration of Zn21 ions increased with increasing thickness of the ZnO films, and medium collected from cells grown on 500-cycle ZnO thin films contained the highest concentrations of Zn21 ions. Despite the cycle number-dependent increase in Zn21 concentration, there was less than a 10% change in the total number of cells at any cycle number (Figure 2c), indicating that the Zn21 ions How cells change their shape, biochemical characteristics, and motility in response to environmental cues has been extensively studied. Cells continuously perceive stimuli from the underlying surface and transduce these stimuli into specific intracellular signals to properly respond to changes in their surroundings. Specifically, integrin-based adhesion complexes are known to recognize the surrounding extracellular environment with extreme sensitivity; SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 4 Figure 3 \| Fluorescence images of cell proliferation assayed using EdU. (a) Representative images of 1-hour pulse-labeled EdU staining (green) and nuclear staining (blue, Hoechst 3334). As the charge carrier density increased (high cycle numbers), the density of SF295 cells and percentage of proliferating cells decreased, as reflected by a decrease in the EdU-positive cell population. Scale bar 5 20 mm. (b) Quantification of the ratio of EdU-incorporating cells to total cells. Results Immunofluorescence staining showed that the intensities and shapes of vinculin were different among cells growing on different ZnO thin film substrates. ZnO thin films with high conductivity caused weak and less locally concentrated vinculin staining, indicating decreased FA complex formation at intracellular surfaces (Figure 4c and Supplementary Figures S10 and S11). Consistent with this result, we also observed a decrease in immunofluorescence signals for tyr- osine-phosphorylated FAK in SF295 cells seeded on 500-cycle ZnO thin films (Supplementary Figure 12). These results suggest that conductive ZnO substrates trigger the down-regulation of FA com- plexes containing vinculin and tyrosine-phosphorylated FAK. Moreover, whereas FAs with a highly elongated ellipsoid shape were most common in control cells grown on glass substrates, such FAs were less frequent in cells grown on ZnO thin films with high con- ductivity; these cells were more often round rather than elongated. Results The percentage of proliferating SF295 cells on the 500-cycle ZnO substrate (28%) was less than that in controls cultured on a glass substrate (42%). Error bars denote standard errors of the mean (SEM; n 5 4), and asterisks denote statistical significance based on one-way ANOVA and post hoc Fisher’s LSD analysis. , P , 0.05; NS, not significant. Figure 3 \| Fluorescence images of cell proliferation assayed using EdU. (a) Representative images of 1-hour pulse-labeled EdU staining (green) and nuclear staining (blue, Hoechst 3334). As the charge carrier density increased (high cycle numbers), the density of SF295 cells and percentage of proliferating cells decreased, as reflected by a decrease in the EdU-positive cell population. Scale bar 5 20 mm. (b) Quantification of the ratio of EdU-incorporating cells to total cells. The percentage of proliferating SF295 cells on the 500-cycle ZnO substrate (28%) was less than that in controls cultured on a glass substrate (42%). Error bars denote standard errors of the mean (SEM; n 5 4), and asterisks denote statistical significance based on one-way ANOVA and post hoc Fisher’s LSD analysis. , P , 0.05; NS, not significant. A comparison of the size and shape of FAs, determined by measuring the length (long axis) and width (short axis) of individual vinculin- positive foci (Figure 4c), showed that the long axis/short axis ratio was smaller and more narrowly distributed in 250-cycle ZnO thin films than in 50-cycle ZnO films. These results indicate that con- ductive substrates with a higher charge carrier density prevent the growth of FA complexes in adhesive cells. When adhesive cells attach themselves on a substrate, focal complexes (nascent adhesins) are formed that initially have a rounded shape and a diameter of ,100 nm. On a conventional substrate, such focal complexes evolve into FAs, which are far more elongated and localized at the termini of stress fibers. However, focal complexes cannot mature into FAs on a conducting substrate and they remain as smaller and round focal complexes, as shown in Figure 4c. the biochemical properties, stiffness, and topography of the extra- cellular matrix (ECM) transduce signals into the interior of cells, causing a rearrangement of the cellular cytoskeleton43,44. Four differ- ent types of adhesion complexes are known to exist in fibroblasts: focal complexes, focal adhesions (FAs), fibrillar adhesions and three-dimensional matrix adhesions43. Results Of these complexes, the most important are FAs, which are large protein complexes composed of vinculin, talin, focal adhesion kinase (FAK), and paxillin. FAs are involved in many cellular events, including cell adhesion and migra- tion. Several studies have shown that strong cell adhesion might occur through increases in the FA complex formation, which is highly correlated with the acceleration of cell proliferation. To explore the enhanced adhesion on conductive ZnO films, we exam- ined the types of FA complexes in cells cultured on ZnO thin films by immunofluorescently staining cells with an anti-vinculin antibody. Immunofluorescence staining showed that the intensities and shapes of vinculin were different among cells growing on different ZnO thin film substrates. ZnO thin films with high conductivity caused weak and less locally concentrated vinculin staining, indicating decreased FA complex formation at intracellular surfaces (Figure 4c and Supplementary Figures S10 and S11). Consistent with this result, we also observed a decrease in immunofluorescence signals for tyr- osine-phosphorylated FAK in SF295 cells seeded on 500-cycle ZnO thin films (Supplementary Figure 12). These results suggest that conductive ZnO substrates trigger the down-regulation of FA com- plexes containing vinculin and tyrosine-phosphorylated FAK. Moreover, whereas FAs with a highly elongated ellipsoid shape were most common in control cells grown on glass substrates, such FAs were less frequent in cells grown on ZnO thin films with high con- ductivity; these cells were more often round rather than elongated. the biochemical properties, stiffness, and topography of the extra- cellular matrix (ECM) transduce signals into the interior of cells, causing a rearrangement of the cellular cytoskeleton43,44. Four differ- ent types of adhesion complexes are known to exist in fibroblasts: focal complexes, focal adhesions (FAs), fibrillar adhesions and three-dimensional matrix adhesions43. Of these complexes, the most important are FAs, which are large protein complexes composed of vinculin, talin, focal adhesion kinase (FAK), and paxillin. FAs are involved in many cellular events, including cell adhesion and migra- tion. Several studies have shown that strong cell adhesion might occur through increases in the FA complex formation, which is highly correlated with the acceleration of cell proliferation. To explore the enhanced adhesion on conductive ZnO films, we exam- ined the types of FA complexes in cells cultured on ZnO thin films by immunofluorescently staining cells with an anti-vinculin antibody. Discussions l Higher magnification images of vinculin staining are shown in Supplementary Figure S10. Right panel: FAs were quantified by measuring the long axis length, short axis length, and long axis/short axis ratio. The distribution of FAs became more granular, and their expression level decreased as the charge carrier density increased to the metallic level (Supplementary Figures S10 and S11). (d) Summary of the different electro-conductive properties of ZnO thin films prepared with different ALD cycles and their effects on SF295 cell adhesion and proliferation. Figure 4 \| ZnO film thickness-dependent variations in charge carrier densities differentially affect SF295 cell adhesion and focal adhesion (FA) complex formation. (a) Quantitative assay of SF295 cell attachment assay on insulating substrates with a low charge carrier density (glass and 50-cycle ZnO films) compared with metallic substrates with a high charge carrier density (250- and 500-cycle ZnO films). Trypsin-treated SF295 cells, which adhered to each substrate, were centrifuged at different centrifugal forces (500, 1000, and 2000 3 g), and then the cells were collected and counted (total cell number). More than three independent experiments were performed. Data are presented as the means 6 SD (n 5 3). Statistical significance was tested using one-way ANOVA with post hoc Fisher’s LSD analysis. , P , 0.001; NS, not significant. (b) Changes in SF295 cell morphology after trypsin treatment, and quantification of the degree of cell adhesion on ZnO substrates. Left: DIC images. Right: Quantification, showing histograms of the cell elongation factor. Differences in cell elongation factor values reveal changes in cell morphology after trypsinization for 150 seconds (right histograms, red) compared with those before trypsinization (left histogram, gray). (c) FA complex analysis of SF295 cells grown on ZnO substrates. Left panel: Florescence images of vinculin staining with an Alexa 488-conjugated anti-vinculin antibody. Green rods and spots are vinculin-associated FA complexes, which are less abundant and aligned unidirectionally in cells grown on 250- and 500-cycle ZnO films. White arrows indicate conspicuous FA regions on each substrate. Higher magnification images of vinculin staining are shown in Supplementary Figure S10. Right panel: FAs were quantified by measuring the long axis length, short axis length, and long axis/short axis ratio. The distribution of FAs became more granular, and their expression level decreased as the charge carrier density increased to the metallic level (Supplementary Figures S10 and S11). Discussions l Conventional substrates, such as glass or plastics, are insulating, yet most cells display charges because of their membrane potential. Cells, which are considered charged spheres, may stick to insulating sub- strates through electrostatic interactions. By contrast, the counter charges developed by cells are not stable on metallic substrates; thus, cells are unable to stick to these surfaces and tend to slip away from them. Recently, Li et al. showed that a large-area graphene film transferred to a metallic substrate exhibited antibacterial activity compared with graphene transferred to insulating substrates45. Li and colleagues proposed that charges could be transferred from cells to a metallic (or semiconducting) substrate, thereby causing mem- brane damage. As a rough approximation, we modeled a cell as a dielectric sphere with a negative surface charge corresponding to a membrane potential of ,70 mV, a radius (R) of 5 mm and a SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 5 www.nature.com/scientificreports Figure 4 \| ZnO film thickness-dependent variations in charge carrier densities differentially affect SF295 cell adhesion and focal adhesion (FA) complex formation. (a) Quantitative assay of SF295 cell attachment assay on insulating substrates with a low charge carrier density (glass and 50-cycle ZnO films) compared with metallic substrates with a high charge carrier density (250- and 500-cycle ZnO films). Trypsin-treated SF295 cells, which adhered to each substrate, were centrifuged at different centrifugal forces (500, 1000, and 2000 3 g), and then the cells were collected and counted (total cell number). More than three independent experiments were performed. Data are presented as the means 6 SD (n 5 3). Statistical significance was tested using one-way ANOVA with post hoc Fisher’s LSD analysis. *, P , 0.001; NS, not significant. (b) Changes in SF295 cell morphology after trypsin treatment, and quantification of the degree of cell adhesion on ZnO substrates. Left: DIC images. Right: Quantification, showing histograms of the cell elongation factor. Differences in cell elongation factor values reveal changes in cell morphology after trypsinization for 150 seconds (right histograms, red) compared with those before trypsinization (left histogram, gray). (c) FA complex analysis of SF295 cells grown on ZnO substrates. Left panel: Florescence images of vinculin staining with an Alexa 488-conjugated anti-vinculin antibody. Green rods and spots are vinculin-associated FA complexes, which are less abundant and aligned unidirectionally in cells grown on 250- and 500-cycle ZnO films. White arrows indicate conspicuous FA regions on each substrate. Discussions l (d) Summary of the different electro-conductive properties of ZnO thin films prepared with different ALD cycles and their effects on SF295 cell adhesion and proliferation. endent variations in charge carrier densities differentially affect SF295 cell adhesion and focal adhesion (FA) less abundant and aligned unidirectionally in cells grown on 250- and 500-cycle ZnO films. White arrows indicate conspicuous FA regions on each substrate. Higher magnification images of vinculin staining are shown in Supplementary Figure S10. Right panel: FAs were quantified by measuring the long axis length, short axis length, and long axis/short axis ratio. The distribution of FAs became more granular, and their expression level decreased as the charge carrier density increased to the metallic level (Supplementary Figures S10 and S11). (d) Summary of the different electro-conductive properties of ZnO thin films prepared with different ALD cycles and their effects on SF295 cell adhesion and proliferation. dielectric constant of 100 and investigated the interaction of cells with metallic and insulating substrates. Figure 5 shows schematic diagrams of the cell interactions with insulating and metallic sub- strates and indicates the electric potential distribution between the cell and substrate calculated using a finite element method. In cells with a membrane potential of ,70 mV, the calculated charge density on the cell surface would be 21.2431027 C/m2. When such a cell is in contact with an insulating substrate with a dielectric constant of 5, the attractive force exerted on the cell is calculated to be 4.91310215 N, whereas that of cells in contact with a metallic substrate with dielectric constant of ‘ would be 3.03310215 N. Therefore, the adhesion force for cells on an insulating substrate is much larger than that on a metallic substrate. However, if the contact area between the cell and metallic substrate becomes larger through deformation of the cell, the adhesion force of the metallic substrate increases and becomes even larger than the values obtained for the insulatingsubstrate. Because cells grown on a metallic substrate exhibit a more elongated shape (see Figure 1a), the cellsmayrearrangetheircytoskeletontoadheretothemetallicsubstrate. Becauseofcytoskeletondeformation,theadhesionstrengthofdeformed cells grown on a metallic substrate could be larger than that of cells grown on an insulating substrate. Additionally, as noted by Li and colleagues, cells may not activate the normal FA pathway, which involves the formation and subsequent maturation of the FA complex, owing to charge transfer from the cell membrane to the metallic sub- strate. SCIENTIFIC REPORTS \| 65: 9974 \| DOI: 10.1038/srep09974 Materials & Methods (Invitrogen, CA, USA). Prior to seeding SF295 cells, ZnO thin film substrates (1.4 3 1.4 cm in 12-well plates) were sterilized in ethanol, washed with phosphate- buffered saline (PBS), and then pre-incubated in growth medium for 1–2 hours. To analyze cell morphology, SF295 cells grown on each substrate were fixed with 4% paraformaldehyde after 3–4 days of growth and then imaged using an Olympus IX81 inverted microscope system (Olympus, Japan). After culturing for 7 days, cells on each substrate were quantified using MTS assays as described by the manufacturer (Promega, CA, USA). Deposition of ZnO thin films. The ZnO thin films were deposited by ALD in a Lucida D-100 chamber using diethylzinc (DEZ, electronic grade; Sigma-Aldrich, MO, USA) and H2O as the reactant and oxidant, respectively. All deposition schemes were performed under full saturation conditions, with DEZ-purge-H2O-purge cycles controlled at 0.5 seconds-10 seconds-0.1 second-30 seconds. The deposition temperature was fixed at 150uC. The canister temperature for both the source and oxidant was controlled by a Peltier device and was maintained at 15uC and 10uC for DEZ and H2O, respectively. This setting resulted in a peak pressure of ,1.5 torr during injection periods at a working pressure of ,1.36 torr. Zn21 ion cytotoxicity test. The cytotoxic effects of Zn21 ions released from ZnO thin films were assessed by incubating two sets of each ZnO substrate in a 12-well plate in growth medium for 24 hours after sterilization. Conditioned medium from one set was used to determine the concentration of Zn21 ions released from each ZnO substrate using duo inductively coupled plasma atomic emission spectroscopy (ICP-AES) (iCAP 6500; Thermo Scientific, Waltham, MA, USA). Conditioned medium from the second set was transferred to plates containing properly spread and growing SF295 cells. After 24 hours, cell viability under Zn21 ion-rich conditions was assessed by MTS assay. Fabrication and characterization of ZnO thin film field effect transistors. ZnO thin film transistors were fabricated using standard photolithographic processes in which an electrode of Al (100 nm) was deposited by thermal evaporation. The active channel in the device was defined by photolithographic patterning followed by etching with diluted nitric acid and had device dimensions of 40 mm (width) 3 100 mm (length). Transport measurements of the devices were conducted under ambient conditions using a Keithley 4200-SCS semiconductor characterization system. Discussions l Instead, cells deform their shapes to adhere to substrates with high conductivity; because of the disparity in adhesion mechanisms, cells adhered to a metallic substrate could not be easily removed in trypsin-based cell detachment assays. In conclusion, we have demonstrated the potential of ALD-grown ZnO films as a model system for studying the effect of electrically conductive substrates on cell fate. The complete control of electrical conductivity, encompassing the full range of insulating to semicon- ducting and metal, was achieved with ALD, with minimal changes to other physico-chemical parameters of the ZnO films. Intriguingly, the SF295 glioblastoma cell line cultured on tailored ZnO films exhibited different behaviors depending on the conductivity of the film: cells on highly conductive ZnO displayed decreased prolifera- tion and cytoskeletal rearrangements within the cell body that were clearly distinguishable from those of cells grown on a glass substrate (control) or ZnO substrates with lower conductivity. Moreover, using immunocytochemistry measurements of the adhesion com- plex and simple numerical calculation, we showed that it is difficult for cells to form FAs on conductive substrates, which translates into skeletal changes in the cells and the prevention of proliferation. SCIENTIFIC REPORTS \| 65: 9974 \| DOI: 10.1038/srep09974 6 Figure 5 \| Schematic diagram showing the geometry used in modeling electrostatic forces exerted on a cell in contact with insulating (a) and metallic (b and c) substrates. In (c), the cell is deformed. The cell is modeled as a dielectric body (dielectric constant, 100) with a negatively charged surface (charge density, 21.24 3 1027 C/m2) and a radius (R) of 5 mm. The cell is in medium with an assumed dielectric constant of 70. The dielectric constant of the insulating substrate is 5 and that of the metallic substrate is ‘. (d-f) Electric potential distribution map for the corresponding cases in the top row, simulated by the finite element method. The force on the dielectric body was calculated by integrating the electrostatic force on the dielectric surface over the electric field obtained from the electrostatic potential measurements. (a) When a cell (modeled as a dielectric sphere) is in contact with an insulating substrate with a dielectric constant of 5, the calculated force on the cell is 4.91 3 10215 N (attractive). (b) When a cell is in contact with a metallic substrate, the force is 3.03 3 10215 N (attractive). Discussions l (c) When a cell in contact with a metallic substrate is deformed, as observed in the microscopic images, the calculated force is 21.01 3 10213 N. www.nature.com/scientificreports Figure 5 \| Schematic diagram showing the geometry used in modeling electrostatic forces exerted on a cell in contact with insulating (a) and metallic (b and c) substrates. In (c), the cell is deformed. The cell is modeled as a dielectric body (dielectric constant, 100) with a negatively charged surface (charge density, 21.24 3 1027 C/m2) and a radius (R) of 5 mm. The cell is in medium with an assumed dielectric constant of 70. The dielectric constant of the insulating substrate is 5 and that of the metallic substrate is ‘. (d-f) Electric potential distribution map for the corresponding cases in the top row, simulated by the finite element method The force on the dielectric body was calculated by integrating the electrostatic force on the dielectric surface over Figure 5 \| Schematic diagram showing the geometry used in modeling electrostatic forces exerted on a cell in contact with insulating (a) and metallic (b and c) substrates. In (c), the cell is deformed. The cell is modeled as a dielectric body (dielectric constant, 100) with a negatively charged surface (charge density, 21.24 3 1027 C/m2) and a radius (R) of 5 mm. The cell is in medium with an assumed dielectric constant of 70. The dielectric constant of the insulating substrate is 5 and that of the metallic substrate is ‘. (d-f) Electric potential distribution map for the corresponding cases in the top row, simulated by the finite element method. The force on the dielectric body was calculated by integrating the electrostatic force on the dielectric surface over the electric field obtained from the electrostatic potential measurements. (a) When a cell (modeled as a dielectric sphere) is in contact with an insulating substrate with a dielectric constant of 5, the calculated force on the cell is 4.91 3 10215 N (attractive). (b) When a cell is in contact with a metallic substrate, the force is 3.03 3 10215 N (attractive). (c) When a cell in contact with a metallic substrate is deformed, as observed in the microscopic images, the calculated force is 21.01 3 10213 N. Materials & Methods High field-effect mobility zinc oxide thin film transistors produced at room temperature. J. Non-Crys. Solids 338–340, 806–809 (2004). 20. Fortunato, E. M. C. et al. Wide-bandgap high-mobility ZnO thin-film transistors produced at room temperature. Appl. Phys. Lett. 85, 2541–2543 (2004). 21. Xu, X., Cui, Q., Jin, Y. & Guo, X. Low-voltage zinc oxide thin-film transistors with solution-processed channel and dielectric layers below 150uC. Appl. Phys. Lett. 101, 222114 (2012). 22. Wang, Z. L. Splendid One-Dimensional Nanostructures of Zinc Oxide: A New Nanomaterial Family for Nanotechnology. ACS. Nano 2, 1987–1992 (2008). 23. Guziewicz, E. et al. Extremely low temperature growth of ZnO by atomic layer deposition. J. Appl. Phys. 103, 033515–033515–6 (2008). 22. Wang, Z. L. Splendid One-Dimensional Nanostructures of Zinc Oxide: A New Nanomaterial Family for Nanotechnology. ACS. Nano 2, 1987–1992 (2008). The interfering effect of Zn21 ions released from ZnO substrates on trypsin activity was assessed by treating SF295 cells on each substrate with the diluted trypsin solution under the same experimental conditions as described above for the cell adhesion experiments. After collecting and centrifuging the trypsin-treated solutions, the supernatants were added to SF295 cells in a multiwell plate. Changes in cell morphology were monitored, and the number of detached cells was compared. In addition, trypsin solutions collected from treated metallic substrates were applied to insulating substrates and vice versa. After incubation at room temperature for 10 minutes, detached cells and cells remaining on each substrate were collected and counted. 23. Guziewicz, E. et al. Extremely low temperature growth of ZnO by atomic layer deposition. J. Appl. Phys. 103, 033515–033515–6 (2008). 24. Levy, D. H., Freeman, D., Nelson, S. F., Cowdery-Corvan, P. J. & Irving, L. M. Stable ZnO thin film transistors by fast open air atomic layer deposition. Appl. Phys. Lett. 92, 192101–192101–3 (2008). 25. Rutka, J. T. et al. Establishment and characterization of five cell lines derived from human malignant gliomas. Acta. Neuropathol. 75, 92–103 (1987). 26. Kirson, E. D. et al. Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. Proc. Natl. Acad. Sci. U.S.A. 104, 10152–10157 (2007). Immunofluorescence staining of vinculin and FAK. For immunofluorescence labeling of cell adhesion components, SF295 cells were seeded on ZnO thin film substrates at the indicated densities. Materials & Methods Cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes, washed three times with PBS, and then permeabilized and blocked by incubating with PBS containing 1% horse serum and 0.1% Triton X-100 for 30 minutes. FA complexes were analyzed by incubating overnight at 4uC with anti- vinculin (1:400; Sigma) or phosphospecific anti-FAK [pY397] (1:1000; Invitrogen) antibodies. The next day, the cells were washed three times with blocking solution and then incubated with the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:2000, Jackson Immunoresearch) containing 4’,6-diamidino- 2-phenylindole (DAPI, 1:1000; Sigma) for 3 hours. After washing with blocking solution (PBS/1% horse serum), the samples were mounted on glass slides for subsequent imaging processing. All fluorescent images were acquired on a Nikon inverted fluorescence microscope (Nikon Instruments, Japan). 27. Gutin, P. H. & Wong, E. T. Noninvasive application of alternating electric fields in glioblastoma: a fourth cancer treatment modality. Am. Soc. Clin. Oncol. Educ. Book 126–131 (2012). doi:10.14694/EdBook_AM.2012.32.126 28. Li, Y. et al. Engineering cell alignment in vitro. Biotechnol. Adv. 32, 347–365 (2014). 29. Tandon, N. et al. Electrical stimulation systems for cardiac tissue engineering. Nat. Protoc. 4, 155–173 (2009). 30. Pang, Y., Wang, X., Lee, D. & Greisler, H. P. Dynamic quantitative visualization of single cell alignment and migration and matrix remodeling in 3-D collagen hydrogels under mechanical force. Biomaterials 32, 3776–3783 (2011). 31. Dewey, C. F., Bussolari, S. R., Gimbrone, M. A. & Davies, P. F. The dynamic response of vascular endothelial cells to fluid shear stress. J. Biomech. Eng. 103, 177–185 (1981). 32. Girton, T. S., Barocas, V. H. & Tranquillo, R. T. Confined compression of a tissue- equivalent: collagen fibril and cell alignment in response to anisotropic strain. J. Biomech. Eng. 124, 568–575 (2002). 1. Hamilton, D. W. & Brunette, D. M. The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation. Biomaterials 28, 1806–1819 (2007). 33. Hosseini, V. et al. Fiber-Assisted Molding (FAM) of Surfaces with Tunable Curvature to Guide Cell Alignment and Complex Tissue Architecture. Small 10, 4851–4857 (2014). 2. Nematollahi, M., Hamilton, D. W., Jaeger, N. J. & Brunette, D. M. Hexagonal micron scale pillars influence epithelial cell adhesion, morphology, proliferation, migration, and cytoskeletal arrangement. J. Biomed. Mater. Res. A. 91A, 149–157 (2009). 34. Kalinina, S. et al. Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns. Biomaterials 29, 3004–3013 (2008). 35. Turney, T. W. et al. Materials & Methods Hall measurements were conducted using an HMS-3000 (Ecopia, Korea) in the Van der Pauw configuration, where the induced current was fixed to 1 mA to ensure proper electrical characterization for all cases. A Dimension 3100 atomic force microscope (Veeco, NY, USA) was employed to obtain topographical images and confirm the surface roughness. Cell counting assay. SF295 cells grown on ZnO substrates were counted at specific times after seeding to estimate the effects of the depth of ZnO substrates on the cell proliferation rate. After seeding and culturing SF295 cells on each ZnO thin film, cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with the fluorescent dye, Hoechst 33342, to facilitate cell nuclei counting. Cells were counted 24, 49, 94, 121, and 140 hours after seeding. Total cell populations at each time point were displayed as histograms, and differences in the cell proliferation rate as a function of the depth of ZnO thin films were determined by plotting cell growth curves. Cell culture on ZnO thin films. The culture of SF295 glioblastoma cells was performed according to a standard protocol, with slight modifications. Briefly, SF295 cells were cultured at 37uC in a humidified 5% CO2 atmosphere in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 7 www.nature.com/scientificreports www.nature.com/scientificreports Cell proliferation assay. The proliferation rate of SF295 cells on ZnO films was determined using a Click-iT EdU Alexa Fluor 488 Imaging Kit (Molecular Probes, CA). SF295 cells were seeded on glass (control) or ZnO thin films. The next day, the cells were pulsed-labeled for 1 hour with EdU (10 mM). After replacing the cell growth medium with fresh medium, the label was chased by incubating cells for an additional 7–11 hours. After the chase period, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with Click-iT reaction reagents as described by the manufacturer (Invitrogen, CA). In brief, fixed cells were rinsed twice with PBS/3% bovine serum albumin (BSA) and permeabilized with PBS/0.5% Triton X-100 for 20 minutes. The permeabilization buffer was replaced with 500 mL of Click-iT reaction cocktail to detect proliferating cells. The nuclei were stained with Hoechst 33342 following standard protocols. All staining procedures were performed at room temperature, and all fluorescence images were obtained using a Nikon C1 laser-scanning confocal microscope system equipped with a Nikon inverted fluorescence microscope (Nikon, Japan). Materials & Methods 10. Tuzlakoglu, K. & Reis, R. L. Biodegradable polymeric fiber structures in tissue engineering. Tissue. Eng. Pt. B-Rev. 15, 17–27 (2009). 11. Bhardwaj, N. & Kundu, S. C. Electrospinning: A fascinating fiber fabrication technique. Biotechnol. Adv. 28, 325–347 (2010). 12. Thrivikraman, G., Mallik, P. K. & Basu, B. Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro. Biomaterials 34, 7073–7085 (2013). 13. Jun, I., Jeong, S. & Shin, H. The stimulation of myoblast differentiation by electrically conductive sub-micron fibers. Biomaterials 30, 2038–2047 (2009). 14. Baxter, F. R., Bowen, C. R., Turner, I. G. & Dent, A. C. E. Electrically Active Bioceramics: A Review of Interfacial Responses. Ann. Biomed. Eng. 38, 2079–2092 (2010). 15. Maydanov, A., Calabro, A., Queenan, C., Becker, D. & Kim, D. Investigation of Astrocyte Cell Growth on Metal, Semi-Conductor and Insulator Surfaces. Microsc. Microanal. 17, 158–159 (2011). Trypsin-treated cell adhesion assay. Cell adhesion on ZnO thin films was assessed by treating cells with trypsin followed by centrifugation. SF295 cells were cultured on glass (control), 50-cycle ZnO thin films (insulating), and 250- and 500-cycle ZnO thin films (metallic). After removing the culture medium and washing with PBS, a detachment rate with a gentle slope was achieved during centrifugation by treating each sample set with 0.01% trypsin-EDTA (diluted in PBS from a 0.05% trypsin- EDTA stock solution; Gibco, USA) at room temperature for 10–12 minutes. Culture medium was then added, and each sample was gently transferred to a 5 ml centrifugation tube (Eppendorf, Germany) and centrifuged in a swinging bucket rotor centrifuge (Allegra X-15R Centrifuge; Beckman Coulter Korea, Korea) at different relative centrifugal force (RCF) values (500, 1000, and 2000 3 g) for 5 minutes. After centrifugation, each sample was transferred to a container containing PBS, and cells remaining on the substrates were detached by applying a strong fluid force using a micropipette. Centrifuged cells and forcibly detached cells were collected and counted using a hemocytometer. 16. O¨ zgu¨r, U¨ . et al. A comprehensive review of ZnO materials and devices. J. Appl. Phys. 98, 1301 (2005). 17. Fortunato, E. M. C. et al. Fully Transparent ZnO Thin-Film Transistor Produced at Room Temperature. Adv. Mater 17, 590–594 (2005). 18. Cross, R. B. M. & De Souza, M. M. Investigating the stability of zinc oxide thin film transistors. Appl. Phys. Lett. 89, 263513–263513–3 (2006). 19. Fortunato, E. et al. Materials & Methods Formation of Zinc-Containing Nanoparticles from Zn21 Ions in Cell Culture Media: Implications for the Nanotoxicology of ZnO. Chem. Res. Toxicol. 25, 2057–2066 (2012). 3. Ghrebi, S., Hamilton, D. W., Douglas Waterfield, J. & Brunette, D. M. 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The fate of ZnO nanoparticles administered to human bronchial epithelial cells. ACS. Nano 6, 4921–4930 (2012). 9. Altmann, B. et al. Distinct cell functions of osteoblasts on UV-functionalized titanium- and zirconia-based implant materials are modulated by surface topography. Tissue. Eng. Pt. C-Meth. 19, 850–863 (2013). 9. Altmann, B. et al. Additional information Supplementary information accompanies this paper at http://www.nature.com/ scientificreports Supplementary information accompanies this paper at http://www.nature.com/ scientificreports Acknowledgments g This research was supported by the Creative Allied Program (CAP-12-1) through the Korea Research Council of Fundamental Science and Technology funded by the Ministry of Science, ICT and Future Planning and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF-2012M3A9C1053532 to H.W.K.) Competing financial interests: The authors declare no competing financial interests. How to cite this article: Choi, W.J. et al. Effects of substrate conductivity on cell morphogenesis and proliferation using tailored, atomic layer deposition-grown ZnO thin films. Sci. Rep. , 9974; DOI:10.1038/srep09974 (201 ). 5 5 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Materials & Methods Distinct cell functions of osteoblasts on UV-functionalized titanium- and zirconia-based implant materials are modulated by surface topography. Tissue. Eng. Pt. C-Meth. 19, 850–863 (2013). SCIENTIFIC REPORTS \| 5: 9974 \| DOI: 10.1038/srep09974 8 www.nature.com/scientificreports 42. Mann, B. K. & West, J. L. Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds. J. Biomed. Mater. Res. 60, 86–93 (2002). surface adhesion change in SF295 cells as the thickness of ZnO thin films was modulated. J.J. also performed the cell proliferation assay, cell counting assay, Zn21 ion cytotoxicity test, quantification of released Zn21 ions, and trypsin-treated centrifugation assay. S.L. performed the cell proliferation (EdU) assay, F-actin staining, vinculin and FAK staining experiments. C-S.Y. and Y-S.L. performed and analyzed the numerical calculations. W.J.C., J.J., S.L., Y.J.C, Y.K.L., H.W.K, and J-O.L prepared the figures and wrote the main manuscript text. J.K.P., H.W.K. and J-O.L. supervised the experimental aspects of the project. All authors contributed to the discussion and wrote the manuscript. p y ( ) 43. Berrier, A. L. & Yamada, K. M. Cell–matrix adhesion. J. Cell. Physiol. 213, 565–573 (2007). 44. Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell. Biol. 10, 21–33 (2009). 45. Li, J. et al. Antibacterial activity of large-area monolayer graphene film manipulated by charge transfer. Sci. Rep. 4, 4359 (2014). Author contributions W.J.C. and J-O.L. designed and conducted the experiments employing ZnO thin films W.J.C. and Y.J.C. fabricated the substrates and analyzed the samples using AFM and contact angle and electrical transport measurements. J.J. and H.W.K. made plans for the cell experiments and characterization. J.J. determined the morphology, proliferation, and SCIENTIFIC REPORTS \| : 9974 \| DOI: 10.1038/srep09974 5 9
https://openalex.org/W4381684233	https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/article/download/65/110	Malay	null	Peningkatan Keterampilan Microsoft Word dan Powerpoint Untuk Staf Administrasi Pada Gereja Orthodox Indonesia	Kresna	2,023	cc-by-sa	3,421	Abstrak Kebutuhan lembaga atau organisasi masyarakat di era informasi saat ini adalah bagaimana mampu membuat laporan dalam bentuk digital dan juga mampu menampilkannya dalam bentuk digital. Sayangnya, kebutuhan tersebut masih belum dirasakan oleh masyarakat luas. Mitra pengabdian kepada masyarakat (PKM) saat ini adalah Gereja Orthodox Indonesia, sebuah organisasi masyarakat yang memiliki catatan tertulis yang harus diinventarisasi dengan baik dan suatu saat dapat disajikan dengan menggunakan media komputer. Permasalahan yang dihadapi Gereja Orthodox adalah tidak semua staf administrasi memahami dengan baik penggunaan teknologi komputer, sehingga proses administrasi tidak dapat dilakukan dengan cepat. Sebelum pelatihan nilai rata-rata pre-test peserta sebesar 59, setelah pelatihan nilai rata-rata post-test adalah 85.38. Hal ini membuktikan terjadi peningkatan keterampilan dan pengetahuan staf administrasi setelah mengikuti pelatihan Microsoft Word dan Powerpoint. Kata kunci : Arsip, Komputer, Microsoft Office, Microsoft Word, Microsoft Power Point Improvement of Microsoft Word and Powerpoint Skills for Administrative Staff of the Indonesian Orthodox Church Mardi Hardjianto1, Agnes Aryasanti2, Yan Everhard 3, Jeremy Jonathan4, Putri Hayati5* 12345 Fakultas Teknologi Informasi Universitas Budi Luhur E-mail: 1mardi.hardjianto@budiluhur.ac.id, 2agnes.aryasanti@budiluhur.ac.id, 3yan.everhard@budiluhur.ac.id, 4jeremy.jonathan@budiluhur.ac.id, 5putri.hayati@budiluhur.ac.id ( corresponding author) Mardi Hardjianto1, Agnes Aryasanti2, Yan Everhard 3, Jeremy Jonathan4, Putri Hayati5* 12345 Fakultas Teknologi Informasi Universitas Budi Luhur E-mail: 1mardi.hardjianto@budiluhur.ac.id, 2agnes.aryasanti@budiluhur.ac.id, 3yan.everhard@budiluhur.ac.id, 4jeremy.jonathan@budiluhur.ac.id, 5putri.hayati@budiluhur.ac.id ( corresponding author) Keywords : Archives, Computers, Microsoft Office, Microsoft Word, Microsoft Power Point eywords : Archives, Computers, Microsoft Office, Microsoft Word, Microsoft Power Point KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Abstract The need for community institutions or organizations in the current information age is how to be able to make reports in digital form and also be able to display them in digital form. Unfortunately, this need is still not felt by the wider community. The community service partner (PKM) is currently the Indonesian Orthodox Church, a community organization that has written records that must be properly inventoried and one day can be presented using computer media. The problem faced by PKM partners is that not all administrative staff understand well the use of computer technology, so that administrative processes cannot be carried out quickly. Before the training the average pretest score of the participants was 59, after the training the average posttest score was 85.38. This proves that there has been an increase in the skills and knowledge of administrative staff after participating in Microsoft Word and Powerpoint training. 1. PENDAHULUAN Perkembangan teknologi dalam penggunaan Microsoft Office saat ini sangat dibutuhkan terhadap masyarakat umum sebagai media informasi dan multimedia untuk kebutuhan akan penguasaan berbagai hal yang harus dikuasai. Mitra Pengabdian Kepada Masyarakat (PKM) saat ini adalah Gereja Orthodox Indonesia, merupakan suatu organisasi masyarakat yang memiliki arsip tulisan yang harus diinventariskan dengan baik dan pada kondisi tertentu mampu memaparkan menggunakan media komputer. https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 95 https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Permasalahan yang dihadapi oleh mitra adalah belum semua staf administrasi mengerti dan memahami penggunaan teknologi komputer dalam pekerjaannya. Hal ini mengakibatkan proses administrasi tidak dapat dilakukan dengan cepat karena mengandalkan staf yang mampu mengoperasikan komputer. Atas dasar permasalahan tersebut, maka diadakan pelatihan penguasaan Microsoft Office, khususnya Microsoft Word dan Powerpoint. Harapannya setelah pelatihan, ada peningkatan kemampuan dan keterampilan staf administrasi dalam memanfaatkan Microsoft Word dan Powerpoint secara optimal dalam melakukan pekerjaaan. Berikut ini beberapa kegiatan pelatihan menggunakan Microsoft Office untuk meningkatkan ketrampilan komputer. Kegiatan pelatihan yang dilakukan [1] menghasilkan kemajuan pengetahuan guru dan staf penggunaan Microsoft Powerpoint membuat presentasi yang menarik. [2] melakukan pelatihan membuat surat lamaran pekerjaan menggunakan Microsoft Word, setelah pelatihan didapatkan peningkatan pengetahuan peserta berdasarkan rata-rata nilai sebelum dilakukan pelatihan dengan rata- rata setelah pelatihan, naik sebesar 16.94. Pengenalan dan pelatihan komputer terutama penggunaan Microsoft Office PowerPoint mampu memberikan manfaat sebagai media pembelajaran lebih modern dan menarik, setelah pelatihan para pengajar lebih memahami pentingnya teknologi sebagai media pembelajaran[3]. Sirait[4] melakukan pelatihan hasilnya, peserta dapat menggunakan Microsoft Office (Microsoft Excel, Word, dan Power Point) dalam pembuatan bahan ajar, laporan, undangan, dan administrasi kelas. Pelatihan dilakukan oleh [5], diperoleh hasil dari para peserta sangat antusias, dan didapatkan bahwa 74% mampu untuk mengaplikasikan Microsoft Word. [6] melakukan Pelatihan PowerPoint bagi karyawan agar memiliki kemampuan yang lebih baik, bagus, menarik dan informatif dalam membuat slide presentasi. Hasil evaluasi diatas 90% peserta merasa puas dan mendapatkan manfaat [7] melakukan pelatihan PowerPoint untuk mengedukasi siswa dalam membuat presentasi yang menarik seperti menambah suara, gambar, video maupun teks serta mengatur effect dan background. Hasil yang diperoleh meningkat diatas 60%. Arsyad [8] melakukan pelatihan media pembelajaran berbasis powerpoint untuk Guru dengan tujuan dapat meningkatkan softkill dalam mengoperasikan laptop maupun komputer sehingga media pembelajaran lebih efisien dan fleksibel. Hasil yang diperoleh dari pelatihan sebanyak 83% dari jumlah guru yang berhasil membuat powerpoint secara tuntas. 1. PENDAHULUAN Aditama[9] melakukan pelatihan penggunaan Microsfot Word, Microsoft PowerPoint dan Microsoft Excel untuk lembaga pemerintahan untuk menunjang keterampilan administrasi dalam hal surat menyurat, presentasi yang menarik dan juga untuk menyimpan database. Hasil post-test yang dilakukan sebesar 80% mendapat nilai 90 dan yang mendapat nilai 80 sebanyak 20%. [10] melakukan pelatihan dan sertifikasi kepada siswa untuk meningkatkan keterampilan dalam memanfaatkan software microsoft office untuk membuat laporan kegiatan. Hasil tes dan evaluasi diperolah dengan grade "B" (Baik). Tujuan pelatihan kegiatan pengabdian kepada masyarakat untuk meningkatkan keterampilan staf administrasi mitra dalam menguasai Microsoft Word dan Powerpoint. Hasil evaluasi dari kegiatan ini adalah peserta dapat memiliki pengetahuan lebih luas dalam penggunaan Microsoft Word dan powerpoint, mampu mengaplikasikan secara optimal dalam melaksanakan pekerjaan administrasi. KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 Pemberian soal post-test dilakukan setelah selesai melakukan pelatihan. Pada tahap ini, dibandingkan hasil pre-test dan post-test untuk mengetahui hasil dari peningkatan kemampuan dan ketrampilan peserta setelah mengikuti kegiatan pelatihan. Pada tahap ketiga, para peserta akan diberikan kuisioner yang menjadi bahan evaluasi tim pelaksana terhadap kegiatan yang telah dilakukan. Hasil kuisioner dihitung dengan rumus: f. Penyusunan Laporan Tahap ini dilakukan setelah seluruh rangkaian kegiatan telah selesai dilaksanakan. Tim pelaksana membuat laporan yang diserahkan ke universitas sebagai persyaratan administratif. y p Tahap ini dilakukan setelah seluruh rangkaian kegiatan telah selesai dilaksanakan. Tim pelaksana membuat laporan yang diserahkan ke universitas sebagai persyaratan administratif. KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 a. Analisis Kondisi Obyek PKM Tim pelaksanaan kegiatan melakukan survey pada Gereja Orthodox yang menjadi target pelaksanaan kegiatan. Pada proses ini tim melakukan wawancara terhadap Kepala mitra, menggali apa saja permasalahan yang dihadapi dan mengidentifikasi kebutuhan. Selanjutnya, tim memberikan solusi terhadap permasalahan mitra serta menentukan target peserta yang akan diikutsertakan dalam pelatihan. a. Analisis Kondisi Obyek PKM Tim pelaksanaan kegiatan melakukan survey pada Gereja Orthodox yang menjadi target pelaksanaan kegiatan. Pada proses ini tim melakukan wawancara terhadap Kepala mitra, menggali apa saja permasalahan yang dihadapi dan mengidentifikasi kebutuhan. Selanjutnya, tim memberikan solusi terhadap permasalahan mitra serta menentukan target peserta yang akan diikutsertakan dalam pelatihan. b. Pembuatan Proposal Langkah berikutnya setelah menentukan solusi yang akan diberikan, tim pelaksana membuat proposal PKM yang ditujukan pada lembaga atau universitas tempat tim pelaksana bernaung. c. Pembuatan Materi Tim Pelaksana melakukan pembuatan materi Microsoft Word dan Powerpoint, soal latihan, tugas, soal pre-test, post-test dan kuisioner. c. e buata ate Tim Pelaksana melakukan pembuatan materi Microsoft Word dan Powerpoint, soal latihan, tugas, soal pre-test, post-test dan kuisioner. d l ih d. Pelatihan Pelaksanaan kegiatan pelatihan dirancang untuk peserta agar materi yang diberikan secara komprehensif dapat dipahami dengan baik. Para peserta diharapkan dapat mengimplementasi secara aplikatif dalam dunia kerja. Metode pelaksanaan kegiatan pelatihan ini diproses dengan melakukan pendekatan pembelajaran secara teori dan praktikum. Perbandingan pembelajaran praktek dan pembelajaran teori yang diberikan sekitar 1.5:1. Metode pembelajaran teori meliputi: pengajaran, tanya jawab, dan diskusi. Metode yang digunakan pada praktikum adalah tanya jawab dan praktek tugas/latihan. p g e. Evaluasi Pelatihan Evaluasi pelatihan yang dilakukan tiga tahap. Pertama dengan memberikan soal pre-test sebelum kegiatan pelatihan dilakukan, hal ini dilakukan sebagai tolak ukur kesiapan peserta maupun tingkat pengetahuan peserta sebelum dilakukan pelatihan. Tahap kedua dengan memberikan soal post-test. Pemberian soal post-test dilakukan setelah selesai melakukan pelatihan. Pada tahap ini, dibandingkan hasil pre-test dan post-test untuk mengetahui hasil dari peningkatan kemampuan dan ketrampilan peserta setelah mengikuti kegiatan pelatihan. Pada tahap ketiga, para peserta akan diberikan kuisioner yang menjadi bahan evaluasi tim pelaksana terhadap kegiatan yang telah dilakukan. Hasil kuisioner dihitung dengan rumus: e. Evaluasi Pelatihan Evaluasi pelatihan yang dilakukan tiga tahap. Pertama dengan memberikan soal pre-test sebelum kegiatan pelatihan dilakukan, hal ini dilakukan sebagai tolak ukur kesiapan peserta maupun tingkat pengetahuan peserta sebelum dilakukan pelatihan. Tahap kedua dengan memberikan soal post-test. 2. METODE PENELITIAN Waktu yang dibutuhkan pelaksanaan keseluruhan program ini selama dua setengah bulan. Tahap pelaksanaan kegiatan ditunjukkan pada Gambar 1. Gambar 1: Langkah-langkah Pelaksanaan Kegiatan Gambar 1: Langkah-langkah Pelaksanaan Kegiatan https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 96 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 3.1 Pelaksanaan Kegiatan Kegiatan Pelatihan dilaksanakan di lokasi Gereja Orthodox, Peserta yang hadir berjumlah 8 orang staf administrasi mitra. Pelatihan ini dibagi menjadi empat sesi, yaitu: a. Persiapan pelatihan Sebelum pelatihan dimulai, tim pelaksana melakukan pengecekkan terhadap kesediaan ruang pelatihan, software dan hardware yang digunakan sebagai infrastruktur pelatihan. Tim melakukan troubleshooting pada perangkat pelatihan serta menyiapkan modul materi pelatihan. b. Pelaksanaan Pelatihan Pelatihan dilaksanakan mulai jam 09:00 hingga berakhir di jam 18:00. Pelatihan dibagi dua sesi, sesi pertama pelatihan microsoft yang dilaksanakan pukul 09:00 sampai pukul 12:15. Kemudian dilanjutkan istirahat makan siang. Sesi kedua mulai pukul 13:30 hingga pukul 16:00. Kemudian evaluasi dilakukan selama satu jam. Pelaksanaan kegiatan pelatihan ditunjukkan Gambar 2. https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 97 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Gambar 2: Pelaksanaan Kegiatan Pengabdian Kepada Masyarakat KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 A: Jurnal Riset dan Pengabdian Masyarakat E-ISSN: 2809-6509 Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 Gambar 2: Pelaksanaan Kegiatan Pengabdian Kepada Masyarakat Gambar 2: Pelaksanaan Kegiatan Pengabdian Kepada Masyarakat Pelatihan dimulai dengan sambutan dan pengenalan dari ketua tim pelaksana dan dari kepala mitra. Tim memberikan pengarahan dan menjelaskan rundown kegiatan pelatihan kepada peserta. Selanjutnya, sebelum kegiatan dimulai, soal pre-test diberikan oleh Tim. Waktu yang diberikan dalam pengerjaan soal pretest selama 30 menit. Pertanyaan yang diberikan antara lain: bagaimana mengatur halaman, mengganti font, membuat tabel di Microsoft Word, menyisipkan gambar, memberikan warna dan efek. Peserta diajari juga bagaimana cara membuat header dan footer, daftar isi, nomor halaman menggunakan table of content, dan mencetak dokumen. Materi pelatihan yang diberikan pada sesi pertama adalah membuka Microsoft Word, menyimpan dokumen, mengatur page setup, mengedit teks, menyisipkan gambar, membuat tabel, memberikan nomor halaman, dan membuat daftar isi menggunakan tabulasi maupun menggunakan table of content. Pada praktikum, peserta diajarkan membuat dokumen surat undangan dan laporan kegiatan. Penyampaian materi Microsoft Word ditunjukkan Gambar 3. Sesi kedua pelatihan dibuka dengan pengerjaan soal pre-test, pertanyaan-pertanyaan yang diberikan antara lain: bagaimana cara menambahkan slide baru, transisi slide, layer layout, menampilkan slide, menyembunyikan slide, menyisipkan gambar, membuat video, animasi slide, mencetak slide. Dalam mengerjakan soal pre-test, setiap peserta diberikan waktu 30 menit. Selanjutnya masuk ke sesi materi yang disampaikan oleh instruktur. Peserta dijelaskan bagaimana cara membuat presentasi yang baik dan menarik. Pemanfaatan template pada Microsoft Powerpoint, penggunaan transisi slide, menambahkan link dan animasi supaya slide presentasi lebih menarik. 3.1 Pelaksanaan Kegiatan Peserta juga diajarkan bagaimana membuat video menggunakan fasilitas yang ada pada Microsoft Powerpoint. Pemaparan materi Microsoft Powerpoint ditunjukkan pada Gambar 4. Gambar 3: Penyampaian Materi Microsoft Word Gambar 3: Penyampaian Materi Microsoft Word 98 https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Gambar 4: Pemaparan Materi Microsoft Powerpoint Gambar 4: Pemaparan Materi Microsoft Powerpoint c. Evaluasi Setelah pelatihan berakhir, tim pelaksana memberikan soal post-test dan kuisioner dengan waktu yang diberikan selama 1 jam. 3.2 Hasil Evaluasi Selama kegiatan pelatihan, peserta memberikan respon positif. Peserta antusias mengikuti keseluruhan rangkaian kegiatan dan mampu mengikuti maupun menyelesaikan tugas-tugas yang diberikan oleh Instruktur. Kondisi dan situasi selama pelatihan juga kondusif, disertai dengan fasilitas infrastruktur yang memadai dari Gereja Orthodox. y g j Hasil Evaluasi Kegiatan dari tiga jenis, yaitu hasil pre-test, post-test dan kuesioner, sebagai berikut: a. Hasil Pre-test dan Post-test Hasil Pre-test didapatkan nilai terendah dari peserta adalah 50, range 0 sampai 100 dan Nilai tertinggi 80 ditunjukkan gambar 5. Nilai terendah hasil post-test yaitu 77 dan nilai tertinggi sebesar 100, ditunjukkan Gambar 6. Peserta mengalami peningkatan sebesar 85.38 dalam nilai rata-rata post-test, naik sebesar 26,38 dari nilai rata-rata pre-test 59. Nilai perbandingan dapat dilihat pada Gambar 7. Gambar 5. Hasil Pre Test 0 10 20 30 40 50 60 70 80 90 Peserta 1 Peserta 2 Peserta 3 Peserta 4 Peserta 5 Peserta 6 Peserta 7 Peserta 8 NILAI PRE-TEST NILAI PRE-TEST Gambar 5. Hasil Pre Test https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 99 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Gambar 6. Hasil Post Test Gambar 7: Perbandingan Nilai Pre Test dan Post Test 0 20 40 60 80 100 120 Peserta 1 Peserta 2 Peserta 3 Peserta 4 Peserta 5 Peserta 6 Peserta 7 Peserta 8 NILAI POST TEST 59 85,38 Perbandingan Rata-rata Nilai Pre-Test dan Post Test Pre-Test Post Test Jurnal Riset dan Pengabdian Masyarakat Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Gambar 6. Hasil Post Test 0 20 40 60 80 100 120 Peserta 1 Peserta 2 Peserta 3 Peserta 4 Peserta 5 Peserta 6 Peserta 7 Peserta 8 NILAI POST TEST KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 E-ISSN: 2809-6509 Gambar 6. Hasil Post Test Gambar 6. Hasil Post Test Gambar 7: Perbandingan Nilai Pre Test dan Post Test 59 85,38 Perbandingan Rata-rata Nilai Pre-Test dan Post Test Pre-Test Post Test Perbandingan Rata-rata Nilai Pre-Test dan Post Test Gambar 7: Perbandingan Nilai Pre Test dan Post Test aldrpm.budiluhur.ac.id/index.php/Kresna/ 100 b. Hasil Kuesioner . Hasil Kuesioner Tabel 1 menunjukkan respon dari koresponden terhadap pertanyaan-pertanyaan kuesioner. Tabel 1 menunjukkan respon dari koresponden terhadap pertanyaan-pertanyaan kuesioner. Tabel 1 menunjukkan respon dari koresponden terhadap pertanyaan-pertanyaan kuesioner. Tabel 1. Hasil Respon Koresponden No. Pertanyaan SS S KS TS STS Total Responden 1 Program pelatihan Microsot Office yang diberikan menambah wawasan dan kesempatan Bapak/Ibu untuk kemajuan karir 0 5 3 0 0 8 2 Program pelatihan yang diberikan kepada Bapak/Ibu dapat meningkatkan ketrampilan dan produktifitas dalam pekerjaan 1 5 2 0 0 8 3 Atasan mendukung program pelatihan yang diikuti Bapak/Ibu 4 4 0 0 0 8 4 Program pelatihan dapat meningkatkan produktivitas Bapak/Ibu 1 4 3 0 0 8 5 Bapak/Ibu lebih menguasai pekerjaan yang diberikan setelah dilakukan pelatihan. 0 3 5 0 8 6 Lama waktu pelaksanaan sebanding dengan materi pelatihan 1 5 2 0 0 8 7 Pelatihan-pelatihan sangat dibutuhkan bagi karyawan agar dapat meningkatkan prestasi kerja. 1 5 2 0 0 8 8 Materi yang diberikan dalam pelatihan membuat tugas dan pekerjaan saya sangat terbantu. 1 5 2 0 0 8 https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 100 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Keterangan SS – Sangat Setuju, S – Setuju, KS – Kurang Setuju, TS – Tidak Setuju, STS – Sangat Tidak Setuju Skor Ideal = Bobot tertinggi x jumlah responden = 5 x 8 = 40 9 Materi yang diberikan pada saat pelatihan, belum pernah saya dapat sebelumnya 4 4 0 0 0 8 10 Program pelatihan ini dirasakan sangat bermanfaat bagi saya dalam meningkatkan produktivitas serta kualitas. 2 5 1 0 0 8 11 Perlunya diadakan pelatihan lanjutan agar kemampuan saya dapat dtingkatkan dalam melaksanakan tugas. 5 3 0 0 0 8 12 Program pelatihan seperti ini, sebaiknya dilaksanakan secara terus menerus. 4 4 0 0 0 8 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Keterangan SS – Sangat Setuju, S – Setuju, KS – Kurang Setuju, TS – Tidak Setuju, STS – Sang Tidak Setuju Skor Ideal = Bobot tertinggi x jumlah responden = 5 x 8 = 40 gg j p Tabel 2. Perhitungan Hasil Kuesioner Peserta Pelatihan No. b. Hasil Kuesioner K SS S KS TS STS Pembobotan Skor Aktual Prosentase Skor Aktual SS x 5 S x 4 KS x 3 SS x 2 SS x 1 (Skor Aktual/skor Ideal)*100% K1 0 5 3 0 0 0 20 9 0 0 29 73% K2 1 5 2 0 0 5 20 6 0 0 31 78% K3 4 4 0 0 0 20 16 0 0 0 36 90% K4 1 4 3 0 0 5 16 9 0 0 30 75% K5 0 3 5 0 0 0 12 15 0 0 27 68% K6 1 5 2 0 0 5 20 6 0 0 31 78% K7 1 5 2 0 0 5 20 6 0 0 31 78% K8 1 5 2 0 0 5 20 6 0 0 31 78% K9 4 4 0 0 0 20 16 0 0 0 36 90% K10 2 5 1 0 0 10 20 3 0 0 33 83% K11 5 3 0 0 0 25 12 0 0 0 37 93% K12 4 4 0 0 0 20 16 0 0 0 36 90% Rata-rata Presentase Skor Aktual 80.83% Keterangan: No.K-Nomor Pertanyaan, K1-Pertanyaan nomor 1, K2-Pertanyaan nomor 2, K3- Pertanyaan nomor 3 dst. Tabel 2. Perhitungan Hasil Kuesioner Peserta Pelatihan terangan: No.K-Nomor Pertanyaan, K1-Pertanyaan nomor 1, K2-Pertanyaan nomor 2, K3- rtanyaan nomor 3 dst. Tabel 3. Range Presentase Range Prosentase Kategori Prosentase 0% - 19,9% Buruk. 20% - 39,9% Tidak Baik 40% - 59,9% Kurang Baik 60% - 79,9% Baik 80% - 100% Sangat Baik Tabel 3. Range Presentase https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 4. KESIMPULAN Berdasarkan hasil dari evaluasi, maka dapat ditarik beberapa simpulan sebagai berikut: program pengabdian kepada masyarakat telah mampu memberikan manfaat bagi staf administrasi Gereja Orthodox Indonesia Jakarta. Tingkat pengetahuan peserta tentang penggunaan Microsoft Word dan https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ 101 KRESNA: Jurnal Riset dan Pengabdian Masyarakat Volume 3, Nomor 1, Mei, 2023, Hal: 95-102 E-ISSN: 2809-6509 Powerpoint sebelum dilaksanakan pelatihan masih kurang. Hal ini berdasarkan dengan nilai rata-rata hasil pretest sebesar 59. Setelah dilakukan pelatihan, tingkat pengetahuan dan keterampilan staf administrasi meningkat. Hasil post-test yang dilakukan oleh peserta, diperoleh nilai rata-rata sebesar 85,38, naik sebesar 26,38. Penyampaian materi tentang penggunaan Microsoft Word dan Powerpoint kepada peserta pelatihan dapat meningkatkan pengetahuan dan ketrampilan peserta, sehingga nantinya mampu digunakan dalam pekerjaaan. DAFTAR PUSTAKA [1] S. Rudiarto, S. Wardhana, S. Dwiasnati, and Zulfah, “Pelatihan Penggunaan Microsoft Power Point untuk Guru SMP 215 Jakarta,” PEMANAS: Jurnal Pengabdian Masyarakat Nasional, vol. 1, no. 1, pp. 8–17, 2021. [1] S. Rudiarto, S. Wardhana, S. Dwiasnati, and Zulfah, “Pelatihan Penggunaan Microsoft Power Point untuk Guru SMP 215 Jakarta,” PEMANAS: Jurnal Pengabdian Masyarakat Nasional, vol. 1, no. 1, pp. 8–17, 2021. [2] A. Aryasanti, R. Pradana, R. Irawati, D. Achadiani, and H. Irawan, “Pelatihan Pembuatan Surat Lamaran Kerja Berbasis Digital Branding,” JAM-TEKNO (Jurnal Pengabdian Kepada Masyarakat TEKNO), vol. 2, no. 2, pp. 1–6, 2021. [3] M. Wijana, Y. Muhyidin, and A. Wibowo, “Pelatihan Aplikasi Microsoft Office PowerPoint untuk Metode Pembelajaran,” Jurnal AbdiMU : Pengabdian Kepada Masyarakat, vol. 1, no. 2, pp. 48–52, 2021, doi: 10.32627. [4] K. Sirait et al., “Pelatihan Microsoft Office pada Pegawai Kantor KPU Medan,” Jurnal Pengabdian Pada Masyarakat METHABDI, vol. 1, no. 2, pp. 114–118, 2021. [5] F. A. S. N. Haq and K. Asmar, “Pelatihan Penggunaan Microsoft Word dalam Membua Pembelajaran,” JMS: Jurnal Masyarakat Siber, vol. 1, no. 1, pp. 27–32, 2022. [6] A. A. A. Ushud, “Pelatihan Powerpoint Bagi Karyawan Hassana Boga Sejahtera Powerpoint Trainning for Hassana Boga Sejahtera Employees,” KRESNA: Jurnal Riset dan Pengabdian Masyarakat, vol. 2, no. 2, pp. 288–294, 2022. [7] O. Sarta, M. Purba, A. Gunawan, E. Tri, and E. Handayani, “Pelatihan Dasar Penggunaan Microsoft Powerpoint Di MTS Asyafi’iyah 04 Jakarta,” Jurnal PKM: Pengabdian kepada Masyarakat, vol. 06, no. 01, 2023, [Online]. Available: www.DeepL.com/Translator y p [8] M. N. Arsyad and T. Ifianti, “Pelatihan Membuat Media Pembelajaran Berbasis Powerpoint Bagi Guru-guru Madrasah Ibtidaiyah”. [9] P. W. Aditama, I. G. A. Indrawan, I. K. A. G. Wiguna, and K. J. Atmaja, “Pelatihan Penggunaan Microsoft Office dan Email untuk Administrasi Surat Menyurat di Dinas Perhubungan Provinsi Bali,” Widya Laksmi, vol. 1, no. 1, pp. 37–42, 2021. , y pp [10] O. Alexander and E. N. Isnaini, “Pelatihan dan Sertifikasi Microsoft Office Pada SMK Karya Guna Bhakti 1,” JOURNAL OF EMPOWERMENT, vol. 2, no. 1, pp. 46–61, 2021. 102 https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/ https://jurnaldrpm.budiluhur.ac.id/index.php/Kresna/
https://openalex.org/W2275096063	https://pubs.rsc.org/en/content/articlepdf/2016/ra/c6ra90016j	English	null	Correction: Phase transformation-controlled synthesis of CuO nanostructures and their application as an improved material in a carbon-based modified electrode	RSC advances	2,016	cc-by	467	aKey Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou, 730070, China. E-mail: xuezh@nwnu.edu.cn; luxq@nwnu.edu.cn; Fax: +86 9317971323; Tel: +86 9317975276 bLanzhou City University, Lanzhou, 730070, China RSC Advances RSC Advances This journal is © The Royal Society of Chemistry 2016 Correction: Phase transformation-controlled synthesis of CuO nanostructures and their application as an improved material in a carbon- based modiﬁed electrode Correction: Phase transformation-controlled synthesis of CuO nanostructures and their application as an improved material in a carbon- based modiﬁed electrode Correction: Phase transformation-controlled synthesis of CuO nanostructures and their application as an improved material in a carbon- based modiﬁed electrode Zhonghua Xue,a Mengqian Li,a Honghong Rao,b Bo Yin,a Xibin Zhou,a Xiuhui Liua and Xiaoquan Lua Correction for ‘Phase transformation-controlled synthesis of CuO nanostructures and their application as an improved material in a carbon-based modiﬁed electrode’ by Zhonghua Xue et al., RSC Adv., 2016, 6, 12829–12836. The authors regret that in the original article the structures of glucose and gluconolactone in Scheme 2 are incorrect. A corrected version of Scheme 2, in which the orientations of a number of the hydroxyl groups have been corrected, is presented herein. Scheme 2 The schematic representation of the mechanism of the glucose oxidation based on CuO/GCE. Cite this: RSC Adv., 2016, 6, 20520 DOI: 10.1039/c6ra90016j www.rsc.org/advances Open Access Article. Published on 19 February 2016. Downloaded on 10/24/2024 6:48:37 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Correction for ‘Phase transformation-controlled synthesis of CuO nanostructures and their application as an improved material in a carbon-based modiﬁed electrode’ by Zhonghua Xue et al., RSC Adv., 2016, 6, 12829–12836. The authors regret that in the original article the structures of glucose and gluconolactone in Scheme 2 are incorrect. A corrected version of Scheme 2, in which the orientations of a number of the hydroxyl groups have been corrected, is presented herein. Scheme 2 The schematic representation of the mechanism of the glucose oxidation based on CuO/GCE. Open Access Article. Published on 19 F This article is licensed un Scheme 2 The schematic representation of the mechanism of the glucose oxidation based on CuO/GCE. Open Access Article. Published on 19 F This article is licensed un eme 2 The schematic representation of the mechanism of the glucose oxidation based on CuO/GCE. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. aKey Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou, 730070, China. E-mail: xuezh@nwnu.edu.cn; luxq@nwnu.edu.cn; Fax: +86 9317971323; Tel: +86 9317975276 bLanzhou City University, Lanzhou, 730070, China This journal is © The Royal Society of Chemistry 2016 20520 \| RSC Adv., 2016, 6, 20520 20520 \| RSC Adv., 2016, 6, 20520
https://openalex.org/W3041513180	https://hal.archives-ouvertes.fr/hal-03353129/document	English	null	Performance of Notched Connectors for CLT-Concrete Composite Floors	Buildings	2,020	cc-by	15,725	To cite this version: Minh-Van Thai, Sylvain Menard, Sidi Mohammed Elachachi, Philippe Galimard. Performance of Notched Connectors for CLT-Concrete Composite Floors. Buildings, 2020, 10 (7), pp.122. 10.3390/buildings10070122. hal-03353129 Distributed under a Creative Commons Attribution 4.0 International License Received: 2 June 2020; Accepted: 4 July 2020; Published: 8 July 2020 Abstract: CLT-concrete composite ﬂoor systems are a solution for timber buildings with a long-span ﬂoor. It yields a reduction of carbon footprint and even eco-friendly structure at the end of its service life. This study will evaluate the structural performance of notched connectors in the CLT-concrete composite ﬂoor, comprised of the serviceability stiﬀness, maximum load, and behavior at failure. The parameters of the test plan are the loaded edge length, the notch depth, the concrete thickness, and the screw length. Other secondary variables are also assessed, such as diﬀerent loading sequences, speed of test, and timber moisture content. Experimental results prove that the performance of the connector depends signiﬁcantly but not linearly on the notch depth and the length of the loaded edge. The connector with a deeper notch and a shorter heel will be stiﬀer and more robust, but it also tends to have a brittle rupture. The test results also help validate a solution for deconstructable connector systems. A nonlinear ﬁnite element model of the connector is built and validated versus the experimental results. It yields reasonably good predictions in terms of resistance and can capture the load-slip relationship. Keywords: shear test; CLT-concrete; notched connector; ﬁnite element model; deconstructable composite ﬂoor; timber-concrete composite ﬂoor; screw Performance of Notched Connectors for CLT-Concrete Composite Floors Composite Floors Minh Van Thai 1,2,, Sylvain Ménard 1, Sidi Mohammed Elachachi 2 and Philippe Galimard 2 1 Applied Sciences Department, University of Québec in Chicoutimi, Saguenay, QC G7H 2B1, Canada; sylvain_menard@uqac.ca 2 Civil and Environmental Engineering Department, University of Bordeaux, 33000 Bordeaux, France; sidi-mohammed.elachachi@u-bordeaux.fr (S.M.E.); philippe.galimard@u-bordeaux.fr (P.G.) Correspondence: minh-van.thai1@uqac.ca Minh Van Thai 1,2,, Sylvain Ménard 1, Sidi Mohammed Elachachi 2 and Philippe Galimar 1 Applied Sciences Department, University of Québec in Chicoutimi, Saguenay, QC G7H 2B1, Canada; sylvain_menard@uqac.ca 2 Civil and Environmental Engineering Department, University of Bordeaux, 33000 Bordeaux, France; sidi-mohammed.elachachi@u-bordeaux.fr (S.M.E.); philippe.galimard@u-bordeaux.fr (P.G.) Correspondence: minh-van.thai1@uqac.ca 1 Applied Sciences Department, University of Québec in Chicoutimi, Saguenay, QC G7H 2B1, Canada; sylvain_menard@uqac.ca 2 Civil and Environmental Engineering Department, University of Bordeaux, 33000 Bordeaux, France; sidi-mohammed.elachachi@u-bordeaux.fr (S.M.E.); philippe.galimard@u-bordeaux.fr (P.G.) * Correspondence: minh-van.thai1@uqac.ca Received: 2 June 2020; Accepted: 4 July 2020; Published: 8 July 2020 www.mdpi.com/journal/buildings buildings buildings buildings buildings Article Performance of Notched Connectors for CLT-Concrete Composite Floors Minh Van Thai 1,2,, Sylvain Ménard 1, Sidi Mohammed Elachachi 2 and Philippe Galimard 2 1 Applied Sciences Department, University of Québec in Chicoutimi, Saguenay, QC G7H 2B1, Canada; sylvain_menard@uqac.ca 2 Civil and Environmental Engineering Department, University of Bordeaux, 33000 Bordeaux, France; sidi-mohammed.elachachi@u-bordeaux.fr (S.M.E.); philippe.galimard@u-bordeaux.fr (P.G.) Correspondence: minh-van.thai1@uqac.ca Buildings 2020, 10, 122; doi:10.3390/buildings10070122 HAL Id: hal-03353129 https://hal.science/hal-03353129v1 Submitted on 23 Sep 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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 Keywords: shear test; CLT-concrete; notched connector; ﬁnite element model; deconstructable composite ﬂoor; timber-concrete composite ﬂoor; screw 1. Introduction French engineer Pierre-Eugene Gauthier developed the idea of cross-laminated timber (CLT), then patented it in 1952 [1]. It was redeveloped in Austria in early 1990 [2] and saw much broader usage in Europe by the 2000s. CLT is particularly suitable for ﬂoor system applications. There have been many successful applications of CLT in the construction of mid-rise and high-rise buildings. Brock Commons (Vancouver, BC, Canada) is the highest only residential building in timber construction with 17 stories in CLT panels. Origine Project (Quebec City, QC, Canada) is a 12-story building based on a concrete podium, constructed using CLT panels and glued-laminated timber beams. The idea of timber-concrete composite (TCC) was used for bridge structures in the 1940s and, recently, for the renovation of timber structures [3]. TCC has been an objective for many extensive studies regarding short-term [4], and long-term behavior [5,6], ﬁre resistance performance [7], and prefabricated solutions [3,8]. These studies emphasized the advantages of the TCC solution: resistance and rigidity, but also ﬁre and seismic resistance, easy and rapid installation, dry site, prefabrication capacity, acoustic and thermal isolation, environmental and in deconstruction. From an economic and environmental standpoint, TCC is an excellent balancing solution, rather than using an all reinforced concrete or all timber ﬂoor. CLT-concrete composite (CCC) structures inherit the advantage of a TCC one. In the case of mid- and high-rise buildings, CCC is notably more beneﬁcial than a TCC system, Buildings 2020, 10, 122; doi:10.3390/buildings10070122 2 of 21 Buildings 2020, 10, 122 i.e., wooden beam−concrete slab, because we could lose up to 30 cm for each TCC ﬂoor as compared to a CCC one. This means one ﬂoor per 10 story building for a deﬁned total height. The uses of CLT in timber-concrete composite structures are still in development. Though, there is a new successful application of the CCC at the Design Building, University of Massachusetts (Amherst, MA, USA). In this building, the ﬂoor span varied from 6 to 8 m, the ﬂoor section comprised a 175 mm of 5 ply CLT panel, 25 mm of rigid insulation, and 100 mm of reinforced concrete. The connector composite was the patented HBV system [9]. p y The connector system is the means to obtain the mentioned composite action. Since the timber-concrete connector would be deformable rather than inﬁnitely stiﬀ, the full composite section is impossible to achieve. 1. Introduction Slip between timber and concrete layer results in a partial composite action. Many connector solutions for TCC are available, such as shear interlock, bolts, screw, metal plates [10]. They have been widely studied for four decades [11–14]. These solutions are all applicable for CCC structures; some require a few minor modiﬁcations. There were many recent studies dedicated to CCC structures, as well as their connector systems. Gerber [15] tested two types of connectors: screws, and HBV mesh, on timber-concrete composite panels (CLT, LVL, LSL). The study showed that the analytical expressions could reasonably predict dynamic properties. Mai et al. [16] conducted shear tests on the individual screw connectors and applied them to the full-scale CLT-concrete beams. The authors observed a higher dynamic and static performance on composite structure than on bare CLT ﬂoor [17]. In the Oregon State University report [18], Higgins et al. carried out short-term and long-term tests on a full-scale CLT-concrete composite ﬂoor. They found that HBV mesh possesses a superior performance. Recently, Jiang and Crocetti [19] studied the performance of a single notched connector and a full-scale CLT-concrete beam. Lag screws and steel stirrup reinforced the notched connectors. The authors conﬁrmed that this type of solution is reliable, robust, stiﬀ, and inexpensive. Timber material has undeniable advantages in terms of carbon footprint; however, some sustainability issues remain when considering the end of service life. Since we opt for a composite solution due to the mentioned reasons, a TCC ﬂoor with permanent connector systems would spawn an amount of solid waste with an incredibly low possibility of reusing after the dismantling. This would cause a mixture of concrete and timber and hence make timber lose its environmental-friendly characteristic. Hence, an adaptation for the deconstructable connector will facilitate the dismantling of the CCC structure and enhance the reusability of materials [20]. In this study, we conducted a shear test on a reinforced notched connector of CCC structures. The main objectives are to investigate and compare the inﬂuence of many variables on the load-bearing capacity, the stiﬀness modulus, and the post-peak behavior that could be associated in some way to its ductility. The solution of a deconstructable connector for CCC structures is proposed and tested. A ﬁnite element model was also built in this study to understand the involved mechanisms, and the experimental result would validate this model. 2.1. Materials Properties For an application on-site, once we control this unknown variable, the removal of this film (if it is a case) would not cause any significant difference between the laboratory test and real behavior (a) (b) (c) Figure 1. Specimen with preinstalled polyethylene film (a), vertical attachment part with a threaded screw, counter-sunk washer and plastic sleeve installed (b), reinforcing steel mat of the specimens (c). Figure 1. Specimen with preinstalled polyethylene ﬁlm (a), vertical attachment part with a threaded screw, counter-sunk washer and plastic sleeve installed (b), reinforcing steel mat of the specimens (c). (a) (c) (b) (a) (b) (c) Figure 1. Specimen with preinstalled polyethylene film (a), vertical attachment part with a threaded screw, counter-sunk washer and plastic sleeve installed (b), reinforcing steel mat of the specimens (c). Figure 1. Specimen with preinstalled polyethylene ﬁlm (a), vertical attachment part with a threaded screw, counter-sunk washer and plastic sleeve installed (b), reinforcing steel mat of the specimens (c). A local supplier provided the concrete material with the indicated class of C35. The nominal aggregate size was 14 mm to assure a complete fill of concrete in the minimum notch’s depth of 20 mm. There was also water-reducing admixture in the mix to achieve the same objective. Standard ASTM C39 [23], the test method for compressive strength of cylindrical concrete specimens, was used for the measurements of the compression strength and the modulus of elasticity of concrete. The last column of Table 1 indicates the properties of the concrete material. To prevent the uplift phenomenon, screws or bolts were adopted as the vertical attachment [19]. For deconstructable composite floors, these screws must be easy to uninstall. According to Gutkowski et al. [25], some adjustments were made to simplify the implementation. The vertical attachment systems comprised a screw, a plastic sleeve, and a washer (Figure 1b). ASSY VG countersunk head screws (My-Ti-Con Ltd., Surrey, BC, Canada), with a diameter of 8 mm and full-length threaded, were used in the tests. They had an identical outer diameter and facilitated the installation of the sleeve. A 90° washer put A local supplier provided the concrete material with the indicated class of C35. The nominal aggregate size was 14 mm to assure a complete ﬁll of concrete in the minimum notch’s depth of 20 mm. There was also water-reducing admixture in the mix to achieve the same objective. 2.1. Materials Properties ** Experimentally measured on five cylindrical specimens according to the ASTM C39/C39M—18 standard [23]. After each shear test, moisture content of the timber was measured at six different randomly chosen locations on the two sides of the CLT part. The average moisture content of all tested The humidity exchange between timber and concrete is an open-ended question. Roughly speaking, if the timber absorbs water too much from concrete or vice versa, the stiﬀness and strength of both materials will be aﬀected [24]. Polyethylene ﬁlm was considered in the design since our knowledge of the humidity exchange between timber and concrete is limited for the case of CLT-concrete; hence, an unknown variable. Humidity isolation, in this case, is necessary. The thickness of the crystal polyethylene ﬁlm was about 50 µm (Figure 1a). This ﬁlm would also reduce a certain amount of friction between timber and concrete and compensate for the phenomenon of eccentricity. In the full-scale ﬂoor, this friction phenomenon is minor, and therefore the necessity of this layer lies in its capacity of humidity isolation. For an application on-site, once we control this unknown variable, the removal of this ﬁlm (if it is a case) would not cause any signiﬁcant diﬀerence between the laboratory test and real behavior. p g specimens had a mean of 15.7%, with a coefficient of variation (CoV) of 14%. The high moisture content of the wood is because the specimens were stored in an uncontrolled environment, and the measurement technique only allowed sampling up to 1 cm from the timber surface. The humidity exchange between timber and concrete is an open-ended question. Roughly speaking, if the timber absorbs water too much from concrete or vice versa, the stiffness and strength of both materials will be affected [24]. Polyethylene film was considered in the design since our knowledge of the humidity exchange between timber and concrete is limited for the case of CLT- concrete; hence, an unknown variable. Humidity isolation, in this case, is necessary. The thickness of the crystal polyethylene film was about 50 µm (Figure 1a). This film would also reduce a certain amount of friction between timber and concrete and compensate for the phenomenon of eccentricity. In the full-scale floor, this friction phenomenon is minor, and therefore the necessity of this layer lies in its capacity of humidity isolation. 2.1. Materials Properties The CLT material used in this study, provided by Nordic Structures® (Montréal, QC, Canada), was a 5-ply CLT and had a thickness of 175 mm. Its lamella conﬁguration is 35L-35T-35L-35T-35L, where “35” is the thickness in mm while “L” and “T” are the longitudinal and transversal directions, respectively. The timber specimens were E1 grade, complying with the standard ANSI/APA PRG-320-2019 [21]. E1 grade CLT should have 1950f-1.7E (the term “1950f-1.7E” represents the bending stress parallel to grain, 1950 lb/in2, and the modulus of elasticity, 1.7 × 106 lb/in2) Spruce-pine-ﬁr MSR lumber in all parallel (longitudinal) layers and No. 3 Spruce-pine-ﬁr lumber in all perpendicular (transversal) layers. The panel was face-glued by Purbond HD E202 adhesive (Henkel Canada Corporation, Mississauga, ON, Canada) and not edge-glued [22]. Table 1 presents the characteristic values of the CLT material. Buildings 2020, 10, 122 3 of 21 Table 1. Properties of CLT and concrete. Table 1. Properties of CLT and concrete. Property Unit CLT Longitudinal Layer * CLT Transversal Layer * Concrete ** Compression strength, fc MPa 23.6 8.5 36.8 Modulus of elasticity, E GPa 11.7 9.0 22.3 Density kg/m3 514 514 2262 * Standard properties of CLT given by the manufacturer [22]. ** Experimentally measured on ﬁve cylindrical specimens according to the ASTM C39/C39M—18 standard [23]. Buildings 2020, 10, x FOR PEER REVIEW 3 of 20 Table 1. Properties of CLT and concrete. Property Unit CLT Longitudinal Layer * CLT Transversal Layer * Concrete ** C i t th f MP 23 6 8 5 36 8 * Standard properties of CLT given by the manufacturer [22]. ** Experimentally measured on ﬁve cylindrical specimens according to the ASTM C39/C39M—18 standard [23]. Property Unit CLT Longitudinal Layer * CLT Transversal Layer * Concrete ** After each shear test, moisture content of the timber was measured at six diﬀerent randomly chosen locations on the two sides of the CLT part. The average moisture content of all tested specimens had a mean of 15.7%, with a coeﬃcient of variation (CoV) of 14%. The high moisture content of the wood is because the specimens were stored in an uncontrolled environment, and the measurement technique only allowed sampling up to 1 cm from the timber surface. Modulus of elasticity, E GPa 11.7 9.0 22.3 Density kg/m3 514 514 2262 * Standard properties of CLT given by the manufacturer [22]. 2.2. Test Specimens Buildings 2020, of Specimens Heel Length (a) in mm Notch Depth (b) in mm Screw Length (c) in mm Concrete Thickness (d) in mm A 5 400 20 160 80 B 5 400 20 220 100 C 5 400 35 160 100 D 5 400 35 220 80 E 5 300 20 160 100 F 5 300 20 220 80 G 5 300 35 160 80 H 5 300 35 220 100 I 6 400 25 220 80 J 3 300 25 220 80 K 3 350 20 220 80 L 3 350 35 220 80 M 5 350 25 220 80 Figure 2. Diagram of a typical specimen, with indicated (a) heel length, (b) notch depth, (c) screw length, (d) concrete thickness. Figure 2. Diagram of a typical specimen, with indicated (a) heel length, (b) notch depth, (c) screw length, (d) concrete thickness. Table 2. Parameters of test series, in mm. Serie No. of Specimens Heel Length (a) in mm Notch Depth (b) in mm Screw Length (c) in mm Concrete Thickness (d) in mm A 5 400 20 160 80 B 5 400 20 220 100 C 5 400 35 160 100 D 5 400 35 220 80 E 5 300 20 160 100 F 5 300 20 220 80 G 5 300 35 160 80 H 5 300 35 220 100 I 6 400 25 220 80 J 3 300 25 220 80 K 3 350 20 220 80 L 3 350 35 220 80 M 5 350 25 220 80 p p p g y p into thirteen series and tested in two phases, the first phase comprised of eight series from A to H, and five series from I to M in the second phase. Table 2 and Figure 2 present the detailed parameters of each series. Table 2. Parameters of test series, in mm. Serie No. 2.2. Test Specimens Buildings 2020, The “connector” is the zone that transfers the force from one material to another. The “connector” term in this study was used interchangeably with the “connector system,” which implied the assembly of the timber female part, the male concrete part, and screws. on each screw head was to compensate for the bearing load lost due to the sleeve. The head of the screw was at the same level as the top surface of the concrete layer. The plastic sleeve prevented contact between the screws and the concrete. This solution provided easy access to the head of the CLT panels of 175 mm thickness were all cut into pieces with a dimension of 300 mm by 750 mm. A rectangular notch was then cut into the CLT specimen by using a round drill bit. The four corners of the notch were round at the radius of 20 mm. The concrete layer had a thickness of 80 mm or 100 mm. In this study, the part in front of the loaded edge was called the heel of the connector. This is the timber part that supported the compression and shear force transferred from the male concrete part. In our study, the heel part usually comprised more than two lamellas and the round edge of the loaded side. Heel length had three possible levels: 300, 350, and 400 mm. Notch depth varied in three-level: 20, 25, and 35 mm, without exceeding the maximum thickness of the ﬁrst CLT layer. Two self-tapping screws reinforced each connector. The length of the screw was 160 mm or 220 mm. A reinforcing steel mat of 150 mm of the square spacing and a diameter of 6 mm were put in place to prevent a premature crack in concrete due to shrinkage. Sixty specimens were distributed into thirteen series and tested in two phases, the ﬁrst phase comprised of eight series from A to H, and ﬁve series from I to M in the second phase. Table 2 and Figure 2 present the detailed parameters of each series. screw to uninstall it. 2.2. Test Specimens The “connector” is the zone that transfers the force from one material to another. The “connector” term in this study was used interchangeably with the “connector system,” which implied the assembly of the timber female part, the male concrete part, and screws. 2.1. Materials Properties Standard ASTM C39 [23], the test method for compressive strength of cylindrical concrete specimens, was used for the measurements of the compression strength and the modulus of elasticity of concrete. The last column of Table 1 indicates the properties of the concrete material. To prevent the uplift phenomenon, screws or bolts were adopted as the vertical attachment [19]. For deconstructable composite ﬂoors, these screws must be easy to uninstall. According to Gutkowski et al. [25], some adjustments were made to simplify the implementation. The vertical attachment systems comprised a screw, a plastic sleeve, and a washer (Figure 1b). ASSY VG countersunk head screws (My-Ti-Con Ltd., Surrey, BC, Canada), with a diameter of 8 mm and full-length threaded, were used in the tests. They had an identical outer diameter and facilitated the installation of the sleeve. A 90◦washer put on each screw head was to 4 of 21 Buildings 2020, 10, 122 compensate for the bearing load lost due to the sleeve. The head of the screw was at the same level as the top surface of the concrete layer. The plastic sleeve prevented contact between the screws and the concrete. This solution provided easy access to the head of the screw to uninstall it. 2.2. Test Specimens Buildings 2020, CLT panels of 175 mm thickness were all cut into pieces with a dimension of 300 mm by 750 mm. A rectangular notch was then cut into the CLT specimen by using a round drill bit. The four corners of the notch were round at the radius of 20 mm. The concrete layer had a thickness of 80 mm or 100 mm. In this study, the part in front of the loaded edge was called the heel of the connector. This is the timber part that supported the compression and shear force transferred from the male concrete part. In our study, the heel part usually comprised more than two lamellas and the round edge of the loaded side. Heel length had three possible levels: 300, 350, and 400 mm. Notch depth varied in three-level: 20, 25, and 35 mm, without exceeding the maximum thickness of the first CLT layer. Two self-tapping screws reinforced each connector. The length of the screw was 160 mm or 220 mm. A reinforcing steel mat of 150 mm of the square spacing and a diameter of 6 mm were put in Table 2. Parameters of test series, in mm. Serie No. of Specimens Heel Length (a) in mm Notch Depth (b) in mm Screw Length (c) in mm Concrete Thickness (d) in mm A 5 400 20 160 80 B 5 400 20 220 100 C 5 400 35 160 100 D 5 400 35 220 80 E 5 300 20 160 100 F 5 300 20 220 80 G 5 300 35 160 80 H 5 300 35 220 100 I 6 400 25 220 80 J 3 300 25 220 80 K 3 350 20 220 80 L 3 350 35 220 80 M 5 350 25 220 80 place to prevent a premature crack in concrete due to shrinkage. Sixty specimens were distributed into thirteen series and tested in two phases, the first phase comprised of eight series from A to H, and five series from I to M in the second phase. Table 2 and Figure 2 present the detailed parameters of each series. Table 2. Parameters of test series, in mm. Serie No. 2.3. Test Setups The conﬁguration adopted for the shear test is an asymmetrical specimen system. This type of conﬁguration was cheaper to fabricate than the symmetrical one, and a more signiﬁcant number of the specimens could be tested. The diﬀerence is that the asymmetrical test would estimate the shear stiﬀness and strength higher than the symmetrical one [12]. The eccentricity moment occurred when the testing machine applied on the timber part; this would generate compression force and hence the friction between timber and concrete on the upper part of the specimen [11]. For TCC connector systems, Lukaszewska [11] estimated that the stiﬀness and strength diﬀerence between an asymmetric specimen and a symmetric one was about 10%, depending on the specimen dimensions. In this study, since there was a thin polyethylene ﬁlm at the material interface, the eﬀect of this phenomenon might be lessened. The bench adaptor comprised of two I-proﬁles welded perpendicular to each other. The specimen was placed on a ﬁxed metal plate, only by the concrete part. The moving part of the testing machine transferred the load on a metal plate, then distributed the charge to the timber part of the specimen. Teﬂon plate was put on the vertical I-proﬁle to reduce the friction between the sample and the I-proﬁle (Figure 3). The testing machine, driven by a deﬁned displacement rate, could produce a maximum charge of about 445 kN. Two analog laser sensors (ALSs), were ﬁxed on two sides of the sample to capture the relative displacements between the two layers. The ALSs were ﬁxed on the timber part by screws. The install area of the two ALSs was at about 150 cm to the edge of the sample. This location was where the loaded edge of the notched commences. The ALSs were connected to ampliﬁer units before transmitting the data to the computer. Another displacement sensor was integrated into the testing machine to measure the total displacement of the timber part. The vertical charge applied to the specimen was measured by a sensor of the testing machine. Standard EN 26891 [26] proposed a protocol for the determination of resistance characteristics of the timber connector subjected to static loading. The total time for testing was about 15 minutes. The condition for the ending of the test was whether the charge has a signiﬁcant drop, or the relative displacement reached 15 mm. 2.2. Test Specimens Buildings 2020, • The notch depth was a critical parameter since the characteristic of CLT is sensitive in the depth direction, i.e., one layer is perpendicular adjacent to the other. • Since the notch depth was not exceeded the thickness of the ﬁrst layer of the lamella, and the characteristic of this layer was uniform transversally. A ﬁxed notch’s width of 200 mm was chosen. • The thickness of the concrete layer was included in the testing plan. This variable was to verify the inﬂuence of the eccentricity (between axial force in timber and in concrete). • We chose to use 5-ply-CLT and hence ﬁxed the thickness of the timber layer to 175 mm. This is because a thinner (three plies) or thicker (seven plies) CLT would not be suitable for our future application of long-span composite ﬂoor systems (~9 m span). • We chose to use 5-ply-CLT and hence ﬁxed the thickness of the timber layer to 175 mm. This is because a thinner (three plies) or thicker (seven plies) CLT would not be suitable for our future application of long-span composite ﬂoor systems (~9 m span). 2.2. Test Specimens Buildings 2020, of Specimens Heel Length (a) in mm Notch Depth (b) in mm Screw Length (c) in mm Concrete Thickness (d) in mm A 5 400 20 160 80 B 5 400 20 220 100 C 5 400 35 160 100 D 5 400 35 220 80 E 5 300 20 160 100 F 5 300 20 220 80 G 5 300 35 160 80 H 5 300 35 220 100 I 6 400 25 220 80 J 3 300 25 220 80 K 3 350 20 220 80 L 3 350 35 220 80 M 5 350 25 220 80 Figure 2. Diagram of a typical specimen, with indicated (a) heel length, (b) notch depth, (c) screw length, (d) concrete thickness. Figure 2. Diagram of a typical specimen, with indicated (a) heel length, (b) notch depth, (c) screw length, (d) concrete thickness. Table 2. Parameters of test series, in mm. p p p g y p into thirteen series and tested in two phases, the first phase comprised of e and five series from I to M in the second phase Table 2 and Figure 2 present Table 2. Parameters of test series, in mm. sted in two phases, the first phase comprised of M in the second phase Table 2 and Figure 2 presen Figure 2. Diagram of a typical specimen, with indicated (a) heel length, (b) notch depth, (c) screw length, (d) concrete thickness. Figure 2. Diagram of a typical specimen, with indicated (a) heel length, (b) notch depth, (c) screw length, (d) concrete thickness. 5 of 21 Buildings 2020, 10, 122 Many variables were considered for the experimental plan. They were the design dimension one of the notched connectors: the length of the heel part, the length, depth, width of the notch, and thickness of the timber and concrete layer: • To study the inﬂuence of the screw and to validate the unscrewing option, the screw length was included in the plan. • To study the inﬂuence of the screw and to validate the unscrewing option, the screw length was included in the plan. • A study in the LVL-concrete individual notched connector [24] showed that the length of the notch is not a signiﬁcant issue in terms of stiﬀness and strength. • The aspect of eccentricity and the derived compression between timber-concrete was much related to the length of the heel part. 2.3. Test Setups Once the sample was in place and well-positioned, the speed test was set by turning the dial manually. The loading speed of the test was averagely 1.3 mm per minute, and it varied greatly due In this study, we used the notation K1 and K2 as the slip modulus of the ﬁrst loading sequence and second loading sequence, respectively (Figure 4). By applying the linear regression y = βy + α on all the data points between the modiﬁed displacement, K1 and K2 were obtained as the slope of the regression line. The maximum load Fmax was the load reached at the curve peak or 15 mm of displacement. Both K1 and K2 were deﬁned over the range from 10% to 40% of the maximum load. The modulus K1 had the same meaning as ks, although we used the regression slope instead of an arbitrated displacement point for the determination of the modulus. In the other hand, K2 had no counterpart in the standard in terms of physical meaning (modulus of second loading sequence). From our point of view, K2 is more suitable for the calculation of vibration behavior. Buildings 2020, 10, x FOR PEER REVIEW 7 of 20 manual handling. However, with the order of magnitude of loading speed, the result shows no relation between it and the stiffness or the resistance. After the tests, we measured the moisture ntent and then dismantled some specimens for further study. Specimen disassembling helped us a closer look at the failure mechanism and define the type of failure. According to Standard EN 26891, the slip modulus of the service state limit 𝑘௦ is defined on the tial modified displacement, from 10% to 40% of the maximum load. This definition was proposed iquely for the first loading sequence. The range from 0% to 10% of the maximum load comprised very first loading sequence; the contacts between connector elements were not assured. In this study, we used the notation 𝐾ଵ and 𝐾ଶ as the slip modulus of the first loading sequence d second loading sequence, respectively (Figure 4). By applying the linear regression 𝑦= 𝛽𝑦+ n all the data points between the modified displacement, 𝐾ଵ and 𝐾ଶ were obtained as the slope the regression line. The maximum load 𝐹௠௔௫ was the load reached at the curve peak or 15 mm of placement. 2.3. Test Setups We used a customized forklift to put the specimen into the bench adaptor. Once the sample was in place and well-positioned, the speed test was set by turning the dial manually. The loading speed of the test was averagely 1.3 mm per minute, and it varied greatly due 6 of 21 Buildings 2020, 10, 122 to manual handling. However, with the order of magnitude of loading speed, the result shows no correlation between it and the stiﬀness or the resistance. After the tests, we measured the moisture content and then dismantled some specimens for further study. Specimen disassembling helped us get a closer look at the failure mechanism and deﬁne the type of failure. Buildings 2020, 10, x FOR PEER REVIEW 6 of 20 Figure 3. Adaptor for the asymmetrical CLT-concrete specimen. Figure 3. Adaptor for the asymmetrical CLT-concrete specimen. Figure 3. Adaptor for the asymmetrical CLT-concrete specimen. Figure 3. Adaptor for the asymmetrical CLT-concrete specimen. Two analog laser sensors (ALSs), were fixed on two sides of the sample to capture the relative displacements between the two layers. The ALSs were fixed on the timber part by screws. The install area of the two ALSs was at about 150 cm to the edge of the sample. This location was where the loaded edge of the notched commences. The ALSs were connected to amplifier units before According to Standard EN 26891, the slip modulus of the service state limit ks is deﬁned on the initial modiﬁed displacement, from 10% to 40% of the maximum load. This deﬁnition was proposed uniquely for the ﬁrst loading sequence. The range from 0% to 10% of the maximum load comprised the very ﬁrst loading sequence; the contacts between connector elements were not assured. g p transmitting the data to the computer. Another displacement sensor was integrated into the testing machine to measure the total displacement of the timber part. The vertical charge applied to the specimen was measured by a sensor of the testing machine. Standard EN 26891 [26] proposed a protocol for the determination of resistance characteristics of the timber connector subjected to static loading. The total time for testing was about 15 minutes. The condition for the ending of the test was whether the charge has a significant drop, or the relative displacement reached 15 mm. We used a customized forklift to put the specimen into the bench adaptor. 2.3. Test Setups Both 𝐾ଵ and 𝐾ଶ were defined over the range from 10% to 40% of the maximum load. e modulus 𝐾ଵ had the same meaning as 𝑘௦, although we used the regression slope instead of an bitrated displacement point for the determination of the modulus. In the other hand, 𝐾ଶ had no unterpart in the standard in terms of physical meaning (modulus of second loading sequence). (a) (b) Figure 4. Theoretical loading procedure from EN 26891 (a) and stiffness K1 (red) and K2 (green) determination of specimen A2 (blue) on raw data representation (b). Figure 4. Theoretical loading procedure from EN 26891 (a) and stiﬀness K1 (red) and K2 (green) determination of specimen A2 (blue) on raw data representation (b). g relation between it and the stiffness or t tent and then dismantled some specime a closer look at the failure mechanism an According to Standard EN 26891, the s ial modified displacement, from 10% to quely for the first loading sequence. The very first loading sequence; the contacts In this study, we used the notation 𝐾ଵ second loading sequence, respectively n all the data points between the modifi he regression line. The maximum load 𝐹 placement. Both 𝐾ଵ and 𝐾ଶ were define d l 𝐾h d h i (a) e resistance. After the tests, we measured the moistur s for further study. Specimen disassembling helped u d define the type of failure. p modulus of the service state limit 𝑘௦ is defined on th 0% of the maximum load. This definition was proposed ange from 0% to 10% of the maximum load comprised etween connector elements were not assured. nd 𝐾ଶ as the slip modulus of the first loading sequenc Figure 4). By applying the linear regression 𝑦= 𝛽𝑦+ d displacement, 𝐾ଵ and 𝐾ଶ were obtained as the slop ௔௫ was the load reached at the curve peak or 15 mm o over the range from 10% to 40% of the maximum load 𝑘 l h h d h i l i d f (b) e e 𝐾ଵ an (a) % to 40 (b) modulus 𝐾ଵ had the same meaning as 𝑘௦, although we used the regression slope instead of a rated displacement point for the determination of the modulus. In the other hand, 𝐾ଶ had n terpart in the standard in terms of physical meaning (modulus of second loading sequence Figure 4. 2.3. Test Setups Theoretical loading procedure from EN 26891 (a) and stiffness K1 (red) and K2 (green) determination of specimen A2 (blue) on raw data representation (b). Figure 4. Theoretical loading procedure from EN 26891 (a) and stiﬀness K1 (red) and K2 (green) determination of specimen A2 (blue) on raw data representation (b). 7 of 21 7 of 21 Buildings 2020, 10, 122 3.1. Overview Three diﬀerent failure types were observed: ductile failure of the specimen due to the compression in the timber contact zone (type timber ductile - TD), brittle failure of the specimen due to the shearing-oﬀof the timber lamellas in the heel (type timber brittle - TB). Furthermore, there was a combination of type TD and TB. The specimen had crushed timber and a part of its lamella sheared-oﬀ (type TD + TB). Brittle failure of the specimen due to the shear failure of the concrete part was noted as CB (type concrete brittle). The average stiﬀness and strength of the 13 series are presented in Table 3, with the corresponding coeﬃcient of variation (CoV). Table 3. Summary of tested series. Series K1 K2 Fmax Mean CoV Mean CoV Mean CoV kN/mm % kN/mm % kN % A 224 13 294 5 127 10 B 213 18 303 15 140 9 C 248 8 311 8 221 5 D 238 12 315 5 221 11 E 199 16 258 7 140 4 F 202 4 256 6 151 7 G 208 9 274 11 211 7 H 195 19 254 13 217 9 I 242 13 326 13 177 4 J 208 1 286 7 169 5 K 202 1 270 13 158 7 L 205 7 291 17 238 6 M 212 12 311 14 175 10 Table 3. Summary of tested series. As compared to the results of other studies [15,16,27,28], stiﬀness of the notched connector was on average higher than for the screw connector, but still far lower than HBV mesh. A pair of screws could have a shear stiﬀness that varies from 0.14 kN/mm2 to 0.3 kN/mm2, while that of HBV mesh was about 0.825 kN/mm2. The experimental results of this study showed that the stiﬀness varied from 0.34 to 0.43 kN/mm2. The unit kN/mm2 indicates the shear stiﬀness (kN/mm) per connector length (mm). Figure 5 presents the load-displacement curves of 60 specimens distinguished by their series. The resistance varied from 108 kN to 253 kN depend on the notch geometry, especially the depth of the connector. 8 of 21 erials to 8 of 20 Buildings 2020, 10, 122 length (Figures 5b di i t th h Buildings 2020, 10, x FOR Figure 5. 3.1. Overview Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Influence of Heel Length Figure 6 exhibits the relationship between the heel length and the variables of interest, namely K2, and Fmax. One can see an increase of about 15% of the stiffness K1, K2, when the heel length Figure 5. Cont. gth (Figures 5b and 6c). This is because the shorter heel length specimens have few materials to ipate the charge and more likely sustain the timber softening. Figure 5. Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Influence of Heel Length Figure 5. Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. There were distinct gaps between the curve family with the heel length of 400 mm (Figure 5a). h k b h l h f d h l l h Figure 5. Cont. th (Figures 5b and 6c). This is because the shorter heel length specimens have few materials ipate the charge and more likely sustain the timber softening. Figure 5. Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Influence of Heel Length Figure 5. Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Figure 5. Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Influence of Heel Length Figure 5. Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Figure 5. 3.1. Overview Load-displacement curves of specimens that have (a) 400 mm, (b) 350 mm, (c) 300 mm heel length, curves in red indicate 20 mm notch depth specimens, 25 mm in green, and 35 mm in blue. Figure 6 exhibits the relationship between the heel length and the variables of interest, namely K1, K2, and Fmax. One can see an increase of about 15% of the stiffness K1, K2, when the heel length increases from 300 mm to 400 mm (Figure 6a,b). Heel length was assumed not to influence either the stiffness or strength of the connector. This slight increase was probably because of the asymmetrical 3.2. Influence of Heel Length Figure 6 exhibits the relationship between the heel length and the variables of interest, namely K1, K2, and Fmax. One can see an increase of about 15% of the stiffness K1, K2, when the heel length increases from 300 mm to 400 mm (Figure 6a,b). Heel length was assumed not to influence either the There were distinct gaps between the curve family with the heel length of 400 mm (Figure 5a). However, the post-peak behavior was less consistent in other series of 350 mm and 300 mm heel length (Figures 5b and 6c). This is because the shorter heel length specimens have few materials to dissipate the charge and more likely sustain the timber softening. Figure 6 exhibits the relationship between the heel length and the variables of interest, namely K1, K2, and Fmax. One can see an increase of about 15% of the stiffness K1, K2, when the heel length increases from 300 mm to 400 mm (Figure 6a,b). Heel length was assumed not to influence either the stiffness or strength of the connector. This slight increase was probably because of the asymmetrical 3.2. Influence of Heel Length Figure 6 exhibits the relationship between the heel length and the variables of interest, namely K1, K2, and Fmax. One can see an increase of about 15% of the stiffness K1, K2, when the heel length increases from 300 mm to 400 mm (Figure 6a,b). Heel length was assumed not to influence either the There were distinct gaps between the curve family with the heel length of 400 mm (Figure 5a). However, the post-peak behavior was less consistent in other series of 350 mm and 300 mm heel length (Figures 5b and 6c). 3.3. Influence of Notch Depth 3.2. Inﬂuence of Heel Length the sample size, and 𝑡௡ିଵ is of freedom. Since the stand f f p Figure 7a,b show the evolution of slip modulus when the notch depth increases. A deeper cut did not yield a stiffer connector. In most of the cases, the notch with 25 mm depth had the highest stiffness. In the notch with 35 mm of depth, the timber material of the first layer of the lamella was extracted entirely, and the second layer, which laid in the direction perpendicular to the first one, was weaker in terms of modulus perpendicular to the grain. The transversal timber lamellas were also not glued edgewise. They could be the reason for the “peak” trend of the slip modulus curves. Figure 6 exhibits the relationship between the heel length and the variables of interest, namely K1, K2, and Fmax. One can see an increase of about 15% of the stiﬀness K1, K2, when the heel length increases from 300 mm to 400 mm (Figure 6a,b). Heel length was assumed not to inﬂuence either the stiﬀness or strength of the connector. This slight increase was probably because of the asymmetrical properties of the test. The lengthy heel magniﬁed the eccentricity and the friction between concrete and timber. The resistance of the connector of diﬀerent notch depth distinguished clearly from each other’s; they developed almost independently regarding their heel length. Modulus K2 was more consistent than K1 since the specimen was stabilized after the ﬁrst loading sequence. In terms of the eﬀect of heel length on the failure type, a specimen with shorter heel tended to have its lamellas sheared-oﬀat failure. The error bars in the graphs represent the 95% conﬁdence interval of the mean value x. They are calculated as x ± tn−1.s/ √n, with s is the standard deviation of the sample, n is the sample size, and tn−1 is the upper (1 −0.95)/2 critical value for the t distribution with (n −1) degrees of freedom. Since the standard error was signiﬁcant in some average data points, the evolution of stiﬀness K1, K2, was challenging to be veriﬁed. stiffness K1, K2, was challenging to be verified. Figure 6. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) of different notch depths. 3.1. Overview This is because the shorter heel length specimens have few materials to dissipate the charge and more likely sustain the timber softening. Buildings 2020, 10, 122 p of freedom. Since 9 of 21 g ution of 9 of 21 g ution of Figure 6. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) of different notch depths. Figure 6. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) of diﬀerent notch depths. Buildings 2020, 10, x FOR PEER REVIEW 9 of 20 properties of the test. The lengthy heel magnified the eccentricity and the friction between concrete and timber. The resistance of the connector of different notch depth distinguished clearly from each other’s; they developed almost independently regarding their heel length. Modulus K2 was more consistent than K1 since the specimen was stabilized after the first loading sequence. In terms of the effect of heel length on the failure type, a specimen with shorter heel tended to have its lamellas sheared-off at failure. The error bars in the graphs represent the 95% confidence interval of the mean value 𝑥̅. They are calculated as 𝑥̅ ± 𝑡௡ିଵ. 𝑠/√𝑛, with 𝑠 is the standard deviation of the sample, 𝑛 is th l i d 𝑡 i th (1 0 95)/2 iti l l f th t di t ib ti ith ( 1) d Buildings 2020, 10, x FOR PEER REVIEW properties of the test. The lengthy and timber. The resistance of the c other’s; they developed almost ind consistent than K1 since the specim effect of heel length on the failure sheared-off at failure The error bar 9 of 20 and the friction between concrete h distinguished clearly from each el length. Modulus K2 was more loading sequence. In terms of the heel tended to have its lamellas pe, a specimen with shorter the graphs represent the 95% Figure 6. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) of different notch depths. Figure 6. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) of diﬀerent notch depths. g p p They are calculated as 𝑥̅ ± 𝑡௡ିଵ. 𝑠/√𝑛, with 𝑠 is the standard deviation of the sam 3.3. Inﬂuence of Notch Depth 3.3. Influence of Notch Depth Figure 7. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) of different heel lengths. Figure 8a,b show the stiffness K1 and K2 per notch depth. The modulus gained per millimeter of notch depth was higher in the less deep notches. The shallow notch used the material more Figure 7a,b show the evolution of slip modulus when the notch depth increases. A deeper cut did not yield a stiﬀer connector. In most of the cases, the notch with 25 mm depth had the highest stiﬀness. In the notch with 35 mm of depth, the timber material of the ﬁrst layer of the lamella was extracted entirely, and the second layer, which laid in the direction perpendicular to the ﬁrst one, was weaker in terms of modulus perpendicular to the grain. The transversal timber lamellas were also not glued edgewise. They could be the reason for the “peak” trend of the slip modulus curves. Figure 7a,b show the evolution of slip modulus when the notch depth increases. A deeper cut did not yield a stiffer connector. In most of the cases, the notch with 25 mm depth had the highest stiffness. In the notch with 35 mm of depth, the timber material of the first layer of the lamella was extracted entirely, and the second layer, which laid in the direction perpendicular to the first one, was weaker in terms of modulus perpendicular to the grain. The transversal timber lamellas were also not glued edgewise. They could be the reason for the “peak” trend of the slip modulus curves. effectively in terms of stiffness, and further extraction of material in the topmost longitudinal laye would reduce the effectiveness of the connector. The linear correlations between the stiffness pe depth and the notch depth were also observed. The resistance of the connector is higher for the deeper cut (Figure 8c). An increase of th resistance of about 50% was observed when the cut more profound. The correlation between notch Figure 7. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) of different heel lengths. Figure 7. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) of diﬀerent heel lengths. and further extraction of materia of the connector. The linear cor also observed. 3.3. Inﬂuence of Notch Depth 3.3. Influence of Notch Depth Hence, the curves of these specimens had a more prolonged post-peak displacement that varied from 10 to 15 mm. Optimization of the notch depth will have to balance between the performance and the post-peak behavior. (i.e., modulus K1 and K2). It meant that the experimental measurement of stiffness was difficult, and the maximum load of the notch would be more straightforward to be predicted by the variable of notch depth. A shallower notch connector tended to have the loaded edge crushed rather than the shear-off lamellas (cf. Section 3.7). Hence, the curves of these specimens had a more prolonged post- peak displacement that varied from 10 to 15 mm. Optimization of the notch depth will have to balance between the performance and the post-peak behavior. For a CCC notched connector, Jiang et al. [19] reported a serviceability stiffness per 25 mm notch- depth of 15.3 kN/mm2 and the resistance per depth of 7.1 kN/mm, while the corresponding results of our study were 12.5 kN/mm2 and 7.0 kN/mm. The notch connectors in this study featured a round corner at the loaded edge while Jiang et al. tested a full-width square notch. This detail generated a transverse component of the applied force exerted on the notch. It might be the reason for the less stiff connector observed in this study. Figure 8. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) per notch depth of different heel Figure 8. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) per notch depth of diﬀerent heel lengths. Figure 8. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) per notch depth of different heel Figure 8. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) per notch depth of diﬀerent heel lengths. lengths. 3.4. Influence of Concrete Thickness and Screw Length Figure 9 shows some minor changes (about 10% maximum) of the stiffnesses and the resistance of the connector in terms of concrete thickness and screw length. It could be concluded that these two variables do not influence the overall performance of individual notch connectors. Since the implementation of the deconstructable screw part yielded no difference in terms of the structural For a CCC notched connector, Jiang et al. 3.3. Inﬂuence of Notch Depth 3.3. Influence of Notch Depth ector is higher for the deeper c erved when the cut more profou effectively in terms of stiffness, would reduce the effectiveness depth and the notch depth were The resistance of the conn resistance of about 50% was obs al in the topmost longitudinal lay rrelations between the stiffness p cut (Figure 8c). An increase of th und. The correlation between notc Figure 7. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) of different heel lengths. Figure 7. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) of diﬀerent heel lengths. Buildings 2020, 10, 122 10 of 21 Figure 8a,b show the stiﬀness K1 and K2 per notch depth. The modulus gained per millimeter of notch depth was higher in the less deep notches. The shallow notch used the material more eﬀectively in terms of stiﬀness, and further extraction of material in the topmost longitudinal layer would reduce the eﬀectiveness of the connector. The linear correlations between the stiﬀness per depth and the notch depth were also observed. Buildings 2020, 10, x FOR PEER REVIEW 10 of 20 depth and the maximum load Fmax was almost linear. The notch depth had a more significant effect on maximum load, as compared to the effect of heel length in Figure 6c. The coefficient of variations of mean data points of the maximum load was considerably smaller than the other two responses (i e modulus K1 and K2) It meant that the experimental measurement of stiffness was difficult and The resistance of the connector is higher for the deeper cut (Figure 8c). An increase of the resistance of about 50% was observed when the cut more profound. The correlation between notch depth and the maximum load Fmax was almost linear. The notch depth had a more signiﬁcant eﬀect on maximum load, as compared to the eﬀect of heel length in Figure 6c. The coeﬃcient of variations of mean data points of the maximum load was considerably smaller than the other two responses (i.e., modulus K1 and K2). It meant that the experimental measurement of stiﬀness was diﬃcult, and the maximum load of the notch would be more straightforward to be predicted by the variable of notch depth. A shallower notch connector tended to have the loaded edge crushed rather than the shear-oﬀlamellas (cf. Section 3.7). 3.3. Inﬂuence of Notch Depth 3.3. Influence of Notch Depth [19] reported a serviceability stiﬀness per 25 mm notch-depth of 15.3 kN/mm2 and the resistance per depth of 7.1 kN/mm, while the corresponding results of our study were 12.5 kN/mm2 and 7.0 kN/mm. The notch connectors in this study featured a round corner at the loaded edge while Jiang et al. tested a full-width square notch. This detail generated a transverse component of the applied force exerted on the notch. It might be the reason for the less stiﬀconnector observed in this study. i p e e tatio o t e deco st uctab e sc ew pa t performance of the connector, this solution was 3.4. Inﬂuence of Concrete Thickness and Screw Length uninstalling of the screws was carried out without any difficulty (Figure 10) by using a wired screwdriver. Figure 9 shows some minor changes (about 10% maximum) of the stiﬀnesses and the resistance of the connector in terms of concrete thickness and screw length. It could be concluded that these two variables do not inﬂuence the overall performance of individual notch connectors. Since the implementation of the deconstructable screw part yielded no diﬀerence in terms of the structural performance of the connector, this solution was possible for CLT-concrete ﬂoors systems. The uninstalling of the screws was carried out without any diﬃculty (Figure 10) by using a wired screwdriver. Buildings 2020, 10, 122 p uninstalling of the 11 of 21 a wired 11 of 21 a wired Figure 9. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) in terms of screw length and concrete thickness. Figure 9. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) in terms of screw length and concrete thickness. Figure 9. Stiffness K1 (a), K2 (b), and maximum load Fmax (c) in terms of screw length and concrete thickness. Figure 9. Stiﬀness K1 (a), K2 (b), and maximum load Fmax (c) in terms of screw length and concrete thickness. 3.5. Inﬂuence of Loading Sequence The modulus of the second loading sequence K2 was on average 35% higher than for K1. This is because the contact between the component of the connector was well established after the ﬁrst loading sequence. Any possible gaps between them were closed, and the material is stabilized. Modulus K2 in terms of the heel length and the notch depth had almost the same and more transparent tendencies, as compared to modulus K1. Although it is worth noting that the conﬁdence interval of the mean values was still considerable. The stiﬀness results were not more consistent after the ﬁrst loading-unloading cycle. The loading speed was manually set for each specimen and constant in the loading sequences. This variable was massively varied but did not have any signiﬁcant eﬀect on the stiﬀness and the maximum load. Buildings 2020, 10, x FOR PEER REVIEW 11 of 20 Figure 10. Screw uninstallation. Figure 10. Screw uninstallation. Figure 10. Screw uninstallation. Figure 10. Screw uninstallation. 3.5. Influence of Loading Sequence 3.6. Inﬂuence of Moisture Content of Timber The modulus of the second loading sequence K2 was on average 35% higher than for K1. This is because the contact between the component of the connector was well established after the first loading sequence. Any possible gaps between them were closed, and the material is stabilized. Modulus K2 in terms of the heel length and the notch depth had almost the same and more transparent tendencies, as compared to modulus K1. Although it is worth noting that the confidence interval of the mean values was still considerable. The stiffness results were not more consistent after The moisture content of timber was measured after the test, as it was considered as an essential variable. Some specimens showed minor color change spots and no signiﬁcant deformations of the upmost timber layer. Most of the series had stiﬀness K1 reduced when the timber humidity increased (eight over 13 series). The same phenomenon could be observed in K2 (10 over 13 series) and Fmax (eight over 13 series). Only the correlations between stiﬀness K1 and moisture content are shown in Figure 11. 12 of 21 hown in Buildings 2020, 10, 122 (eight over 13 series Figure 11. Correlation between stiffness K1 and moisture content of timber of different series. 20 mm notch depth specimens are represented by the red points, 25 mm in green, and 35 mm in blue. Figure 11. 3.5. Inﬂuence of Loading Sequence Correlation between stiﬀness K1 and moisture content of timber of diﬀerent series. 20 mm notch depth specimens are represented by the red points, 25 mm in green, and 35 mm in blue. Figure 11. Correlation between stiffness K1 and moisture content of timber of different series. 20 mm notch depth specimens are represented by the red points, 25 mm in green, and 35 mm in blue. Figure 11. Correlation between stiﬀness K1 and moisture content of timber of diﬀerent series. 20 mm notch depth specimens are represented by the red points, 25 mm in green, and 35 mm in blue. 3 7 Failure Types 3.7. Failure Types 3.7. Failure Types Three principal failure types were observed. Figure 12 shows the typical load-slip curves of different failure types. Figure 13 presents photos of these different failure types. Most of the tested Three principal failure types were observed. Figure 12 shows the typical load-slip curves of diﬀerent failure types. Figure 13 presents photos of these diﬀerent failure types. Most of the tested specimens in this study had the failure type with a compression zone principally in the heel (47 over 60 tested samples). They were classiﬁed as type TD. Buildings 2020, 10, x FOR PEER REVIEW 12 of 20 specimens in this study had the failure type with a compression zone principally in the heel (47 over 60 tested samples). They were classified as type TD. Figure 12. Load-slip curves of different failure types. Figure 12. Load-slip curves of diﬀerent failure types. Figure 12. Load-slip curves of different failure types. Figure 12. Load-slip curves of diﬀerent failure types. 13 of 21 Buildings 2020, 10, 122 TD TB CB TD + TB Figure 13. Typical different failure types. Figure 13. Typical diﬀerent failure types. TD TB CB TD + TB TD + TB Figure 13. Typical different failure typ Figure 13. Typical diﬀerent failure types. The failure type TB occurred when the lamellas of the heel got sheared-oﬀat the displacement from 2 mm to 3 mm. The full measured curves of type TB could reach a signiﬁcant displacement at a relatively high load. This is because, ﬁrstly, when the testing machine pushed to the timber part, it held the sheared-oﬀlamellas in place. The metal plate, designated for distributing the load from the testing machine, covered the surface of the timber upper part. When the timber lamellas of the heel part were sheared-oﬀ, the metal plate held the lamellas in place and caused a load increase in the load-displacement curve. Secondly, at a signiﬁcant slip, only the screws bear the load. The sleeve between screws and concrete would prevent the contribution of the screws to the shear loading from initial up to 2 mm of displacement. Regarding this artifact, we considered that the specimen failed at the ﬁrst drop in terms of load. In the specimens classiﬁed as type TD + TB, there was a minor drop in the load after the sample reaches its peak load at the slip of about 2 mm to 6 mm. 3 7 Failure Types 3.7. Failure Types A plastic hinge was observed in the screw at the interface location. However, this only contributed to the post-peak behavior and helped extend the slip. The reasons are, firstly, that an individual screw was much less rigid than the notch itself. Secondly, there was a gap between the screw and the concrete due to the plastic sleeve; it prevented the screw from contributing to the connector rigidity. Figure 14 the failure types distribution for only one variable at a time; the data labels represent the number of specimens. The percentage of failure type TD reduced from 73% to 61% when the heel length decreased from 400 mm to 300 mm. The timber imperfection was likely to be manifested heavily by a brittle failure in the specimens with a shorter heel. In terms of notch depth, one could observe a signiﬁcant reduction from 87% at 20 mm depth to 48% at 35 mm depth. g g y Figure 14 the failure types distribution for only one variable at a time; the data labels represent the number of specimens. The percentage of failure type TD reduced from 73% to 61% when the heel length decreased from 400 mm to 300 mm. The timber imperfection was likely to be manifested heavily by a brittle failure in the specimens with a shorter heel. In terms of notch depth, one could observe a significant reduction from 87% at 20 mm depth to 48% at 35 mm depth. Figure 14. Number of specimens with different failure types. Figure 14. Number of specimens with diﬀerent failure types. Figure 14 Number of specimens with different failure types Figure 14. Number of specimens with diﬀerent failure types. The deeper notch must withstand greater shear force, and more likely failed in a brittle manner. This shows differences in load transmission depending on heel length and notch depth. As lamellas were not edgewise glued, the sheared area must be significant enough not to be the weakest (brittle) link versus the compressed (ductile) parallel-to-grain area. Potential future design rules will have to prevent brittle failure. One could see that the thickness of the concrete layer did not influence the distribution of failure type. The longer screw might have caused a brittle rupture of the connector, from 15% to 35% of the specimen’s number (three over 20 specimens versus 14 over 40 specimens). 3 7 Failure Types 3.7. Failure Types The specimens still were carrying on and achieved a signiﬁcant displacement (10 mm or more) because there was a portion of the heel still glued Buildings 2020, 10, 122 In the specime reaches its peak lo 14 of 21 sample on and to the transversal layer, got crushed at the loaded edge, and held the charge. Hence, we considered this type as a sub-category of type TD. achieved a significant displacement (10 mm or more) because there was a portion of the heel still glued to the transversal layer, got crushed at the loaded edge, and held the charge. Hence, we considered this type as a sub-category of type TD. In the specimens classiﬁed as type CB, the notched concrete part was sheared-oﬀwhile the load was still increasing. After the failure, the screws held the residual charge. It is worth noting that the concrete has rebar steel mesh, and the two screws rigidly attached the two parts altogether. Therefore, the connector in this study was less likely to have the failure type CB (two over 60 specimens). In the specimens classified as type CB, the notched concrete part was sheared-off while the load was still increasing. After the failure, the screws held the residual charge. It is worth noting that the concrete has rebar steel mesh, and the two screws rigidly attached the two parts altogether. Therefore, the connector in this study was less likely to have the failure type CB (two over 60 specimens). Almost every specimen had diagonal cracks at failure. These cracks took place in the corner between timber and concrete shortly before the load reaches its peak. They developed at an angle from 30◦to 45◦to the concrete’s surface. A plastic hinge was observed in the screw at the interface location. However, this only contributed to the post-peak behavior and helped extend the slip. The reasons are, ﬁrstly, that an individual screw was much less rigid than the notch itself. Secondly, there was a gap between the screw and the concrete due to the plastic sleeve; it prevented the screw from contributing to the connector rigidity. y y yp p Almost every specimen had diagonal cracks at failure. These cracks took place in the corner between timber and concrete shortly before the load reaches its peak. They developed at an angle from 30° to 45° to the concrete’s surface. 3 7 Failure Types 3.7. Failure Types Further investigation is needed to confirm this phenomenon The deeper notch must withstand greater shear force, and more likely failed in a brittle manner. This shows diﬀerences in load transmission depending on heel length and notch depth. As lamellas were not edgewise glued, the sheared area must be signiﬁcant enough not to be the weakest (brittle) link versus the compressed (ductile) parallel-to-grain area. Potential future design rules will have to prevent brittle failure. One could see that the thickness of the concrete layer did not inﬂuence the distribution of failure type. The longer screw might have caused a brittle rupture of the connector, from 15% to 35% of the specimen’s number (three over 20 specimens versus 14 over 40 specimens). Further investigation is needed to conﬁrm this phenomenon. 4.1.1. Timber Timber was modeled as an orthotropic elastic-perfect plastic material. According to Dias et al. [29], Hill’s criterion, which is an extension of von Mises’s function, could be used for orthotropic material to characterize the yielding phenomenon: f(σ) = F(σ22 −σ33)2 + G(σ33 −σ11)2 + H(σ11 −σ22)2 + 2Lσ2 23 + 2Mσ2 31 + 2Nσ2 12 (1 (1) where F, G, H, L, M, and N are constants determined experimentally: where F, G, H, L, M, and N are constants determined experimentally: F = σ02 2   1 σ2 22 + 1 σ2 33 −1 σ2 11  ; (2) G = σ02 2   1 σ2 33 + 1 σ2 11 −1 σ2 33  ; (3) H = σ02 2   1 σ2 11 + 1 σ2 22 −1 σ2 33  ; (4) L = 3 2 τ0 σ23 !2 ; (5) M = 3 2 τ0 σ13 !2 ; (6) N = 3 2 τ0 σ12 !2 . (7) F = σ02 2   1 σ2 22 + 1 σ2 33 −1 σ2 11  ; (2) G = σ02 2   1 σ2 33 + 1 σ2 11 −1 σ2 33  ; (3) H = σ02 2   1 σ2 11 + 1 σ2 22 −1 σ2 33  ; (4) L = 3 2 τ0 σ23 !2 ; (5) M = 3 2 τ0 σ13 !2 ; (6) N = 3 2 τ0 σ12 !2 . (7) (2) G = σ02 2   1 σ2 33 + 1 σ2 11 −1 σ2 33  ; (3) H = σ02 2   1 σ2 11 + 1 σ2 22 −1 σ2 33  ; (4) G = σ02 2   1 σ2 33 + 1 σ2 11 −1 σ2 33  ; (3) (3) H = σ0 2   1 σ2 11 + 1 σ2 22 −1 σ2 33  ; (4) (4) L = 3 2 τ0 σ23 !2 ; (5) M = 3 2 τ0 σ13 !2 ; (6) N = 3 2 τ0 σ12 !2 . (7) (5) (6) N = 3 2 τ0 σ12 !2 . 4. Finite Elements Model Validation Many authors have used diﬀerent techniques to model timber material in terms of connection, whether for notched or dowel connectors. Dias et al. [29] used Hill’s criterion to characterize the anisotropy of timber material. The method was useful in terms of modeling elastic-plastic behavior and was later employed in CCC by Jiang et al. [19] for notched connector and Mai et al. [16] for screw connectors. Models with damage mechanics were also adopted in the literature, such as using continuum damage mechanics to obtain brittle and ductile failure modes (Sandhaas and van de Kuilen [30]), using Hashin failure criterion for the laterally loaded nail of CLT bracket connection (Hollenbeck [31]), cohesive elements to simulate cracking in timber bolted connection (Franke and Quenneville [32]). User-deﬁned features (such as subroutine in Abaqus) is required for such applications and raises concerns about the computational cost. Buildings 2020, 10, 122 15 of 21 4.1. Materials 4.1. Materials 4.1. Materials 4.1.1. Timber 4.1.2. Concrete 4.1.2. Concrete Concrete was modeled as an isotropic elastic material without any plasticity properties. The experimental test provided the modulus of elasticity of concrete (cf. Table 1). The impact of plasticity characteristics of concrete was considered minimal to the model based on preliminary modeling. This simpliﬁcation helped reduce the computational cost and increased the possibility of convergence of the model. 4.1.1. Timber (7) (7) The deﬁnition of the potential coeﬃcients Rij is: The deﬁnition of the potential coeﬃcients Rij is: R11, R22, R33, R12, R13, R23 = σ11 σ0 ; σ22 σ0 ; σ33 σ0 ; σ12 τ0 ; σ13 τ0 ; σ23 τ0 (8) (8) where σij is the measured yield stress value when σij is applied as the only nonzero stress component; σ0 is the reference yield stress and τ0 = σ0/ √ 3. The problem was deﬁning the yield stress σ0 and the yield stress ratio Rij. Considering σ0 as the yield strength in the direction parallel to the grain of the timber, Dias et al. [29] proposed the coeﬃcients Rij for spruce R11 = 1; R22 = R33 = 0.19 and R12 = R13 = R23 = 0.38. The Wood Handbook [33] suggests R11 = 1; R22 = R33 = 0.11 and R12 = R13 = R23 = 0.39 (mean value) for Canadian SPF. The model used value from this reference [33]. The preliminary modeling showed that the resistance of the notched connector depends heavily on the “shear” ratio R12, R13 rather than the “orthogonal” ratio R22, R33. The lamellas of the third and ﬁfth layers of CLT specimen were extracted and subjected to the compression tests, based on standard ASTM D143-2014 [34] for a small clear specimen of timber. Table 4 presents the extreme case value of σ0 and Exx obtained from the experimental tests [35]. Other properties Eyy, Ezz, Gxy, Gzx, Gyz were derived from Exx as the indications of EN 338 [36] for softwoods: Exx = 30Eyy = 30Ezz (9) 16Gxy = 16Gxz = 0.5 Exx + Eyy (10) 16Gyz = 0.5 Eyy + Ezz (11) (9) (10) (11) Buildings 2020, 10, 122 16 of 21 16 of 21 Table 4. Summary of the timber parameters of the FEM model. Parameters Value Parameters Value Parameters Value σ0 37.5–25.8 Exx 18200–9340 νxy 0.4 R11 1 Eyy, Ezz 607–311 νxz 0.3 R22, R33 0.11 Gxy, Gzx 588–302 νyz 0.3 R12, R13, R23 0.39 Gyz 59–30 (–) (–) 4.1.2. Concrete Table 4. Summary of the timber parameters of the FEM model. 4.2. Models The ﬁnite element model of the notched connector was performed with the commercial software Abaqus [37]. The tie constraint was used for the glued contact between the lamellas (rigid contact). The non-glued one and the one between timber and concrete had “hard” contact in the normal direction and “frictionless” in the tangential direction. The ﬁnite elements were eight-node cubic with reduced integration C3D8R. The reduced integration minimized the computational eﬀort but raised some problems in terms of “hourglass.” Hourglass is nonphysical, zero-energy modes of deformation that produce zero strain and no stress. This occurs in the reduced integration elements, with only 1 integration point in the middle (e.g., C3D8R). To minimize it, we used the default hourglass control algorithm of Abaqus. The ﬁne mesh of 4 mm-element was applied to the contact zone of concrete and timber down to the second layer. The coarse mesh of 12 mm was for the rest of the model (Figure 14). The local failure criteria of the materials were not implemented. We imposed a criterion of global relative displacement of 10 mm between timber and concrete. Hill’s criteria were used for the sake of yielding phenomenon in orthotropic material, i.e., timber in our case. The screws were omitted because of their minimal impact on the overall performances of the connector. Only half of the specimen was modeled to reduce the computational cost. The charge was placed on the concrete part. The boundary condition was set to imitate the behavior of the specimen on the bench test. The CLT part was limited in the translational displacement Uy and Ux, while the concrete part was constrained in the Uy of the loaded end (Figure 15). The model was built based on the standard/implicit calculation regime. Abaqus would check for the convergence of the model at each iteration. The convergence of the model was quite good for displacement from 0 to 4 mm. Beyond this, there were some cases where the model aborts the calculation due to the divergence. The excessive displacement of the element at the loaded edge of the notch might be the reason for this divergence. 17 of 21 16 of 20 16 of 20 Buildings 2020, 10, 122 Buildings 2020 10 x FO Buildings 2020 10 x FOR Figure 15. Mesh definition of timber and concrete part and boundary conditions. Figure 15. Mesh deﬁnition of timber and concrete part and boundary conditions. 4.2. Models Figure 15. Mesh definition of timber and concrete part and boundary conditions. Figure 15. Mesh definition of timber and concrete part and boundary conditions. Figure 15. Mesh deﬁnition of timber and concrete part and boundary conditions. Figure 15. Mesh definition of timber and concrete part and boundary conditions. 4.3. Results 4.3. Results 4.3. Results These results showed that the initial loading and the unloading step of the test might impact the specimen (close the contact and stabilize the material). However, it exhibited an opposite trend in terms of the heel length. The experimental stiffness (both K1 and K2) decreased for a smaller length; the stiffness of the model increases. It could be explained that our model assumed the same modulus of elasticity for both compression and traction behavior of timber. F th i l d th d l ld h t i th t d i t f t h d th d h l For the maximum load, the model could characterize the trend in terms of notch depth and heel length (Figure 17). The envelope of maximum load created by the model was consistent in terms of heel length and increased when the depth of the cut is more profound. The model over-estimated the load even though we used the experimental compression data of lamellas of the tested CLT. For the maximum load, the model could characterize the trend in terms of notch depth and heel length (Figure 17). The envelope of maximum load created by the model was consistent in terms of heel length and increased when the depth of the cut is more profound. The model over-estimated the load even though we used the experimental compression data of lamellas of the tested CLT. For the maximum load, the model could characterize the trend in terms of notch depth and heel length (Figure 17). The envelope of maximum load created by the model was consistent in terms of heel length and increased when the depth of the cut is more profound. The model over-estimated the load even though we used the experimental compression data of lamellas of the tested CLT. Figure 17. FEM envelop of maximum load Fmax per notch depth. Figure 17. FEM envelop of maximum load Fmax per notch depth. Figure 17. FEM envelop of maximum load Fmax per notch depth. Figure 17. FEM envelop of maximum load Fmax per notch depth. Figure 17. FEM envelop of maximum load Fmax per notch depth. Figure 17. FEM envelop of maximum load Fmax per notch depth. The comparison between the experimental and modeled load-displacement curves of series I is shown in Figure 18. 4.3. Results 4.3. Results 4.3. Results For the stiffness of the connector, the model could capture the trend in terms of notch depth. Figure 16 shows that the stiffness per depth decreases when the notch depth increase. The model yields a better prediction for K2 than for K1. For the stiﬀness of the connector, the model could capture the trend in terms of notch depth. Figure 16 shows that the stiﬀness per depth decreases when the notch depth increase. The model yields a better prediction for K2 than for K1. For the stiffness of the connector, the model could capture the trend in terms of notch depth. Figure 16 shows that the stiffness per depth decreases when the notch depth increase. The model yields a better prediction for K2 than for K1. Figure 16. FEM envelop of stiffness K1 and K2 per depth of multiple heel length. Figure 16. FEM envelop of stiffness K1 and K2 per depth of multiple heel length. Figure 16. FEM envelop of stiﬀness K1 and K2 per depth of multiple heel length. Figure 16. FEM envelop of stiffness K1 and K2 per depth of multiple heel length. Figure 16. FEM envelop of stiffness K1 and K2 per depth of multiple heel length. Figure 16. FEM envelop of stiﬀness K1 and K2 per depth of multiple heel length. Buildings 2020, 10, 122 B ildi 2020 10 FOR Buildings 2020, 10, x FOR 18 of 21 17 of 20 17 of 20 These results showed that the initial loading and the unloading step of the test might impact the specimen (close the contact and stabilize the material). However, it exhibited an opposite trend in terms of the heel length. The experimental stiﬀness (both K1 and K2) decreased for a smaller length; the stiﬀness of the model increases. It could be explained that our model assumed the same modulus of elasticity for both compression and traction behavior of timber. These results showed that the initial loading and the unloading step of the test might impact the specimen (close the contact and stabilize the material). However, it exhibited an opposite trend in terms of the heel length. The experimental stiffness (both K1 and K2) decreased for a smaller length; the stiffness of the model increases. It could be explained that our model assumed the same modulus of elasticity for both compression and traction behavior of timber. 4.3. Results 4.3. Results 4.3. Results The model strength was overestimated when using the upper bound of the reference yield strength 𝜎଴ (37.5 MPa, cf. Table 4) while it showed some agreement up to the displacement of 6 mm at lower strength bound (25.8 MPa). The comparison between the experimental and modeled load-displacement curves of series I is shown in Figure 18. The model strength was overestimated when using the upper bound of the reference yield strength σ0 (37.5 MPa, cf. Table 4) while it showed some agreement up to the displacement of 6 mm at lower strength bound (25.8 MPa). The comparison between the experimental and modeled load-displacement curves of series I is shown in Figure 18. The model strength was overestimated when using the upper bound of the reference yield strength 𝜎଴ (37.5 MPa, cf. Table 4) while it showed some agreement up to the displacement of 6 mm at lower strength bound (25.8 MPa). Figure 18. Load-displacement curves comparison between experimental and FEM of series I. Figure 18. Load-displacement curves comparison between experimental and FEM of series I. Figure 18. Load-displacement curves comparison between experimental and FEM of series I. Figure 18. Load-displacement curves comparison between experimental and FEM of series I. Figure 18. Load-displacement curves comparison between experimental and FEM of series I. Figure 18. Load-displacement curves comparison between experimental and FEM of series I. 5 Conclusions 5. Conclusions 5. Conclusions (2) By comparing the results of diﬀerent conﬁguration, the geometry variables, heel length, and notch depth had signiﬁcant inﬂuences on the stiﬀness and the maximum load of the connector than other variables. The performance of the connector was not increased proportionally with a deeper notch cut. Screw length and concrete thickness only had minor inﬂuences that are diﬃcult to spot since the number of tests was limited. (2) By comparing the results of diﬀerent conﬁguration, the geometry variables, heel length, and notch depth had signiﬁcant inﬂuences on the stiﬀness and the maximum load of the connector than other variables. The performance of the connector was not increased proportionally with a deeper notch cut. Screw length and concrete thickness only had minor inﬂuences that are diﬃcult to spot since the number of tests was limited. (3) The ﬁnite element model could capture the tendency of both stiﬀness and maximum load in terms of notch depth. For a better prediction of stiﬀness, the model would need to characterize diﬀerent timber modulus of elasticity for compression and traction. Such implementation requires user-coded material in Abaqus, which is out of the scope of this study. (4) The results showed that the length of the screw did not impact the overall results. After the test, the specimens were successfully disassembled and separated by using a simple screwdriver. This connector system could be employed if there are concerns about the reusability at the end of the structure service life. This study gave more information about the performance of individual notched connectors of diﬀerent geometry conﬁgurations. The ﬁnite-element model could produce a reasonably good prediction, though, it could not entirely capture the experimental phenomenon due to an over-simpliﬁed timber material. Since our next step would focus on the vibrational behavior of full-scale specimens, this study provided a basis to create a simpliﬁed parametrical model of the connector for a global optimization application. For future research, full-scale testing of long-span CCC beams will adopt this type of connector. The presented ﬁndings on individual behavior (strength, stiﬀness) and many other aspects such as group eﬀect, number of connectors, the distance between the connectors will be considered. One of the objectives of our research project is to propose a solution to a deconstructable connector. It involved a vision at the scale of beams, ﬂoors, and entire structures. This solution will also be validated in-depth on a full-scale beam test. 5 Conclusions 5. Conclusions 5. Conclusions Author Contributions: Methodology, M.V.T., and P.G.; validation, M.V.T.; investigation, M.V.T.; data curation, M.V.T.; writing—original draft preparation, M.V.T.; writing—review and editing, M.V.T., S.M., S.M.E., and P.G.; visualization, M.V.T.; supervision, S.M., and S.M.E.; funding acquisition, S.M. All authors have read and agreed to the published version of the manuscript. Funding: The authors are grateful to Natural Sciences and Engineering Research Council of Canada for the ﬁnancial support through its IRC and CRD programs (IRCPJ 461745-18 and RDCPJ 524504-18) as well as the industrial partners of the NSERC industrial chair on eco-responsible wood construction (CIRCERB). Conﬂicts of Interest: The authors declare no conﬂict of interest. 5 Conclusions 5. Conclusions 5. Conclusions 5. Conclusions We have investigated and compared the influence of four variables on the overall mechanical performance of the notched connector. The variables tested were heel length, notch depth, screw length, and thickness of the concrete layer. The following conclusions were drawn from the results of the study: We have investigated and compared the influence of four variables on the overall mechanical performance of the notched connector. The variables tested were heel length, notch depth, screw length, and thickness of the concrete layer. The following conclusions were drawn from the results of the study: We have investigated and compared the inﬂuence of four variables on the overall mechanical performance of the notched connector. The variables tested were heel length, notch depth, screw length, and thickness of the concrete layer. The following conclusions were drawn from the results of the study: y 1) Based on the load-displacement behavior of the specimens, the notched connector with reinforced screws could withstand a load from 120 kN to 240 kN. The resistance of connectors 1) Based on the load-displacement behavior of the specimens, the notched connector with reinforced screws could withstand a load from 120 kN to 240 kN. The resistance of connectors d d d th t fi ti Th tiff f th t h d t hi h th (1) Based on the load-displacement behavior of the specimens, the notched connector with reinforced screws could withstand a load from 120 kN to 240 kN. The resistance of connectors depended on y 1) Based on the load-displacement behavior of the specimens, the notched connector with reinforced screws could withstand a load from 120 kN to 240 kN. The resistance of connectors 1) Based on the load-displacement behavior of the specimens, the notched connector with reinforced screws could withstand a load from 120 kN to 240 kN. The resistance of connectors d d d th t fi ti Th tiff f th t h d t hi h th (1) Based on the load-displacement behavior of the specimens, the notched connector with reinforced screws could withstand a load from 120 kN to 240 kN. The resistance of connectors depended on Buildings 2020, 10, 122 19 of 21 the geometry conﬁguration. The stiﬀness of the notched connector was higher than the screw connectors but lower than HBV mesh. Most of the specimens exhibited ductile rupture with signiﬁcant post-peak displacement. References Composite concrete-timber structures. Prog. Struct. Eng. Mater. 2002, 4, 264–275. [CrossRe 11. Lukaszewska, E.; Johnsson, H.; Fragiacomo, M. Performance of connections for prefabricated timber–concrete composite ﬂoors. Mater. Struct. 2008, 41, 1533–1550. [CrossRef] 12. Van der Linden, M. Timber-Concrete Composite Floor Systems; University of Technology Delft: Delft, The Netherlands, 1999. 13. Yeoh, D.; Fragiacomo, M.; Aldi, P.; Mazzilli, M.; Kuhlmann, U. Performance of Notched Coach Screw Connection for Timber-Concrete Composite Floor System. In Proceedings of the 10th World Conference on Timber Engineering, Miyazaki, Japan, 2–5 June 2008; paper 221. pp. 1–8. 4. Dias, A.M.P.G.; Cruz, H.M.P.; Lopes, S.M.R.; Van De Kuilen, J.W. Stiﬀness of dowel-type fastener timber–concrete joints. Struct. Build. 2010, 163, 257–266. [CrossRef] 15. Gerber, A.R. Timber-Concrete Composite Connectors in Flat-Plate Engineered Wood Products. Master’s Thesis, University of British Columbia, Vancouver, BC, Canada, 2016. 16. Mai, K.Q.; Park, A.; Lee, K. Experimental and numerical performance of shear connections in CLT–concrete composite ﬂoor. Mater. Struct. 2018, 51, 84. [CrossRef] 17. Mai, K.Q.; Park, A.; Nguyen, K.T.; Lee, K. Full-scale static and dynamic experiments of hybrid CLT–concrete composite ﬂoor. Constr. Build. Mater. 2018, 170, 55–65. [CrossRef] 18. Higgins, C.; Barbosa, A.R.; Blank, C. Structural Tests of Concrete Composite-Cross-Laminated Timber Floors; School of Civil and Construction Engineering: Corvallis, OR, USA, 2017; p. 76. 19. Jiang, Y.; Crocetti, R. CLT-concrete composite ﬂoors with notched shear connectors. Constr. Build. Mater. 2019, 195, 127–139. [CrossRef] 20. Khorsandnia, N.; Valipour, H.; Schänzlin, J.; Crews, K. Experimental Investigations of Deconstructable Timber–Concrete Composite Beams. J. Struct. Eng. 2016, 142, 04016130. [CrossRef] 21. ANSI/APA PRG-320-2019 Standard for Performance-Rated Cross-Laminated Timber; APA—The Engineered Wood Association: Tacoma, WA, USA, 2019. 22. NRC—CNRC Evaluation Listing CCMC 13654-L Nordic X-Lam; National Research Council of Canada: Ottawa, ON, Canada, 2013. 23. ASTM C39/C39M—18 Test Method for Compressive Strength of Cylindrical Concrete Specimens; ASTM C39/C39M—18; ASTM International: West Conshohocken, PA, USA, 2018. 24. Boccadoro, L. Timber-Concrete Composite Slabs Made of Beech Lami Connection. Ph.D. Thesis, ETH Zurich, Zürich, Switzerland, 2016. 24. Boccadoro, L. Timber-Concrete Composite Slabs Made of Beech Laminated Veneer Lumber with Notched Connection. Ph.D. Thesis, ETH Zurich, Zürich, Switzerland, 2016. 25. Gutkowski, R.M.; Brown, K.; Shigidi, A.; Natterer, J. Investig Connections. J. Struct. Eng. 2004, 130, 1553–1561. [CrossRef] 25. Gutkowski, R.M.; Brown, K.; Shigidi, A.; Natterer, J. Investigation of Notched Composite Wood–Concrete Connections. J. Struct. Eng. 2004, 130, 1553–1561. [CrossRef] 26. References 1. Gauthier, P.-E. Charpente en Bois, Notamment Pour Ossature de Hangar Agricole. 1952. Available online: https://bases-brevets.inpi.fr/fr/document/FR1009171/publications.html (accessed on 7 July 2020). 1. Gauthier, P.-E. Charpente en Bois, Notamment Pour Ossature de Hangar Agricole. 1952. Available online: https://bases-brevets.inpi.fr/fr/document/FR1009171/publications.html (accessed on 7 July 2020). p p p y 2. Karacabeyli, E.; Gagnon, S. (Eds.) Canadian CLT Handbook, 2019th ed.; Special Publication SP-532E; FPInnovations: Point-Claire, QC, Canada, 2019; ISBN 978-0-86488-590-6. 2. Karacabeyli, E.; Gagnon, S. (Eds.) Canadian CLT Handbook, 2019th ed.; Special Publication SP-532E; FPInnovations: Point-Claire, QC, Canada, 2019; ISBN 978-0-86488-590-6. 3. Lukaszewska, E. Development of Prefabricated Timber-Concrete Composite Floors. Ph.D. Thesis, Luleå University of Technology, Luleå, Sweden, 2009. 3. Lukaszewska, E. Development of Prefabricated Timber-Concrete Composite Floors. Ph.D. Thesis, Luleå University of Technology, Luleå, Sweden, 2009. 4. Dias, A.M.P.G.; Jorge, L. The eﬀect of ductile connectors on the behaviour of timber–concrete composite beams. Eng. Struct. 2011, 33, 3033–3042. [CrossRef] 4. Dias, A.M.P.G.; Jorge, L. The eﬀect of ductile connectors on the behaviour of timber–concrete composite beams. Eng. Struct. 2011, 33, 3033–3042. [CrossRef] 5. Fragiacomo, M.; Lukaszewska, E. 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[CrossRef] Buildings 2020, 10, 122 21 of 21 21 of 21 33. Laboratory, F.P. Wood Handbook: Wood as an Engineering Material; USDA Forest Service: Madison, WI, USA, 1999; Volume 113, p. 509. 34. ASTM D143—14 Test Methods for Small Clear Specimens of Timber; ASTM International: West Conshohocken, PA, USA, 2014. 35. Thai, M.V.; Ménard, S.; Elachachi, S.M.; Galimard, P. Experimental Report on the Push-Out Test of CLT-Concrete Shear Notched Connector (Phase 1); University of Bordeaux: Bordeaux, France, 2019. 36. EN 338—2009 Structural Timber—Strength Classes; European Committee for Standardization (CEN): Brussels, Belgium, 2009. 37. Abaqus 6.14 Online Documentation; Dassault Systèmes: Velizy-Villacoublay, France, 2014; Available online: http://ivt-abaqusdoc.ivt.ntnu.no:2080/v6.14/books/usb/default.htm (accessed on 7 July 2020). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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W4385953024.txt	https://cjme.springeropen.com/counter/pdf/10.1186/s10033-023-00905-6	en		NuBot: A Magnetic Adhesion Robot with Passive Suspension to Inspect the Steel Lining	Chinese journal of mechanical engineering/Chinese Journal of Mechanical Engineering	2,023	cc-by	10,620	Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 https://doi.org/10.1186/s10033-023-00905-6 Chinese Journal of Mechanical Engineering Open Access ORIGINAL ARTICLE NuBot: A Magnetic Adhesion Robot with Passive Suspension to Inspect the Steel Lining Hao Xu1, Youcheng Han1, Mingda He1, Yinghui Li1 and Weizhong Guo1* Abstract The steel lining of huge facilities is a significant structure, which experiences extreme environments and needs to be inspected periodically after manufacture. However, due to the complexity (crisscross welds, curved surface, etc.) of their inside environments, high demands for stable adhesion and curvature adaptability are put forward. This paper presents a novel wheeled magnetic adhesion robot with passive suspension applied in nuclear power containment called NuBot, and mainly focuses on the following aspects: (1) proposing the wheeled locomotion suspension to adapt the robot to the uneven surface; (2) implementing the parameter optimization of NuBot. A comprehensive optimization model is established, and global optimal dimensions are properly chosen from performance atlases; (3) determining the normalization factor and actual dimensional parameters by constraints of the steel lining environment; (4) structure design of the overall robot and the magnetic wheels are completed. Experiments show that the robot can achieve precise locomotion on both strong and weak magnetic walls with various inclination angles, and can stably cross the 5 mm weld seam. Besides, its maximum payload capacity reaches 3.6 kg. Results show that the NuBot designed by the proposed systematic method has good comprehensive capabilities of surfaceadaptability, adhesion stability, and payload. Besides, the robot can be applied in more ferromagnetic environments and the design method offers guidance for similar wheeled robots with passive suspension. Keywords Wheeled robot, Passive suspension, Magnetic adhesion, Steel lining, Design method 1 Introduction Currently, the nuclear power industry takes large adoption of steel lining construction as the inner wall of security containment, and that is the last barrier to prevent the radioactive substance from leaking in the nuclear accident [1]. Hence, it is very essential to inspect the sealing safety and welding reliability periodically, considering that the steel lining is a huge sealed container welded by thousands of weld seams for hundreds of curved steel plates. Traditional method for inspecting the steel lining *Correspondence: Weizhong Guo wzguo@sjtu.edu.cn 1 State Key Lab of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China weld is mainly relying on the worker’s manual examination standing on the scaffold, bringing the demerits of long-period time, great difficulty, and high cost. Hence, it is significant and challenging to develop a robot platform for the (semi-)automatic inspection of steel lining welds. The relevant research priorities/problems are: The wall-climbing and surface-adaptive locomotion mechanism to accommodate and move on the containment steel lining. The steel lining has a large curved surface with massive crisscross arc welds and obstructed surroundings, demanding that the robot should possess a capable locomotion mechanism and reasonable setup of degrees of freedom (DOF). In previous researches, the climbing robots include: a) the crawler robots need additional mechanisms to ensure the whole track fits the wall shape. Seo et al. [2] adopted active compliant joints © The Author(s) 2023. 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/. Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 2 of 18 Table 1 Qualitative comparison of different types of climbing robots. Adhesion Magnetic Example Locomotion mechanism Wall type Adhesion force Velocity Payload Terrainadaptable Wall-harmless Build-in power NuBot (ours) Wheel Steel √ √ √ √ √ Track √ ○ ○ Magnetic crawler [3] √ ○ √ √ × ○ Smooth surface √ √ √ √ × ○ Rough surface × × × × √ × × √ Leg Flat surface √ ○ √ ○ ○ × Wheel-leg Smooth surface ○ ○ ○ √ √ √ HIT robot [11] Leg Pneumatic MultiTrack [14] Track W-Climbot [4] Leg Mechanical SpinyBot II [15] Leg CSU robot [17] Wheel Electrostatic SJTU robot [19] Gecko-like ○ to increase the preload on the front track; Gao et al. [3] adopted the linkage-spring system to connect two track modules. However, the active joint increases the number of actuators, and the hard metal track scratches the steel lining plating; b) the legged robots possess a larger workspace with better adaptability. Guan et al. [4] imitated an inchworm and proposed a biped serial climbing robot with five DOFs. Liu et al. [5] presented a robot with eight footpads and one active DOF, which could adapt to the wall curvature by a passive fourbar mechanism. However, the legged locomotion produces 19051270000dynamic adhesion-separation cycles between the footpad and terrain surface, resulting in a discrete gait slowing the speed; c) the wheeled robots with no more than three wheels [6–8] can always keep each wheel in contact with the wall. However, on one hand, more wheels are preferred to provide stronger adhesion force and larger driving force; on the other hand, more wheels can make many of them separate from the wall, unless the robot is designed with an adaptive suspension or compliant mechanism: Eto et al. [9] developed a passive rocker arm suspension and spherical wheels to adapt to the wall shape; Guo et al. [10] proposed a robot with an electromagnetically driven compliant beam to effectively negotiate corners. In conclusion, the wheeled robot with passive suspension is a feasible option, which offers sufficient DOFs to ensure that the wheels adapt to the wall without increasing actuators. (1) The stable and practical adhesive approach to attach to the containment steel lining. Adhesion principles are the direct factor that determines the adhesion stability and payload. Table 1 compares five types of adhesive approaches in various aspects of performances, where √, ×, ○ indicate respectively that the robot can, basically can, cannot meet the current demands. Thereinto, magnetic √ √ × ○ ○ × √ √ × × × √ ○ adhesion [3, 6–8, 11–13] works well on a ferromagnetic surface, which is an effective choice for the steel lining. Generally, the adhesion force depends on the magnet material, size, shape, and distance from the wall. In contrast, pneumatic adhesion [4, 14] can be employed for more occasions. However, the required pump limits the minimum size and weight, and the adhesion force will decrease when the gap occurs between the adhesion mechanism and the curved wall. Mechanical adhesion [15–17] imitates the structure of insects in nature, such as claws, hooking tiny grooves on rough walls. Nevertheless, they cannot adapt to the steep and smooth wall of containment due to insufficient adhesion force. Electrostatic adhesion [18, 19] generates adhesion and actuation force by energizing the soft material. However, the current conducted to the wall increases the risk to cause nuclear accidents. Gecko-like adhesion [20, 21] relies on the van der Waals force between the footpads and the wall. However, this method is not workable because the dust on the surface of the containment affects the adhesion. Considering environmental constraints, the NuBot is expected to be endowed with good comprehensive capabilities of surface adaptability, adhesion stability, steel-lining surface harmlessness, locomotion velocity, lightweight with high integration of build-in power and other mechatronic devices. Based on the above analysis and demands, magnetic adhesion is the optimal option for the steel lining. (2) The systematic design method to develop the robot platform under the comprehensive operation demands. In the environment of nuclear power containment, the adhesion robot is required with a big adaptive workspace for the varying terrains, good payload and stiffness, small size and weight. In (2023) 36:90 This paper proposes a magnetic adhesion robot called NuBot to inspect the steel lining weld on the nuclear power containment. As aforementioned, the magnetic adhesion wheeled robot with passive suspension is the optimal option under the special environment. Hence, this research will be mainly focused on the demand-oriented systematic design method for the NuBot from type identification to topology parameter optimization for the NuBot first, and then demonstrate the experimental validation based on the semi-automatic mechatronics system utilizing a proportional integral (PI) control strategy. The rest of this work is organized as follows. Section 2 presents an overview of the robot platform and the design strategy. Section 3 describes the type design of the suspension, its kinematic model, and the overall robot dynamics. Section 4 presents the optimization design process. Overall parameters are obtained from non-dimensional to dimensional, from partial to global utilizing PCbDM. In Section 5, the unit design is completed and the prototype is developed. Locomotion and payload experiments are conducted in Section 6. Section 7 presents the comparison between several climbing robots. Finally, the conclusion is presented in Section 8. 2 Overview of the Robot Platform and Design Method Figure 1(a) shows the nuclear power plant containment [1], which has a cylindrical ferromagnetic steel lining with a diameter of 37 m and a height of 61.6 m. Its surface is distributed with massive crisscross arc welds with a maximum height of 5 mm. The support structure inside the steel lining is only 0.2 m away from the wall, which limits the height of the robot. Besides, the adhesion force must be reliable because it is not allowable to attach the protective rope inside the containment. And the load of NuBot is required to be no less than 0.4 kg, including the controller, camera, communication equipment, battery, etc. Figures 1(b, c) show the structure of NuBot, including two passive suspensions, six magnetic adhesion wheels, the chassis, an image capture system, and a control system. When inspecting the weld seam inside the containment, the technician controls NuBot to move and monitor the images captured by the camera. During locomotion, the 3-DOF suspension ensures all wheels are in contact with the complex surface of steel lining, and adapt to the surrounding environment like support structure. Figure 2 shows the structure of the magnetic adhesion wheel. Each wheel is driven independently with a builtin permanent magnet unit and a motor-reducer-encoder unit. It is connected to the suspension through a passive revolute joint, whose axis is perpendicular to the wheel (a) 11 m the climbing robot field, Zhang et al. [22] designed a passive adaptive moving mechanism utilizing the magnetic circuit optimization, and ensures the allowable motor torque in a stable operation mode; Liu et al. [23] optimized the footpad of a verticalclimbing robot for various curvatures according to the experiment-based Taguchi method; Song et al. [6] optimized the water-jetting wall-climbing robot with the optimum structure of magnetic adhesion system, etc. One can see there does not exist a systematic method for the overall optimization design of the climbing robot, considering the coupling relations among the size, workspace, adhesion force, driving force, stability, etc. Some works in the mechanism optimization field can provide a few references: Hoff et al. [24] optimized the bat-inspired biological robot for better flight characteristics after employing the principal component analysis; Liu et al. [25] proposed the performance-chart-based design methodology (PCbDM) as a general optimization approach to designing the less-parameter mechanism, obtained optimized parameters of a 3-DOF parallel manufacturing module [26], and investigated the 2T1R-type parallel mechanisms applying the Grassmann line geometry method [27]; thereafter, Li et al. [28] analyzed the influence of limb arrangements on the parasitic motion of 3-PRS parallel mechanism, and generated the singularity-free and high-performance workspace for a 3-DOF 2-CRRR-CRR parallel manipulator [29]; Han et al. [30] solved the sixteen-parameter optimization for the reconfigurable legged mobile lander, under the demands of multiple operation modes and multiple performance criteria, etc. Page 3 of 18 (b) ķ ĸ ϕ37 m 50.6 m Xu et al. Chinese Journal of Mechanical Engineering Ľ (c) ľ Ĺ Ļ ĺ ļ Ļ ļ ĺ Ļ Ŀ ĺ Figure 1 Structures of the nuclear power containment and NuBot. a Nuclear power containment, ① a plate of steel lining, ② weld, ③ support structure; b Axonometric view of the NuBot; c Exploded view of the NuBot, ④ magnetic adhesion wheel, ⑤ suspension, ⑥ support, ⑦ shell, ⑧ camera, ⑨ chassis Xu et al. Chinese Journal of Mechanical Engineering Revolute joint Hub (2023) 36:90 Page 4 of 18 Anti-slip rubber Gear motor Hall encoder Yoke iron Arc magnet Bearing Magnet support Reflective stripe Figure 2 Exploded view of the magnetic adhesion wheel axis. The magnet inside the wheel is installed under the motor, so the magnetic force will not change periodically as the wheel rotates. A systematic design method is proposed to design and optimize the magnetic adhesion robot by comparison of various performance indices and specific application occasions: (1) Design the suspension type considering the adaptability, payload, and compliance. (2) Non-dimensional optimization of the suspension. Based on its kinematic model, proper indices such as workspace, payload, and stiffness are chosen to illustrate the performance in workspace, payload, and stiffness capacity. Eventually, optimum parameters with desired comprehensive performance in atlases are selected. (3) Non-dimensional optimization of the overall robot (including the wheel radius). Based on the dynamic model of the robot, the optimum parameters are selected through the adhesion stability on the steel lining. Thus, all non-dimensional parameters of the suspension and overall robot can be determined. (4) Obtaining actual dimension parameters considering the weld-seam traverse capability to determine the wheel radius, the smooth locomotion, and better curvature adaptability to derive the size of suspension and overall robot, and the interference-free conditions to constrain the bottom and top of the overall robot. (5) Magnet unit design in Maxwell, and overall structure and topological optimization are completed. (6) Experimental validation of the capabilities of surface-adaptability, adhesion stability, and payload of NuBot. 3 Type Design, Kinematics and Dynamics Figure 3 Type synthesis of the suspension mechanism based on the passive compliance utilizing cylindrical spring rather than active compliance. It should utilize the energy component (cylindrical spring, coil spring, etc.) to passively match the deformation demands, and not utilize the motor in case of adding much weight and cost. Furthermore, NuBot takes the eudipleural structure on both sides, the base of suspension has a rigid connection with the robot body. So for the type synthesis of passive suspension based on the environmental adaptability of steel lining, the varying surface morphology is regarded as input that makes the length of spring-installed joint change, and the robot body is taken as output which has a significantly reduced fluctuation utilizing the designed suspension. Based on the topological graph and the DOF equation, the number synthesis [31, 32] is implemented initially. Thereinto, Eq. (1) reveals the relations among the limb number k , the independent closed loop number L, the edge number e, the vertex number v ; Eq. (2) reveals the relations among the DOF FD , the motor number in the i th limb qi (i = 1 ∼ n), the limb number k , the active limb number n, and the passive limb number p L = e − v + k, (1) n (2) 3.1 Type Design of the Suspension To ensure good motion and force properties, the design for the wheeled locomotion suspension of NuBot requires to meet multiple demands: (1) Good adaptability to the steel lining with varying geometrical morphology, including adapting to the curvature of the cylindrical wall, and also the uneven weld. The first-line importance is to keep all wheels in contact with the steel lining surface, so the robot can move more smoothly and steadily. (2) Good payload capability. The type design should enhance the load-weight ratio—carry more detection devices while possessing a lightweight structure. (3) Passive compliance FD = i=1 (qi − 1) + k − p. To navigate on the irregular surface of steel lining, the six-wheeled NuBot is a feasible scheme with a good balance of adaptability and lightweight, considering the four-wheeled one reduces stability while the eight-wheeled one adds too much weight. Namely, each suspension should be installed with three wheels. So following the number synthesis, the type results of threewheeled locomotion suspension are listed in Figure 3. Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 They have better adaptability to the irregular surface as the DOF increases along the vertical axis, and the types have more complexity with more loops and links along the horizontal axis. Once many types of suspension have been obtained, the next work should be focused on the identification of the optimal one for practical application. The basic selection rules are as follows. (1) The optimal type should satisfy the navigation demand on a complex surface: 3 DOF with motion characteristics of two translations and one rotation. Moreover, it is more preferred to employ the threewheeled locomotion suspension based on the passive compliance utilizing cylindrical spring. (2) The optimal type should adapt to the surface well and have a good mechanism stiffness. As shown in Figures 3(a, d, g), the wheel is connected to the robot body directly through a cylindrical spring. They just have an open-chain topology while not a closed-loop topology—a lower stiffness and adaptability. (3) The optimal type should have fewer loops and fewer links. On one hand, the type with more loops and links has a strong motion non-linearity and complex force distribution; on the other hand, it produces more weight and doesn’t make any particular significance for engineering practice. Above all, Figure 3(h) is finally identified with a 3 DOF single-loop topology. Compared with the traditional independent suspension, it can guarantee real-time contact between the wheel and the steel lining, as well as a higher stiffness and adaptability. 3.2 Kinematic Modeling of the Suspension After identifying the suspension type, the kinematic and dynamic models can be established. In Figure 4, {O} denotes the suspension coordinate frame. In the spring spring x B l3 A O l6 C θC l2 F l4 D l1 l5 θD y Page 5 of 18 mechanism view, l4 , l5 , l6 are regarded as three inputs, and the relative motions (two translations and one rotation) of suspension with respect to the steel lining are regarded as outputs. Hence, B, C, and E are three independent point positions that can be chosen to install three locomotion wheels, as a reflection of three outputs. Given B, C, and E are all taken as the outputs, so the position kinematics of the suspension can be defined by a loop-closure equation given as   r OA + r AC = r OD + r DC, r OB = r OA + r AB , (3) r = r +r , OE OD DE where r OA is a vector from O to A, and other vectors have the same naming rule. Next, three differential kinematic equations can be derived to indicate the influence of outputs on the inputs. According to the theory of mechanism, the linkage between C and F has three DOFs, while AB and DE are 2-DOF linkages, due to the redundancy of the θC and θD . Inverse Jacobian matrix of C can be obtained as q̇ = l̇4 , l̇5 , l̇6 = J C IK v C , where q denotes the input matrix, J C IK is the inverse Jacobian matrix obtained by Eq. (4), and v C is the velocity of point C.    −tanθC 1 − cosx2Cθ   C     ± � l1 −xC  1 0 ,  JC =  2 −(l −x )2 IK  l   1 C 2  x sinθ 1 C C 0 (4) cosθC  cos2 θC    ẋ C     v C =  ẏC .   θ̇C The inverse Jacobian matrices of linkages AB and DE can be obtained from pseudo-inverse of Jacobian matrices as v B = [ẋB , ẏB ]T = J BFK q̇ , and v E = [ẋE , ẏE ]T = J EFK q̇ , where v B and v E are the velocities of points B and E, and J BFK and J EFK are their Jacobian matrices obtained by Eqs. (5) and (6). κ1 sinθD −κ1 sinθD κ1 cos(θC − θD ) B , J FK = 1 − κ2 sinθD κ2 sinθD −κ2 cos(θC − θD ) (5) κ4 sinθD −κ4 sinθD κ3 sinθD , J EFK = (6) κ4 cosθD 1 + κ4 cosθD −κ3 cosθD where l3 E Figure 4 Sketch of the suspension structure and mechanism Xu et al. Chinese Journal of Mechanical Engineering κ1 = κ2 = κ3 = l3 sinθC l6 sin(θC −θD ) , l3 cosθC l6 sin(θC −θD ) , l3 , l2 sin(θC −θD ) κ4 = cosθD + Hence (2023) 36:90 Page 6 of 18 (a) sinθD tan(θC −θD ) † l3 l2 . † (7) J BIK = (J BFK ) , J EIK = (J EFK ) , (b) Weld seam where J BIK and J EIK represent the inverse Jacobian matrices of wheels B and E, and the symbol † denotes pseudo-inverse. 3.3 Dynamic Modeling of the Overall Robot The dynamic model of the robot describes the relationship between the motor torques and the motion of NuBot, and can be further used to analyze the adhesion stability (Figure 5(a)) and the weld-crossing capacity on the inclined surface (Figure 5(b)). Dynamics of the robot can be expressed by Lagrange formulation. The kinetic energy TOR and the potential energy UOR of the overall robot are obtained by  � � G TG G mj G v Tj G v j + 12 12 TOR = 21 12  j=0 j=0 ωj I j ωj ,   �12  1 �6 T G  UOR = − j=0 mj g pcj − 2 jk =1 ks 2jk , G p = G p + G R Bp , B  c0 cj  � cj � � �    G I = G R B R Pj I G R B R T , B j Pj j B Pj (8) where {B} is the coordinate frame located in the center of gravity (COG) of body, {G} is the global coordinate frame. To arrange the sequence number of overall robot: j = 0 is the robot body, j = 1 ∼ 6 are the wheels, j = 7 ∼ 12 are the suspension parts, and jk = 1 ∼ 6 are the cylindrical springs. Furthermore, ks and jk are the rate and compression of the jk th spring; pcj is the COG position of the jth part; mj , v j , ωj and I j are the corresponding masses, linear velocities, angular velocities, moments of inertia; G R is the orientation matrix of {B} with respect to {G}, B B R is the orientation matrix of the jth link frame P Pj j with respect to {B}, g is the gravitational acceleration. Thus, the Lagrange function can be yielded by LOR = TOR − UOR, indicating the composite effects of kinetic and potential energies, given by  � �  d ∂LOR − ∂LOR = Rw τj − f j , fric ∂qj dt � ∂ q̇j � (9)  d ∂LOR − ∂LOR = ks �j , k ∂qj dt ∂ q̇j k k where qj and qjk are the generalized coordinates composed by the joint variables of wheels and springs respectively; Rw is the wheel radius; τj is the jth motor torque, Figure 5 Dynamic force analysis: (a) Adhesion stability and (b) Weld-crossing ability of the robot j and ffric is the friction between the jth wheel and steel lining surface. 4 Optimization Design Using PCbDM To reveal the relations between the parameters (suspension mechanism and overall robot respectively) and performances of NuBot, the physical model and PCbDM method [25] are utilized for their visualization and global optimization merits. The physical model transforms the design variables from infinite solution space to finite solution space based on the non-dimensional operation, so that it lays the foundation for global optimization and finally requires solving the factor from the non-dimensional solution to the actual dimension parameters. And the PCbDM method provides an atlas-based formulation for the optimization process and result, and facilitates engineering optimization. 4.1 Non‑Dimensional Optimization of the Suspension To establish the optimization model of suspension, the design variables are l1 , l2 , l3 for the sake that they are constants while l4 , l5 , l5 are prismatic joint variables. Moreover, the constraint conditions are translational and rotational ranges of all joints. Last but not least, three criteria (i.e. objective functions) are employed to give a comprehensive performance evaluation on suspension: (1) The workspace area index (WAI) is utilized to describe its motion range which determines the surface adaptability formed by three wheels to traverse the complex surface. The wheels should reach more areas to satisfy the needs of different situations, and can be expressed as Xu et al. Chinese Journal of Mechanical Engineering ηW = S dS = xmax dx xmin (2023) 36:90 ymax ymin dy, V t1 t3 3 y W S where Fi are the eigenvalues of the matrix G T G. (3) The global stiffness index (GSI) is used to evaluate the extreme linear displacement capacity. The suspension should possess larger mechanism stiffness to produce linear displacement in the input joints, so the robot can adapt to the terrain with the displacement of the input joints. Therefore, we use GSIs to evaluate the displacement deformation and hope them to be as larger as possible. The deformation equation is: P = CF , where P is linear displacement deformation of the three prismatic joints, C = J FK J T FK are the compliance matrix. Therefore, the GSIs can be given by � √  Pi \|)dS  ηPmax = S max(\| � , � √ S dS (12) min(\| \|)dS  ηPmin = S � Pi , dS S where Pi are the eigenvalues of the matrix C T C . There are three optimization parameters in the suspension model, including l1, l2 and l3. The mean of the parameters is obtained by: (13) We can get the normalized equation by dividing both sides of equation by T: t1 + t2 + t3 = 3, x (10) where x ∈ [xmin , xmax ] and y ∈ [ymin , ymax ] are the integral variables of the three outputs workspace. (2) The global payload index (GPI) is used to evaluate the extremum payload force that the suspension can bear during locomotion. The suspension bears the most mass of the body and the force from the wheel centers. To obtain the suspension with a larger payload capacity, we use GPIs to evaluate the extremum force the suspension can bear during locomotion on uneven steel lining. The force equation is: F = Gτ , where F is the output general force at wheel centers while τ is the input general force at prismatic joint A, D, and F, and G IK is the inverse force Jacobian matrix. From the relationship G = J −T IK , the GPIs can be given by � √  Fi \|)dS  ηF max = S max(\| � , � √ S dS (11) \|)dS Fi  ηF min = S min(\| � , dS 1 (l1 + l2 + l3 ) = T . 3 Page 7 of 18 (14) t2 O t2 U y 1.5 W x U 0 V 3 t3 t1 Figure 6 Parameter design space of the suspension where t1 = Tl1 , t2 = Tl2 and t3 = Tl3 . Thus, the optimization parameters are converted to ti (i = 1, 2, 3). Considering dimensional constraint l2 < l1, the constraint equations can be given as:   t2 < t1 , 0 < t1 , t3 < 3, (15)  0 < t < 1.5. 2 Eqs. (14) and (15) show the parameter design space of the suspension, as shown in Figure 6. The mapping function of variables between frame O − t1 t2 t3 and frame U − xyz can be given as x = 3−t√1 +t2 , 2 (16) y = 3−t√1 −t2 . 6 Consequently, the ASI, GPI and GSI performance atlases of the suspension from wheel B, C, and E are shown in Figures 7, 8, 9 and 10, respectively. The WAI performance atlases of three wheels are shown in Figure 7, where t1 , t2 , t3 are the nondimensional forms of l1 , l2 , l3 . One can see that wheel B has the smallest motion area, while wheel C has the largest area. Considering a larger output range to better adapt to the complex surface, the solution domain of WAI is assigned as B ≥ 0.2, ηC ≥ 0.5, and ηE ≥ 0.5}, �WAI = {(t1 , t2 , t3 )\|ηW W W C B E where ηW , ηW , ηW denote the WAI of wheels B, C, and E. The GPIs (denoted as ηF max , ηF min ) and GSIs (denoted as ηPmax , ηPmin ) atlases of wheel B are shown in Figure 8. One can see that they show the different distribution principles with the design variables changing. Considering a larger payload capacity of suspension, the solution domain is assigned as �GPI = {(t1 , t2 , t3 )\|ηFBmin ≥ 0.2, ηFCmin ≥ 0.7, ηFE min ≥ 0.5} . Likewise, due to better adaptability to the complex surface, the solution domain is assigned as C E B ≥ 0.3}. ≥ 0.9, ηPmin ≥ 0.5, ηPmin �GSI = {(t1 , t2 , t3 )\|ηPmin Xu et al. Chinese Journal of Mechanical Engineering (a) (2023) 36:90 Page 8 of 18 (b) (c) Figure 7 The suspension WAI performance atlases: (a) Wheel B, (b) Wheel C, (c) Wheel E (a) (b) (c) (d) Figure 8 The suspension performance atlases of wheel B: (a) ηFmax, (b) ηFmin, (c) ηPmax, (d) ηPmin The analysis method is also applicable to wheel C and wheel E. And the above results provide us with credible evidence to compare multiple parameters to satisfy the optimum comprehensive performances for the suspension. Hence, we can conclude that the optimum domain solution of the suspension (denoted as susp) is the intersection set of WAI, GPI and GSI. Further, if we assign each sub-domain (WAI, GPI or GSI) with more strict demands, then the size of susp will be smaller and smaller and tend to be a unique value. In this case, the global optimum non-dimensional solution is finally identified. Besides, it can also be assigned directly in susp according to the designer’s initiative (Figure 11). Following a series of trade-offs among WAI, GPI, and GSI, Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 9 of 18 (a) (b) (c) (d) Figure 9 The suspension performance atlases of wheel C: (a) ηFmax, (b) ηFmin, (c) ηPmax, (d) ηPmin the red star represents our final identification: the global optimum non-dimensional solution with t1 = 1.5 and t2 , t3 = 0.75, that is to say, l1 : l2 : l3 = 2 : 1 : 1. 4.2 Non‑Dimensional Optimization of the Overall Robot The overall robot is comprised of two suspensions in parallel connection, so it is reasonable to be taken as a parallel mechanism; the body is the moving platform and the contact surface of steel lining is the static platform. To establish its optimization model, the design variables (Figure 5) include l1, Rw , and the width of body w0. The adhesion stability is an important performance criterion, and affects the locomotion security of both robot and equipment inside the containment. It can be described as that the robot will not tip over across any line formed by two adjacent wheels. Based on the force-angle stability measure [33], the adhesion stability index (ASI) takes the resultant tipover force, support polygon, COG position, etc. into account simultaneously. Given the reliable magnetic adhesion to the steel lining surface, six wheels form a support polygon, and they together with the COG can eventually form a rectangular pyramid. Figure 5(a) illustrates the geometric analyses (support polygon j and its normal l j, tipover P1 P2 P3 P4 P5 P6, tipover axis a arm d j, tipover angle θj) and force analyses (wheel-surface resultant force f sj, tipover resultant force f r , tipover resultant moment nr , effective tipover resultant force f ∗j ) to evaluate the tipover cost of overall robot, which has an essential significance to move securely on the steel lining. According to the D’Alembert principle in Figure 5(a), the kineto-static equation is j j j f iner = 0, f grav + f mag + f sup + f fric − (17) where j = 1 ∼ 6 refers to the numbers of six wheels; j f grav is the gravitational load; f mag is the magnetic adhej sion force; f sup is the support force applied to the contact points between wheel and surface, and f iner is the j j j inertial force. Notebly, f mag , f sup and f fric can only be applied to the six wheels, while f grav and f iner exist in all parts of the robot. The general tipover resultant force Qr acting on the COG may lead to a tipover instability, given by Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 10 of 18 (a) (b) (c) (d) Figure 10 The suspension performance atlases of wheel E: (a) ηFmax, (b) ηFmin, (c) ηPmax, (d) ηPmin ≥ 0.2 ≥ 0.5 ≥ 0.5 ≥ 0.2 ≥ 0.7 ≥ 0.5 ≥ 0.5 ≥ 0.9 ≥ 0.3 our choice Qr = fr nr = j j is the j th unit tipover axis vector from pj to where a j = pj+1 − pj /�pj+1 − pj �. pj+1 and a Figure 11 The global solution domain and the optimum solution of suspension where pj j = 1 ∼ 6 is the position vectors of each vertex of the support polygon. As the tipover resultant force Qr has various unstable j. Hence, the relative influences on various tipover axes a tipover force component Qrj with respect to the tipover j is extracted and given by axis a � �  � � T � � I − a a fr 3×3 j f rj , � �j Qrj = = (19) T nrj � nr �j a a j f grav + f mag − f iner , j f grav + f mag − f iner pj × (18) Next, nrj is transformed into an equivalent force cou ple l j × nrj /�l j � ( l j is the tipover axis normal with j and l j = I − a T j a respect to a j pj+1) lying in the nor- mal plane of nrj , so as to formulate a unified expression considering both effects of f rj and nrj . Therefore, the Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 11 of 18 effective tipover resultant force f ∗j with respect to the j is given by tipover axis a 1 f ∗j = f rj + l j × nrj . (20) �l j � Herein, the ASI (ηs ) is defined to evaluate the average adhesion stability of the robot in a posture with all configurations of the suspension by µs dW ηs = , µs = min θj · �d j � · �f ∗j � , (21) j dW where µs denotes the FASM; dW is the traversal of the suspension workspace; θj is the tipover angle, and the negative value indicates the robot will tip over along the jth axis directly; d j denotes the tipover radius vector; f ∗j is the resultant tipover force. The means of the optimization parameters l1, w0 , and Rw are obtained by 1 (l1 + w0 + Rw ) = U . 3 (22) We can get the normalized equation by dividing both sides of the equation by U (23) u1 + u2 + u3 = 3, l1 , u2 = wU0 , and u3 = RUw . Thus, the optimizawhere u1 = U tion parameters are converted to u1 , u2 , u3. Considering dimensional constraint Rw < w0 < 2l1, the constraint equations can be given as u3 < u2 < 2u1 , (24) 0 < u3 < 1.2. Eqs. (13) and (14) show the parameter design space of the suspension, as shown in Figure 12. During the locomotion process, the NuBot experiences various postures affected by the complex surface of the steel lining. Thereinto, the complete handstand posture is the most dangerous one. Herein, the mass of the 3 3 0.6 1.2 1 3 0 2 1 2 Figure 12 Parameter design space of the overall robot 2 Figure 13 The ASI performance atlases of the overall robot suspension and the inertial force of the robot are ignored for simplification, and the magnetic force increases gradually until the negative value disappears in the ASI performance. Figure 13 shows the ASI performance atlas, where u1 , u2 , u3 are the non-dimensional parameters corresponding to l1 , w0 , Rw. The ASI atlas shows that there are three ways to improve the adhesion stability of overall robot, i.e., larger length of suspension, larger width of support polygon, and smaller radius of the wheel. We can conclude that u1 = 1, u2 = 2 and u3 = 0 is the optimal choice, that is, w0 = 2l1 and Rw = 0. Although in practice, Rw is limited by the actual size and cannot be zero, this suggests that the height of COG should be minimized as much as possible. 4.3 Identification of Actual Dimensions by Working Condition The above obtain the optimum proportion relations among all design variables of both suspension and overall robot, i.e. l1 : l2 : l3 : w0 = 2 : 1 : 1 : 4 . In the following, we will first solve the normalization factor to transform the non-dimensional solution to actual dimension parameters; according to the working conditions inside the environment of steel lining, we will finally obtain these actual dimension parameters. Three major aspects should be taken into account. (1) The weld-seam traverse capability requires to meet the contact angle condition. Eq. (25) illustrates that a small wheel radius Rw or weld radius rs, or a big weld height hs can make a large contact angle δ between wheel and weld, further resulting to make all wheels slippery and unable to traverse the weld seam. Eq. (26) illustrates that the friction force of Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Figure 14 Relations among the maximum contact angle, wall inclination angle, and yaw angle (a) Page 12 of 18 Figure 16 The orientation fluctuation and minimum radius of curvature when the robot crosses the weld and adapts to steel lining (b) Figure 15 Configurations of the suspension: (a) The middle wheel crosses the weld, (b) The robot moves on curved surface wheel-surface contact has a maximum limit to avoid slip. Rw + rs − hs ≤ δm , δ = arccos (25) Rw + rs j j ffric ≤ µs fsup , (26) where µs is the sliding friction coefficient, µs = 0.5. Moreover, considering the critical slipping condition, a force equilibrium equation can be derived by Eq. (17) regardless of the inertial force term. Thus, the maximum contact angle for a successful traverse under various wall inclination angle is obtained in Figure 14. One can see the minimum δm is 47.2◦ when ϕ is around ±90◦, so the actual contact angle must be no more than δm. Summing up the above extreme conditions for traversing successfully, the maximum arc weld seam has a height hs = 5 mm and radius rs = 11.8 mm, and the allowable δm is assigned to be 31◦. As a result, we can derive Rw = 27 mm according to Eq. (25). (2) The orientation fluctuation quantity caused by the weld-seam height (Figure 15(a)), and the minimum steel lining radius of curvature Rsl that NuBot can adapt to (Figure 15(b)) should be reduced as little as possible. To ensure smooth locomotion and better curvature adaptability, \|θC \| and Rsl is chosen to evaluate the orientation fluctuation and minimum steel lining radius, and smaller values are desired. The relationship between \|θC \|, Rsl and l1 can be derived by geometry relationship. The interferencefree conditions of adjacent wheels can be expressed by   2Rw ≤ l3 + l6 , 2Rw ≤ l2 + l3 ,  2R ≤ (l + l )cosθ − (l + l )cosθ . w 3 6 C 2 3 D (27) Considering each configuration of the suspension, eventually l1 ≥ 54 mm is obtained. After leaving some gap, the travel range of l1 is finally chosen to be 60–140 mm. The results of the above cases are shown in Figure 16. One can see that: when crossing the weld, \|θC \| decreases as l1 increases, while Rsl shows the opposite change when the robot adapts to the curved steel lining. After making a trade-off between the two cases, l1 is chosen to be 100 mm. Hence, the other two lengths of the suspension are given by l2 = l3 = 50 mm, and parameters of the robot are given by w0 = 2l1 = 200 mm. (3) The interference-free conditions considering the weld seam and support structure effects constrain the bottom and top of the overall robot. To avoid interference between the chassis and weld seam, the initial height of the bottom chassis hbc and the travel ranges of l4 and l5 should be as large as possible (Figure 15(a)), which in turn causes higher COG and smaller ASI. Thus, the limit case is considered and hbc , l4 and l5 are respectively chosen to be 15 mm, 10 mm, and 10 mm after trade-off. Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 13 of 18 Figure 17 a Topological optimization results of the chassis, suspension, and wheel hub, b Structure of the magnet unit, c The relationship between magnetic force and wall distance 5 Unit Design and Prototype With all sizes determined, the rest parameters regarding mass, magnet, etc. can be determined through the following steps: (1) obtaining the robot mass after designing and topological optimization of the structure; (2) designing the magnet unit in Maxwell software. (d) Desired velocity Bluetooth Joysck + Velocity PI controller - Encoder Weld Rotational speed n & Scan (1) The overall robot can be designed with corresponding parameters proportion. To reduce weight, all structural components including chassis, suspension, and the wheel hub are obtained by topological optimization and finite element simulation in the software of solidThinking Inspire (Figure 17(a)). Considering the weight of the electronic components, the overall mass of NuBot is around 1 kg. (2) Next, the parameters of magnet can be determined by simulation in Maxwell (Figure 17(b)), including the radius of magnet Rm , rm, the angle of sector magnet θm, the thickness of yoke dy , and the gap distance between the magnet and the wall. Considering the magnetic force is supposed to support the entire robot and ensure certain safety, the design magnetic force is set to be 55 N. Simulation shows that the magnet weight is 75% less than the magnet with a full circle, but the magnetic force only decreases by 15%. To reduce steering friction [6], per middle wheel shares twice the magnetic force of a front or rear wheel. Figure 17(c) shows NuBot Actual velocity Laptop Euler angle, angular IMU velocity, acceleration WIFI camera Figure 18 Schematic of electronic components and the control strategies. a Microcontroller and sensors inside NuBot, b The monitor for displaying weld images and sensor data, c The upper controller for controlling locomotion of NuBot, d The closed-loop that the magnetic force decreases when the gap distance increases, and the 6 mm gap that satisfies the desired force is finally selected. The detailed sizes of the magnet unit are shown in Table 2. So far, all design parameters and specific structures of NuBot can be determined, and the prototype composed of six magnetic wheels, two pairs of suspensions, and a platform is developed, as shown in Figure 18. And the specifications of NuBot are listed in Table 3. The control system is shown in Figure 18(d). The Arduino Uno works as an upper machine, which Table 2 Specifications of the sector magnets Wheel Rm (mm) rm (mm) Front/rear 17.6 9.9 Middle Axial thickness (mm) θm (°) dy (mm) 20 3 30 90 Simulated magnetic force (N) 6.96 13.75 Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 14 of 18 Table 3 Specifications of NuBot platform Items Features Total mass 980 g Partial mass Front/rear wheel: 72 g Middle wheel: 80 g Suspension: 32 g Others: 468 g Length × width × height 260 × 200 × 100 mm3 Length of wheelbase 100 mm Radius of wheel 27 mm Joint limits of suspension l4 , l5 ∈ [0, 10mm] θC ∈ [−15◦ , 15◦ ] Material Carbon fiber board (chassis) 3D printing resin (other body parts) DC motor (encoder inside) Type: GA12-N20 (0.2 Nm) Max. angular speed: 120 r/min Controller Arduino Uno (upper machine) Arduino Nano (lower machine) IMU JY901 (9-axis: accelerometer, gyroscope, and magnetometer) transmits desired speed to the lower machine through Bluetooth. The Arduino Nano works as a lower machine, which processes data from encoders and IMU, and controls driving motors through a speed PI controller. Speeds of the wheels are read from the encoders and the closed-loop control is formed by comparison with the desired speed from the joystick. All the components are powered by an 11.1 V and 3000 mAh battery, and can work continuously for more than 30 min. The camera works independently with the above control system, which will not be described in detail for it is secondary here. Sensor data and weld images can be transmitted to the laptop for monitoring. 6 Experimental Results and Discussion 6.1 Locomotion on Different Types of Walls Figures 19(a, b) show the locomotion on a strong magnetic curved wall with a radius of 2 m. The maximum speed of NuBot is 0.5 m/s, which is around 1.92 times the robot length. Due to the strong magnetic force, the robot can only turn with a radius of 3 m. Please see the Additional file 1: Video. Figure 19(c) shows the locomotion on the weak magnetic blackboard, and the minimum turning radius is 0.2 m. The magnetic force herein is around one-third of that on the curved wall. Locomotion experiments on the blackboard with various inclination angles are conducted in Figures 19(d–f ), which validate the adhesion stability of NuBot. Experiments on the vertical blackboard are conducted to test the precise movement. Given desired Figure 19 Locomotion experiments. a Moving forward, b Steering on a strong magnetic curved wall; c Steering on a weak magnetic blackboard; locomotion on the blackboard with an inclination angle of (d) 90°, (e) 135°, and (f) 180° wheel speed on both sides, the desired trace can be obtained. The actual trace is calculated by accumulating the mileage from encoders inside the left and right wheels. As shown in Figure 20, the robot turns around a circle at a constant speed. The actual trace coincides well with the expected one, and the trace radius error tends to be a minor value. 6.2 Validation of Weld‑crossing Capacity Crossing the welds on the containment surface may cause instability of the robot body, which in turn affects the adhesion stability. Herein we tested this process with 3D printed welds with two typical heights of 3 mm and 5 mm (the maximum weld height), as shown in Figure 21. Results show that the crossing process hardly affects the adhesion stability of the robot. Another function of the suspension is to reduce the fluctuation of the robot body when crossing uneven terrain. Herein, when the robot crosses the weld, the change of its body pitch angle is recorded by IMU, as shown in Figure 22. In general, the theoretical and experimental results fit well before the peak, while after the peak, the suspension makes the body stabilize quickly. And the maximum peak error is within 9%. 6.3 Validation of Payload Capacity Payload capacity is a significant index of the climbing robot. Herein, experiments on payload capacity under different walls are conducted, as shown in Figure 23. The maximum load of the robot on the blackboard is 0.6 kg with a speed of 0.08 m/s (Figure 23(a)), and the wheels will slip when the load continues to increase. Besides, the load on the curved wall reaches 0.8 kg with a speed of 0.065 m/s (Figure 23(b)). In either case, the robot can load enough weight of the equipment for weld inspection. Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Page 15 of 18 Figure 20 Steering motion accuracy experiments. a Comparison between the actual trace and desired trace (desired radius is 375 mm), b Change of left and right wheel speed (desired wheel speed: left 230 mm/s and right 150 mm/s), c Error percentage of trace radius Figure 21 Weld crossing experiments. The robot crosses the weld with a height of (a–c): 3 mm; (d–e): 5 mm 0.8kg 0.6kg (b) (a) (c) 3.6kg δ (e ) weld Rear wheels Mid wheels 1.6kg (d) Figure 22 Comparison of theoretical and experimental results of the body pitch when crossing the weld Besides, extreme payload experiments are conducted to verify the payload capacity (Figures 23(c–f ), namely cases 1 to 4). The case of robot handstand is selected, because the influence of wheel friction can be ignored, and we can focus on the influence of adhesion stability on the robot. Figure 24 illustrates the magnetic force, corresponding ASI, and experimental maximum load under the four cases. As expected, NuBot in case 1 possesses the maximum payload of up to 3.6 kg. Notably, the magnetic force in case 4 is not the smallest, but the payload capacity is the worst, which indicates the asymmetry of magnetic force distribution will reduce the payload capacity. 2kg (f) 1kg Figure 23 Payload capacity experiments: a Locomotion on the vertical blackboard, b Locomotion on curved wall, c Static adhesion to the inverted wall, d–f Crossing the weld on inverted wall by two front, middle, lateral wheels Magnetic force (N) t (s) (e) The ASI / Max. payload (kg) Front wheels Four typical cases Figure 24 The magnetic force under different cases in experiment Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Figure 25 Comparison of payload/mass, mass, and number of driving motors of several typical climbing robots, where the bubble size represents the number of driving motors 6.4 Results and Discussion The experimental results show that NuBot can carry a 0.6 kg load on the weak magnetic wall and a load of no less than 0.8 kg on the strong magnetic wall with a radius of 2 m. The actual trace has a small error compared to the desired one. Besides, the robot reaches a maximum load of 3.6 kg, which is 3.67 times its mass, and the experimental results of its maximum payload are consistent with the analysis of ASI. In addition, the robot can stably cross the simulated welds and move on the walls with different inclination angles. The payload capacity is an important indicator of its performance and can be evaluated by the ratio of the payload to its mass. Robots that can carry more load with less of their mass are favored. Figure 25 shows a comparison of the performances of several climbing robots. Notably, the weight of robots with soft bodies or footpads [2, 5, 21, 34–37] are all less than 1 kg, moreover, robots in [2, 34, 37] are with an excellent payload capacity of more than 2.5 times their weight. By contrast, wheeled [6, 7, 9, 38] or crawler [3, 14, 39, 40] magnetic adhesion robots are heavier due to the mechanical components. Particularly, NuBot obtained under our design strategy has a light body with a good payload capacity, and the number of driving motors ensures it has sufficient driving force. 7 Conclusions This paper proposes a magnetic adhesion robot with passive suspension and six wheels called NuBot, which is used to inspect the steel lining weld of the ferromagnetic wall (nuclear power containment is chosen as an application here). The main contribution of this article is to establish a systematic design method for the wheeled adhesion robot. (1) A 3-DOF suspension with good adaptability to the steel lining, good payload capability, and passive Page 16 of 18 compliance is proposed, which ensures that the robot adapts to the curvature of the steel lining. (2) Building a comprehensive optimization design model for NuBot. Based on the kinematic model of suspension and the dynamic model of the overall robot, the optimization model is established, including the parameters of topological structure and the performance indices of WAI, GPI, GSI, and ASI. Then, the PCbDM is applied to optimize NuBot with five parameters from the suspension (local) to the overall robot (whole). Optimal parameters are properly chosen from the performance atlases perceptually and credibly. (3) The normalization factor and actual dimension parameters are determined by constraints of the working conditions inside the environment of the steel lining. The electronic system with a PI controller is constructed to accomplish a higher control accuracy. Critical experiments are conducted to validate the design and recorded as a video, seen in the Additional file 1. The experimental results show that NuBot meets the comprehensive requirements on size, weight, locomotion, payload, and adhesion stability. Applications of NuBot can be further expanded. On the one hand, the robot can be equipped with more inspection instruments, such as ultrasonic flaw detectors, magnetic flaw detectors, etc. And devices that exceed the robot payload can be equipped by the hard connection of several robots. On the other hand, NuBot can be used on more occasions with ferromagnetic walls, such as oil and gas tanks and pipelines, vessels, and wind turbines. NuBot can be modified to adapt to the specific environment constraints based on the proposed design method. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s10033-023-00905-6. Additional file 1. Experimental video. Acknowledgements The authors sincerely thanks to Professor Shaohong Zhang of Shanghai Nuclear Star Nuclear Power Technology Co., Ltd. for his critical discussion during design of the robot and prototype. Author Contributions HX conducted the detailed study on the theoretical analysis of the robot, and completed the experiment and manuscript writing; YH put forward the idea of theoretical analysis and participated in the writing of manuscript; MH modeled the robot structure and made the prototype. YL participated in the experiment and paper writing; WG gave overall guidance during analysis, design, and experiments of the robot, and writing of the manuscript. All authors read and approved the final manuscript. Xu et al. Chinese Journal of Mechanical Engineering (2023) 36:90 Authors’ Information Hao Xu, born in 1997, received his master degree from State Key Lab of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, China, in 2023. Youcheng Han, born in 1993, is currently a PhD candidate at State Key Lab of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, China. Mingda He, born in 1998, is currently a Mechanical Engineer in Shenzhen Dajiang Innovation Technology Co., Ltd., China. He received his master degree from State Key Lab of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, China, in 2022. Yinghui Li, born in 1999, is currently a PhD candidate at State Key Lab of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, China. Weizhong Guo, born in 1970, is currently a professor and a PhD candidate supervisor at State Key Lab of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, China. His main research interests include modern mechanism, parallel robot, motion planning and design. Funding Supported by Shanghai Nuclear Star Nuclear Power Technology Co., Ltd., National Natural Science Foundation of China (Grant No. 51735009), and State Key Lab of Mechanical System and Vibration Project (Grant No. MSVZD202008). Availability of Data and Materials The datasets supporting the conclusions of this article are included within the article. Page 17 of 18 [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] Declarations Ethics Approval and Consent to Participate Not applicable. [19] [20] Consent for Publication Not applicable. Competing Interests The authors declare no competing financial interests. [21] [22] Received: 29 May 2022 Revised: 5 June 2023 Accepted: 8 June 2023 [23] References [1] H Sun. Third generation nuclear power technology AP1000. Beijing: China Electric Power Press, 2010. [2] T Seo, M Sitti. Tank-like module-based climbing robot using passive compliant joints. IEEE/ASME Transactions on Mechatronics, 2013, 18(1): 397-408. [3] F Gao, J C Fan, L Zhang, et al. 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W4287889745.txt	https://aclanthology.org/2022.naacl-main.46.pdf	en		User-Centric Gender Rewriting	Proceedings of the 2022 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies	2,022	cc-by	9,093	User-Centric Gender Rewriting Bashar Alhafni, Nizar Habash, Houda Bouamor† Computational Approaches to Modeling Language Lab New York University Abu Dhabi † Carnegie Mellon University in Qatar {alhafni,nizar.habash}@nyu.edu, hbouamor@qatar.cmu.edu Abstract output, NLP systems should be designed to produce outputs that are as gender-specific as the users information they have access to. Users information could be either embedded as part of the input or provided externally by the users themselves. In cases where this information is unavailable to the system, generating all gender-specific forms or a gender-neutral form is more appropriate. Producing user-aware outputs becomes more challenging for systems targeting multi-user contexts (first and second persons, with independent grammatical gender preferences), particularly when dealing with gender-marking morphologically rich languages. In this paper, we define the task of gender rewriting in contexts involving two users (I and/or You) – first and second grammatical persons with independent grammatical gender preferences and we focus on Arabic, a gender-marking morphologically rich language. The main contributions of our work are as follows: In this paper, we define the task of gender rewriting in contexts involving two users (I and/or You) – first and second grammatical persons with independent grammatical gender preferences. We focus on Arabic, a gendermarking morphologically rich language. We develop a multi-step system that combines the positive aspects of both rule-based and neural rewriting models. Our results successfully demonstrate the viability of this approach on a recently created corpus for Arabic gender rewriting, achieving 88.42 M2 F0.5 on a blind test set. Our proposed system improves over previous work on the first-person-only version of this task, by 3.05 absolute increase in M2 F0.5 . We demonstrate a use case of our gender rewriting system by using it to post-edit the output of a commercial MT system to provide personalized outputs based on the users’ grammatical gender preferences. We make our code, data, and pretrained models publicly available.1 1 1. We introduce a multi-step gender rewriting system that combines the positive aspects of rule-based and neural models. Introduction Gender bias is a fundamental problem in natural language processing (NLP) and it has been receiving an increasing attention across a variety of core tasks such as machine translation (MT), co-reference resolution, and dialogue systems. Research has shown that NLP systems have the ability to embed and amplify gender bias (Sun et al., 2019), which not only degrades users’ experiences but also creates representational harm (Blodgett et al., 2020). The embedded bias within NLP systems is usually attributed to training models on biased data that reflects the social inequalities of the world we live in. However, even the most balanced of models can still exhibit and amplify bias if they are designed to produce a single text output without taking their users’ gender preferences into consideration. Therefore, to provide the correct user-aware 1 https://github.com/CAMeL-Lab/ gender-rewriting/ 2. We demonstrate our approach’s effectiveness by establishing a strong benchmark on a publicly available multi-user Arabic gender rewriting corpus. 3. We show that our best system yields state-ofthe-art results on the first-person-only version of this task, beating previous work. 4. We demonstrate a use case of our system by post-editing the output of an MT system to match users’ grammatical gender preferences. This paper is organized as follows. We first discuss related work (§2) as well as relevant Arabic linguistic facts (§3). We then define the gender rewriting task in §4 and describe the data we use and the gender rewriting model we build in §5 and §6. Lastly, we present our experimental setup (§7) and results (§8) and conclude in §9. 618 Proceedings of the 2022 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies, pages 618 - 631 July 10-15, 2022 ©2022 Association for Computational Linguistics 2 3 Background and Related Work Substantial research has targeted the problem of gender bias in various NLP tasks such as MT (Rabinovich et al., 2017; Vanmassenhove et al., 2018; Stafanovičs et al., 2020; Savoldi et al., 2021), dialogue systems (Cercas Curry et al., 2020; Dinan et al., 2020; Liu et al., 2020a,b; Sheng et al., 2021), language modeling (Lu et al., 2018; Bordia and Bowman, 2019; Sheng et al., 2019; Vig et al., 2020; Nadeem et al., 2021), co-reference resolution (Rudinger et al., 2018; Zhao et al., 2018a), and named entity recognition (Mehrabi et al., 2019). While the majority of research has focused on tackling gender bias in English by debiasing word embeddings (Bolukbasi et al., 2016; Zhao et al., 2018b; Gonen and Goldberg, 2019; Manzini et al., 2019; Zhao et al., 2020) or by training systems on gender-balanced corpora built using counterfactual data augmentation techniques (Lu et al., 2018; Hall Maudslay et al., 2019; Zmigrod et al., 2019), our work falls under text rewriting through the controlled generation of gender alternatives for morphologically rich languages. Within text rewriting, Vanmassenhove et al. (2021) and Sun et al. (2021) recently presented rule-based and neural rewriting models to generate gender-neutral sentences in English. For morphologically rich languages and specifically Arabic, Habash et al. (2019) introduced the Arabic Parallel Gender Corpus v1.0 (APGC v1.0) of firstperson-singular constructions and designed a twostep gender identification and reinflection system to generate masculine and feminine grammatical gender alternatives. Alhafni et al. (2020) used APGC v1.0 to create a joint gender identification and reinflection model. They treated the problem as a user-aware grammatical error correction task and showed improvements over Habash et al. (2019)’s results. Both efforts modeled gender reinflection using character-level Seq2Seq models. More recently, Alhafni et al. (2022) extended APGC v1.0 to APGC v2.0 by including contexts involving first and second grammatical persons covering singular, dual, and plural constructions; and adding six times more sentences. In our work, we use APGC v2.0 to build a multistep gender rewriting system to generate gender alternatives in multi-user contexts. We also show improvements over both Habash et al. (2019)’s and Alhafni et al. (2020)’s results on APGC v1.0. Arabic Linguistic Facts We highlight two of the many challenges that face Modern Standard Arabic (MSA) NLP systems dealing with gender expressions. Morphological Richness and Complexity Arabic has a rich and complex morphological system that inflects for many morphological features (gender, number, person, case, state, aspect, mood, voice), in addition to several attachable clitics (prepositions, particles, pronouns) (Habash, 2010). Arabic nouns, adjectives, and verbs inflect for gender: masculine (M) and feminine (F), and for number: singular (S), dual (D) and plural (P). Changing the grammatical gender of Arabic words involves either changing the form of the base word, changing the pronominal enclitics that are attached to the base word, or a combination of both. A base word in Arabic refers to the stem along with its attachable affixes (prefixes, suffixes, circumfixes). Changing the base word gender requires either a suffix change, a pattern change, or a lexical change as shown in Table 1(a-c). Arabic also has clitics that attach to the stem after affixes. A clitic is a morpheme that has the syntactic characteristics of a word but shows evidence of being phonologically bound to another word. In this respect, a clitic is distinctly different from an affix, which is phonologically and syntactically part of the word. Proclitics are clitics that precede the word (like a prefix), whereas enclitics are clitics that follow the word (like a suffix). Pronominal enclitics are pronouns that cliticize to previous words (Table 1(d)). It is worth noting that multiple affixes and clitics can appear in a single word in Arabic and changing the grammatical gender of such words requires changing the genders of both the base word and its clitics (Table 1(f-g)). Orthographic Ambiguity Arabic uses diacritics to specify short vowels. However, these optional diacritics are usually omitted in Arabic orthography, leaving readers to infer the meaning of certain words based on the context (Habash, 2010). This increases the degree of word ambiguity as genderspecific words could only differ in terms of dia . ªË lςbt2 can be critics. For instance, the verb IJ diacritized as or as I J. ªË I J. ªË laςibta ‘you [masc.] played’ laςibti ‘you [fem.] played’. 2 Arabic transliteration is in the HSB scheme (Habash et al., 2007). 619 Paired Gender Alternatives ‫ أ‬Âmyr (NOUN.MS) prince ‫ أ‬ÂHmr (ADJ.MS) ‫اء‬ red ‫ أخ‬Âx (NOUN.MS) brother + ‫ أ‬Âmyr+km (NOUN.MS+PRON.2MP) sister + ‫ أ‬Âmyr+kn (NOUN.MS+PRON.2FP) your (FP) prince ‫ أ ات‬ÂmyrAt (NOUN.FP) princesses ‫ أ‬Âmyrt+kn (NOUN.FS+PRON.2FP) + your (MP) prince +‫ أ اء‬ÂmrA’+hm (NOUN.MP+PRON.3MP) princess HmrA’ (ADJ.FS) red ‫ أ‬Âxt (NOUN.FS) your (MP) prince ‫ أ اء‬ÂmrA’ (NOUN.MP) princes + ‫ أ‬Âmyr+km (NOUN.MS+PRON.2MP) Rewrite Type ‫ أ ة‬Âmyrħ (NOUN.FS) your (FP) princess +‫ أ ات‬ÂmyrAt+hn (NOUN.FP+PRON.3FP) their (MP) princes their (FP) princesses Suffix Change (a) Pattern Change (b) Lexical Change (c) Enclitic Change (d) Pattern Change + (e) Suffix Change Suffix Change + (f) Enclitic Change Pattern Change + Suffix Change + (g) Enclitic Change Table 1: Examples of the changes needed to generate gender alternative forms of gender-specific words in Arabic. 4 The Gender Rewriting Task We define the task of gender rewriting as generating alternatives of a given Arabic sentence to match different target user gender contexts (e.g., female speaker with a male listener, a male speaker with a male listener, etc.). This requires changing the grammatical gender (masculine or feminine) of certain words referring to the users (speaker/1st person and listener/2nd person). Previous work done by Habash et al. (2019) and Alhafni et al. (2020) refer to this task as gender reinflection, but we believe that gender rewriting is a more appropriate term given that it goes beyond reinflection.3 Notation We will use four elementary symbols to facilitate the discussion of this task: 1M, 1F, 2M and 2F. The digit part of the symbol refers to the grammatical persons (1st or 2nd ) and the letter part refers to the grammatical genders (masculine or feminine). Additionally, we will use B to refer to invariant/ambiguous gender. We define the sentence-level gender using the following four labels: 1M/2F, 1F/2M, 1M/2F, and 1F/2F. These four labels indicate the grammatical persons and genders of the user contexts we are modeling. We define the word-level gender based on the genders of the word’s base form and its attachable pronominal enclitics (§3) using the notation: base form gender + enclitic gender. This results in 3 Morphological reinflection usually refers to reinflecting either a lemma or an already inflected form to produce a desired form of a particular word (Cotterell et al., 2016, 2017). 25 word-level gender labels (e.g., B+1F, 1F+2M). We use B to refer to gender invariant/ambiguous words. Examples of the word-level gender labels are shown in Table 2. Task Definition Given an Arabic sentence and a sentence-level target gender, the goal is to rewrite the input sentence to match the target users’ gender preferences. Some of the models we explore only use sentence-level gender labels; while other models use word-level gender labels to identify which input words need to be rewritten to match the target users’ gender preferences. 5 Data For our experiments, we use the publicly available Arabic Parallel Gender Corpus (APGC) – a parallel corpus of Arabic sentences with gender annotations and gender rewritten alternatives of sentences selected from OpenSubtitles 2018 (Lison and Tiedemann, 2016). The corpus comes in two versions: APGC v1.0 and APGC v2.0. APGC v1.0 was introduced by Habash et al. (2019) and it contains 12,238 first-person-singular Arabic parallel gender-annotated sentences. Alhafni et al. (2022) expanded APGC v1.0 by including contexts involving first and second grammatical persons covering singular, dual, and plural constructions to create v2.0, which contains 80,326 gender-annotated parallel sentences (596,799 words). Both versions of APGC include the original English parallels of the Arabic sentences. 620 English (a) I want to talk to you (b) Dad, I am here I will tell you [plural] (c) something I am going to my (d) office (e) Because I am an idiot I am glad to know (f) you [plural] Input ‫أن أ ث‬ B B B ‫أ‬ B ‫أر‬ B B ‫أ‬ B 2M+B B Target 1M/2M Target 1F/2M Target 1M/2F Target 1F/2F ‫أر أن أ ث‬ ‫أر أن أ ث‬ ‫أر أن أ ث‬ ‫أر أن أ ث‬ B B ‫أ‬ B B B ‫أ‬ B 2M+B B B ‫أ‬ B B B ‫أ‬ B 2M+B B+2F ‫أ ذا‬ B B+2M ‫أ ذا‬ B B+2M ‫أ ذا‬ B 1M+B B ‫ء‬ ‫و‬ B 1M+B B ‫أ‬ ‫و‬ B 1F+B B ‫ء‬ ‫و‬ 1M+B 1F+B ‫ة‬ 1F+B ‫ة‬ B ‫أ‬ B ‫أ‬ B ‫أ‬ B B ‫أ‬ B B B B ‫أ‬ B 2F+B B+2F ‫أ ذا‬ B 1M+B B ‫أ‬ ‫و‬ 1M+B B ‫أ‬ B+2M 1F+B B B+2M 1M+B B B+2M 1F+B B B+2F 1M+B B B B ‫أ‬ B B ‫أ‬ B 2F+B B B+2F ‫أ ذا‬ B 1F+B B ‫ء‬ ‫و‬ 1F+B ‫ة‬ B ‫أ‬ B+2F 1F+B B Table 2: Examples from the Arabic Parallel Gender Corpus v2.0 including the extended word-level annotations for each sentence and its rewrite to the opposite grammatical gender forms where appropriate. First person gendered words are in purple and second person gendered words are in red. M is Masculine; F is Feminine; and B is invariant. In all of our experiments, we use an extended version of APGC v2.0 to train and test our systems. We also report results on the test set of APGC v1.0 to compare with previous work. Annotations Each sentence in APGC v2.0 has word-level gender labels where each word is labeled as B, 1F, 2F, 1M, or 2M. All sentences containing gender-specific words referring to human participants have parallels representing their opposite gender forms. For the sentences without any gender-specific words, their parallels are trivial copies. Out of the 80,326 sentences in APGC v2.0, 46% (36,980) do not contain any gendered words, whereas sentences with gendered references constitute 54% (43,346). In terms of the word-level statistics, 9.7% (58,066) are gender-specific, whereas 90.3% (538,733) are marked as B. Moreover, APGC v2.0 is organized into five parallel corpora that are fully aligned (1-to-1) at the word level: Input, Target 1M/2M, Target 1F/2M, Target 1M/2F, and Target 1F/2F. All five corpora are balanced in terms of gender, i.e., the number of words marked as 1F and 1M is the same; and the number of words marked as 2F and 2M is the same. The Input corpus contains sentences with all possible word types (B, 1F, 2F, 1M, 2M). The Target 1M/2M corpus contains sentences that consist of B, 1M, 2M words; the Target 1F/2M corpus contains sentences that consist of B, 1F, 2M words; the Target 1M/2F corpus contains sentences that consist of B, 1M, 2F words; and the Target 1F/2F corpus contains sentences that consist of B, 1F, 2F words. The four target corpora are intended to model the target users’ gender preferences for a particular input. Splits We use Alhafni et al. (2022)’s splits: 57,603 sentences (427,523 words) for training (T RAIN), 6,647 sentences (49,257 words) for development (D EV), and 16,076 sentences (120,019 words) for testing (T EST). Preprocessing the Word-Level Annotations Since gender information could be expressed at different parts of Arabic words (§3), we automatically extend the APGC v2.0 word-level annotations to mark the genders of both the base words and their pronominal enclitics. Our preprocessing pipeline considers the labeled gendered words across the five parallel forms of each sentence in APGC v2.0. If the word ends with a gender marking pronominal enclitic, we label the gender of the enclitic based on predefined rules as 1F, 1M, 2F, or 2M. If the gendered word does not end with a gender-marking enclitic, then we label the enclitic as B. Once the enclitic is labeled, we compare the base form of the word across its parallel forms. If the base form is the same, we label it as B. Otherwise, we assign the base form the same label that is provided as part of APGC v2.0. All gender ambiguous words will be labeled as B. Table 2 presents some examples from APGC v2.0 with the extended word-level gender annotations. The extended word-level statistics are presented in Appendix B. We make the extended word-level gender annotations publicly available as a new release of APGC (APGC v2.1).4 621 4 http://resources.camel-lab.com/ 6 Input Sentence The Multi-step Model Approach Most of the recent work on gender rewriting rely on using Seq2Seq models (Habash et al., 2019; Alhafni et al., 2020; Sun et al., 2021; Jain et al., 2021; Vanmassenhove et al., 2021). However, the lack of large gender-annotated parallel datasets presents a challenge when training Seq2Seq models, and especially when dealing with morphologically rich languages. This issue is highlighted by Alhafni et al. (2020), who report that most of the errors (68%) produced by their character-level Seq2Seq model are due to not making any changes to genderspecific words in the input sentences. Given the complexity of the gender rewriting task in Arabic and the relatively small training data size, we model the gender rewriting task using a multi-step system the combines the positive aspects of rule-based and neural models. Our system consists of three components: Gender identification, Out-of-context word gender rewriting, and In-context ranking and selection. 6.1 Gender Identification (GID) We first identify the word-level gender label (base word + pronominal enclitic) for each word in the input sentence. We build a word-level classifier by leveraging a Transformer-based pretrained language model. There are many Arabic monolingual BERT models available such as AraBERT (Antoun et al., 2020), ARBERT (Abdul-Mageed et al., 2021), and QARIB (Abdelali et al., 2021). However, we chose to use CAMeLBERT MSA (Inoue et al., 2021) as it was pretrained on the largest MSA dataset to date. Following the work of Devlin et al. (2019), we fine-tune CAMeLBERT MSA using Hugging Face’s transformers (Wolf et al., 2020) by adding a fully-connected linear layer with a softmax on top of its architecture. During fine-tuning, we use the representation of the first sub-token as an input to the linear layer. 6.2 Out-of-context Word Gender Rewriting Given the desired sentence-level target gender as an input and the identified gender label for each word in the input sentence, we decide if a word-level gender rewrite is needed based on the compatibility between the provided sentence-level target gender and the predicted word-level gender labels. We implement three word-level gender alternative generation models: Corpus-based Rewriter, Morphological Rewriter, and Neural Rewriter. ‫سعيدة حقا بمعرفتكن يا سيدات‬ 1M Really glad to know you ladies Speaker Gender Identification 2M Listener ‫سعيدة حقا بمعرفتكن يا سيدات‬ 2F+B B Word Gender Rewriting B+2F B 1F+B CorpusR MorphR NeuralR Selection ✔︎ ‫ سعيد حقا بمعرفتكم يا سادة‬-17.21 ‫سعيدا حقا بمعرفتكم يا سادة‬ -18.14 Really glad to know you gentlemen Figure 1: The multi-step gender rewriting system. First person gendered words are in purple and second person gendered words are in red. The sentence-level target gender is 1M/2M. The input words glad (1F+B), know you (B+2F), and ladies (2F+B) are rewritten to their masculine forms. Corpus-based Rewriter (CorpusR) We build a simple word-level lookup rewriting model by exploiting the fully aligned words in APGC v2.1. We implement this model as a bigram maximum likelihood estimator: given an input word with its bigram surrounding context (wi , wi−1 ), a gender alternative target word (yi ), and a desired word-level target gender (g), the CorpusR model is built by computing P (yi \|wi , wi−1 , g) over the training examples. During inference, we generate all possible gender alternatives for the given input word (wi ). If the bigram context (wi , wi−1 ) was not observed in the training data, we backoff to a unigram context. If the input word was not observed during training, we pass it to the output as it is. Morphological Rewriter (MorphR) For the morphological rewriter, we use the morphological analyzer and generator provided by CAMeL Tools (Obeid et al., 2020). We extend the Standard Arabic Morphological Analyzer database (SAMA) (Graff et al., 2009) used by the morphological generator to produce controlled gender alternatives. We make our extensions to the database publicly available.5 5 We provide code to reconstruct our extended database from the original SAMA 3.1 database (LDC2010L01) which can be obtained from the LDC. 622 Given an input word and a desired word-level target gender, the morphological generator has the ability to produce gender alternatives by either rewriting the base word, its pronominal enclitics, or both. If an input word does not get recognized by the morphological analyzer and generator, we pass it to the output as it is. It is worth noting that this rewriting model does not require any training data. by one.6 We will refer to the in-context ranking and selection as simply selection throughout the paper. Figure 1 presents an overview of our gender rewriting model. We describe the training settings and the model’s hyperparameters in Appendix A. Neural Rewriter (NeuralR) Inspired by work done on out-of-context morphological reinfection (Kann and Schütze, 2016; Cotterell et al., 2018), we design a character-level encoder-decoder model with attention. Given an input word and word-level target gender, the encoder-decoder model would generate gender alternatives of the input word. For the encoder, we use a two-layer bidrectional GRU (Cho et al., 2014) and for the decoder we use a two-layer GRU with additive attention (Bahdanau et al., 2015). Furthermore, we employ side constraints (Sennrich et al., 2016) to control for the generation of gender alternatives. That is, we add the word-level target gender as a special token (e.g., <1F+B>) to the beginning of the input word and we feed that entire sequence to the model (i.e., <1F+B> YJª). The intuition here is that the attentional encoder-decoder model would be able to learn to pay attention to the side constraints to generate the desired gender alternative of the input word. During inference, we use beam search to generate the top 3-best hypotheses. 7.1 6.3 In-Context Ranking and Selection Since the three word-level gender alternative generation models we implement are out-of-context and given Arabic’s morphological richness, we expect to get multiple output words when generating a single gender alternative for a particular input word. This leads to producing multiple candidate gender alternative output sentences. To select the best candidate output sentence, we rank all candidates in full sentential context based on their pseudo-loglikelihood (PLL) scores (Salazar et al., 2020). We first use Hugging Face’s transformers to fine-tune the CAMeLBERT MSA model on the Input corpus of APGC v2.1 by using a masked language modeling (Devlin et al., 2019) objective. This helps in mitigating the domain shift (Gretton et al., 2006) issue between CAMeLBERT’s pretraining data and APGC v2.1. We then compute the PLL score for each sentence using the fine-tuned CAMeLBERT MSA model by masking the sentence tokens one 7 Experimental Setup Evaluation Metrics We follow Alhafni et al. (2020) by treating the gender rewriting problem as a user-aware grammatical error correction task and use the MaxMatch (M2 ) scorer (Dahlmeier and Ng, 2012) as our evaluation metric. The M2 scorer computes the precision (P), recall (R), and F0.5 by maximally matching phraselevel edits made by a system to gold-standard edits. The gold edits are computed by the M2 scorer based on provided gold references. Moreover and to be consistent with previous work, we also report BLEU (Papineni et al., 2002) scores which are obtained using SacreBLEU (Post, 2018). We report the gender rewriting results in a normalized space for Alif, Ya, and Ta-Marbuta (Habash, 2010). 7.2 Baselines Do Nothing Our first baseline trivially passes the input sentences to the output as they are. This baseline highlights the level of similarity between the inputs and the outputs. Joint Baseline Model Our second baseline uses a variant of the sentence-level linguistically enhanced joint gender identification and rewriting model introduced by Alhafni et al. (2020). The main difference between this model and the one introduced by Alhafni et al. (2020) is that we model four multi-user target genders, whereas they only modeled two single-user target genders (1M, 1F). We implement this joint baseline model using a character-level GRU encoder-decoder with additive attention, where the learned input characterlevel representations are enriched with word-level morphological features obtained from the extended morphological analyzer that is part of CAMeL Tools. The representation of the input sentencelevel target gender (1M/2M, 1F/2M, 1M/2F, 1F/2F) is learned as part of the model and used during decoding when generating gender alternatives. We refer to this baseline as Joint+Morph. 6 We use Salazar et al. (2020)’s implementation to compute the PLL scores: https://github.com/awslabs/ mlm-scoring 623 Extended Joint Baseline Models Our third and fourth baseline models reduce the complexity of the Joint+Morph model by not learning a representation for the input sentence-level target gender as part of the model. Instead, we provide the sentence-level target gender information as side constraints. We add the target gender as a special token to the beginning of the input sentence (e.g., <1M/2F>Input Sentence) when we feed it to the model. Moreover, we explore the effectiveness of enriching the input character representations with word-level morphological features. To do so, we omit the morphological features from the first joint variant, and we use them in the second. We refer to these models as Joint+Side Constraints and Joint+Morph+Side Constraints, respectively. 7.3 Our Multi-step Models We explore five variants of the gender rewriting multi-step model described in §6. All five variants use the same gender identification (GID) and incontext selection models, but they differ in their outof-context word-level gender rewriting generation setup. The first three variants use one word-level gender rewriting model each – CorpusR, MorphR, or NeuralR. The fourth multi-step model uses MorphR as a backoff if the input words that need to be rewritten are not observed by the CorpusR model during training (CorpusR»MorphR). The fifth system uses all three word-level gender alternative generation models in a backoff cascade: CorpusR»MorphR»NeuralR. 7.4 Data Augmentation Given the relatively small size of APGC v2.1 and motivated by recent work on using data augmentation to improve grammatical error correction (Wan et al., 2020; Stahlberg and Kumar, 2021), we investigate adding additional training examples through data augmentation. We randomly selected 800K sentences from the English-Arabic portion of the OpenSubtitles 2018 dataset, which was used to build APGC. We ensured that all extracted pairs include either first or second (or both) person pronouns on the English side: I, me, my, mine, myself, and you, your, yours, yourself. To generate gender alternatives of the selected Arabic sentences, we pass each sentence four times through our best gender rewriting system to generate all four user gender contexts (1M/2M, 1F/2M, 1M/2F, 1F/2F). We add the 800K selected Arabic sentences and their 1M/2M, 1F/2M, 1M/2F, 1F/2F generated gen- der alternatives to the Input, Target 1M/2M, Target 1F/2M, Target 1M/2F, and Target 1F/2F corpora of the training split of APGC v2.1, respectively. At the end, we end up with 857,603 Arabic parallel sentences (6,209,958 words). We make the synthetically generated data publicly available. 8 Results Table 3 presents the D EV set results. Joint+Side Constraints and Joint+Morph+Side Constraints significantly improve over the Joint+Morph baseline with up to 13.87 increase in F0.5 . The best performing system overall is our multi-step model using all rewrite components – Table 3(i), henceforth, Our Best Model. It improves over all the joint models in every compared metric, including a 22.84 increase in F0.5 when compared to the Joint+Morph baseline. Our Best Model’s biggest advantages seem to come from combining the three word-level out-of-context gender alternative generation models in a cascaded setup to deal with OOV words during the generation. Comparing (i) with (e,f,g) in Table 3, we observe improvements ranging from 3.91 to 6.02 F0.5 . We used Our Best Model to conduct the data augmentation experiments as discussed in §7.4. The full set of augmentation experiment results are presented in Appendix C. The best augmented model’s results, which benefits from augmentation in the GID and NeuralR components, are also presented in Table 3(j). However, its increase of 0.19 points in F0.5 is not statistically significant with McNemar’s (McNemar, 1947) test at p > 0.05. The results of our best models on the T EST sets of APGC v2.1 and APGC v1.0 are presented in Table 4. The results on APGC v2.1 T EST show consistent conclusions with the D EV results. Our best augmented model improves over its nonaugmented variant in every compared metric, including a 0.25 absolute increase in F0.5 that is statistically significant with McNemar’s test at p < 0.05. For APGC v1.0, we do not report results with augmentation for fair comparison to previous results. In both T EST sets, we establish new SOTAs. Error Analysis We conducted an error analysis over the output of our best augmented system on APGC v2.1 D EV. In total, there were 1,475 (5.5% out of 26,588) sentences with errors across the four target corpora. Table 5 presents a summary of the error types our best augmented gender rewriting model makes. 624 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) Do Nothing Joint + Morph Joint + Side Constraints Joint + Morph + Side Constraints GID + CorpusR + Selection GID + MorphR + Selection GID + NeuralR + Selection GID + CorpusR » MorphR + Selection GID + CorpusR » MorphR » NeuralR + Selection GIDAug + CorpusR » MorphR » NeuralRAug + Selection P 100.0 64.76 77.10 78.97 88.22 84.48 84.62 88.59 88.46 88.67 R 0.0 67.40 77.71 79.84 71.22 75.29 73.32 85.84 86.74 86.84 F0.5 0.0 65.27 77.22 79.14 84.20 82.47 82.09 88.02 88.11 88.30 BLEU 89.36 93.31 95.60 96.17 96.54 96.96 96.75 97.96 98.04 98.05 Table 3: Results of a number of systems on the D EV set of APGC v2.1. Aug indicates using augmented data. APGC v2.1 Test APGC v1.0 Test Joint + Morph GID + CorpusR GIDAug + CorpusR Habash et al. (2019) Alhafni et al. (2020) GID + CorpusR P R F0.5 + Side Constraints 79.27 80.44 79.50 » MorphR » NeuralR + Selection 88.70 86.13 88.17 » MorphR » NeuralRAug + Selection 88.86 86.69 88.42 77.74 52.06 70.76 78.98 60.32 74.38 » MorphR » NeuralR + Selection 78.57 73.17 77.43 BLEU 96.19 97.98 98.05 98.28 98.49 98.92 Table 4: Gender rewriting results on the T EST sets of APGC v2.1 and APGC v1.0. 1M/2M GID 150 56% Rewrite 69 26% Select 49 18% Total 268 1F/2M 194 70% 50 18% 35 13% 279 1M/2F 325 68% 82 17% 73 15% 480 1F/2F 324 72% 66 15% 58 13% 448 Table 5: Error type statistics of our best augmented system’s performance on APGC v2.1 D EV. Target 1M/2M 1F/2M 1M/2F 1F/2F Google Translate 13.59 13.15 11.38 10.96 Best SystemAug 13.71 13.64 13.30 13.23 Table 6: BLEU results on the post-edited Google Translate output of APGC v2.1 T EST using our best augmented system. The majority of errors (67.3%) were caused by GID which achieves a word-level accuracy of 98.9% on D EV. The gender-rewriting errors constituted 18.1% and selection errors 14.6%. Considering different target corpora, we observe that every time an F target is added, the number of errors increases. The 1M/2M target outputs has the lowest number of errors (268 or 18%), while the 1M/2F targets outputs has the highest number of errors (480 or 33%). Use Case: Post-Editing MT Output We demonstrate next how our proposed gender rewriting model could be used to personalize the output of user-unaware MT systems through post-editing. We use the English to Arabic Google Translate output sentences that are part of APGC v2.1. We evaluate Google Translate’s output against all four target corpora (1M/2M, 1F/2M, 1M/2F, 1F/2F) separately. To re-target Google Translate’s Arabic output for the four user gender contexts we model, we pass each Arabic sentence four times through our best augmented system (Table 3(j)). We present the evaluation in terms of BLEU in Table 6 over APGC v2.1 T EST. All the results are reported in an orthographically normalized space for Alif, Ya, and Ta-Marbuta. Again, we observe that every time an M participant is switched to F, the BLEU scores drop for Google Translate’s output. This is consistent to what have been reported by Alhafni et al. (2022) and highlights the bias the machine translation output has towards masculine grammatical gender preferences. The post-edited outputs generated by our best augmented system improves over Google Translate’s across the four target user contexts, achieving the highest increase in 2.27 BLEU points for 1F/2F. 625 9 Conclusion and Future Work We defined the task of gender rewriting in contexts involving two users (I and/or You), and developed a multi-step system that combines the positive aspects of both rule-based and neural rewriting models. Our best models establish the benchmark for this newly defined task and the SOTA for a previously defined first person version of it. We further demonstrated a use case of our gender rewriting system by post-editing the output of a commercial MT system to provide personalized outputs based on the users’ grammatical gender preferences. In future work, we plan to explore the use of other pretrained models, and to work on the problem of gender rewriting in other languages and dialectal varieties. Ethical Considerations Gender Rewriting Our underlying intention of developing a gender rewriting model for Arabic is to increase the inclusiveness of NLP applications that deal with gender-marking morphologically rich languages. Our work aims at empowering and allowing users to interact with NLP technology in a way that is consistent with their social identities. We acknowledge that by limiting the choice of gender expressions to the grammatical gender choices in Arabic, we exclude other alternatives such as non-binary gender or no-gender expressions. However, we are not aware of any sociolinguistics published research that discusses such alternatives for Arabic. We stress on the importance of adapting Arabic NLP models to new gender alternative forms as they emerge as part of the language usage. We further recognize the limitations of the gender identification component we use as part of our multi-step gender rewriting model as it relies on a language model that was pretrained on a large monolingual Arabic corpus, which could possibly contain biased text. We realize the potential risks of our proposed gender rewriting model if it is intentionally maliciously misused to produce gender alternatives that do not match the target users’ gender preferences. Data We use the publicly available Arabic Parallel Gender Corpus (APGC).7 It is subject to its creators’ own Copy Rights and User Agreement and we strictly adhere to its intended usage. It is worth 7 http://resources.camel-lab.com/ noting that APGC does not contain any heterocentric assumptions as part of its annotations (e.g., the word úk ð P ‘my husband’ is labeled as gender- . ambiguous (B)). Moreover, all proper names are labeled as B, even when they have strong genderspecific association (Alhafni et al., 2022). The Arabic data we use for our data augmentation experiments was randomly sampled from OpenSubtitles 2018 (Lison and Tiedemann, 2016), which is publicly available.8 OpenSubtitles is distributed under a Creative Commons license.9 Acknowledgements We thank Alberto Chierici, Christian Khairallah, Go Inoue, and Ossama Obeid for the helpful discussions. We acknowledge the support of the High Performance Computing Center at New York University Abu Dhabi. Finally, we wish to thank the anonymous reviewers at NAACL 2022 for their feedback. References Ahmed Abdelali, Sabit Hassan, Hamdy Mubarak, Kareem Darwish, and Younes Samih. 2021. 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For the augmentation experiments, we use the same hyperparamters but we run the fine-tuning for 3 epochs. At the end of the fine-tuning, we pick the best checkpoint based on the performance on the D EV set. Our gender identification model has 108,506,901 parameters. In-Context Ranking and Selection We finetune CAMeLBERT MSA on a single GPU for 3 epochs with a learning of 5e-5, batch size of 32, and a seed of 88. The fine-tuned CAMeLBERT MSA model has 109,112,880 parameters. Neural Rewriter (NeuralR) For the characterlevel encoder-decoder neural rewriter model we use a character embedding size of 128, a hidden size of 256, a dropout probability of 0.2 on the outputs of each GRU layer, and gradient clipping with a maximum norm of 1. We train for 50 epochs on a single GPU with early stopping after 6 epochs if the loss does not decrease on the D EV set. We use the Adam (Kingma and Ba, 2014) optimizer with an initial learning rate of 5e-4, decaying by a factor of 0.5 if the loss on the D EV set does not decrease after 2 epochs. We train with greedy decoding and a batch size of 32. We also apply scheduled sampling (teacher forcing) (Bengio et al., 2015) with a constant sampling probability (0.3) during training. During inference, we use beam search with a beam width of 10 to produce the top 3-best hypotheses. Our NeuralR model has 3,287,110 parameters. Joint Models The training settings and the hyperparameters of the joint models are identical to the ones we use in our NeuralR model. The crucial difference between the Joint+Morph model and its extended variants (Joint+Side Constraints and Joint+Morph+Side Constraints) is that the rewriting in the Joint+Morph model is conditioned on the sentence-level target gender. The representation of the sentence-level target gender in the baseline model is learned as an embedding of size 10 during training and only used in the decoder. Our Joint+Morph model has 3,481,178 parameters; the Joint+Side Constraints model has 3,293,258 parameters; and the Joint+Morph+Side Constraints model has 3,480,926 parameters. Training Time The CorpusR model was trained on a single CPU and it took ≈2 minutes to be trained. All our neural models were trained on a single GPU. Fine-tuning CAMeLBERT MSA on the gender identification task took ≈1 hour; finetuning CAMeLBERT MSA on the MLM objective took ≈1 hour. Training the NeuralR model with different settings took ≈12 hours in total. All the baseline joint models took ≈29 hours to be trained. It is worth noting that all the results presented in this work are reported over a single run and the hyperparameters of our neural models were manually tuned based on the performance on the D EV set. B Arabic Parallel Gender Corpus v2.1: Extended Word-Level Annotations Word Gender Train Dev Test Label B 385,693 44,629 108,411 538,733 B+1M 28 5 10 43 B+1F 28 5 10 43 B+2M 1,042 98 279 1,419 B+2F 1,042 98 279 1,419 1M+B 3,490 422 958 4,870 1F+B 3,490 422 958 4,870 2M+B 16,320 1,787 4,548 22,655 2F+B 16,320 1,787 4,548 22,655 1M+1F 0 0 0 0 1F+1M 0 0 0 0 1M+2F 1 0 0 1 1F+2M 1 0 0 1 1M+1M 9 0 1 10 1F+1F 9 0 1 10 1M+2M 1 0 0 1 1F+2F 1 0 0 1 2M+1M 1 0 0 1 2F+1M 1 0 0 1 2M+1F 1 0 0 1 2F+1F 1 0 0 1 2M+2M 22 2 8 32 2F+2F 22 2 8 32 2M+2F 0 0 0 0 2F+2M 0 0 0 0 427,523 49,257 120,019 596,799 Table 7: The statistics of the extended word-level gender annotations of APGC v2.1 across the T RAIN, D EV, and T EST splits. 630 (a) (b) (c) (d) (e) (f) GID GIDAug GID GIDAug GIDAug GIDAug + + + + + + CorpusRAug CorpusR CorpusR CorpusRAug CorpusR CorpusRAug » » » » » » MorphR MorphR MorphR MorphR MorphR MorphR » » » » » » NeuralR NeuralR NeuralRAug NeuralR NeuralRAug NeuralRAug + + + + + + Selection Selection Selection Selection Selection Selection P 88.19 88.63 88.50 88.41 88.67 88.39 R 86.66 86.91 86.64 86.89 86.84 86.87 F0.5 87.88 88.28 88.12 88.10 88.30 88.08 BLEU 98.04 98.05 98.04 98.06 98.05 98.05 Table 8: Results of the data augmentation experiments on the D EV set of APGC v2.1. Aug indicates that the component of the system is trained on the augmented data. C Augmentation Experiments When it comes to the data augmentation experiments, we took the best performing system (Table 3(i)) and explored training its different components on the augmented training data. Evaluation results on the D EV set of APGC v2.1 using data augmentation are presented in Table 8. Starting off with training the CorpusR model on the augmented data (Table 8(a)), we notice a decrease in performance by 0.23 F0.5 compared to Our Best Model (Table 3(i)). This is attributed to the noisy coverage increase in the CorpusR model and can be observed by the decrease in precision (88.19) and recall (86.66). When we train the GID and the NeuralR models on the augmented data (Table 8(b-c)), we get an increase in F0.5 reaching 88.28 and 88.12, respectively. However, using both the augmented GID and CorpusR models (Table 8(d)) decreases the performance slightly as it achieves 88.10 in F0.5 . The best performing system was the one that uses both the augmented GID and NeuralR models (Table 8(e)) as it improves over its non-augmented variant reaching 88.30 (0.19 increase) in F0.5 . Combining the three augmented GID, CorpusR, and NeuralR models (Table 8(f)) achieves 88.08 in F0.5 . 631
https://openalex.org/W4393443557	https://jurnal.unmer.ac.id/index.php/jpkm/article/download/12128/pdf	English	null	Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area	Abdimas	2,024	cc-by-sa	5,305	ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Vol.9(1) February 2024, 56-64 p-ISSN: 2721-138X e-ISSN: 2548-7159 http://jurnal.unmer.ac.id/index.php/jpkm LPPM UNMER MALANG ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Vol.9(1) February 2024, 56-64 p-ISSN: 2721-138X e-ISSN: 2548-7159 http://jurnal.unmer.ac.id/index.php/jpkm LPPM UNMER MALANG Keywords: ©2024 Abdimas: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang This is an open access article distributed under the CC BY-SA 4.0 license (https://creativecommons.org/licenses/by-sa/4.0/) How to cite: Ani, L. S., Hendrayana, M. A., Gandari, N. K. M., Laksmi, I. A. A., Agastiya, I. M. C., Vittala, G., Juniartha, I. G. N., & Dwija, I. B. N. P. (2024). Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area. Abdimas: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang, 9(1), 56-64. https://doi.org/10.26905/abdimas.v9i1.12128 Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani1, Made Agus Hendrayana1, Ni Komang Matalia Gandari2, Ida Ayu Agung Laksmi2, I Made Cahyadi Agastiya2, Govinda Vittala1, I Gusti Ngurah Juniartha1, Ida Bagus Nyoman Putra Dwija1 1Faculty of Medical, Universitas Udayana Jl. P.B. Sudirman, Denpasar, 80232, Indonesia 2Department of Nursing, STIKES Bina Usada Bali Jl. Raya Padang Luwih, Badung, Bali, 80361, Indonesia ARTICLE INFO: Received: 2024-01-07 Revised: 2024-01-28 Accepted: 2024-02-16 Published: 2024-02-29 Tourists who stay at the hotel have a risk of illness and/or experiencing emergency problems that interfere with the sense of security and comfort for tourists. One factor for successful emergency management is first aid proficiency. However, the knowledge and skills of hotel staff are relatively lacking in handling emergency problems for tourists. Based on this, this service was held to train the skills of hotel staff in handling emergencies for tourists. Service activities are carried out by presentation methods and practice on basic life support. The target of this activity is the hotel staff of Ubud Raya Resort. Dedication materials were given in 5 meetings. The training material has been adjusted to travel medicine subjects and based on the results of previous research by the implementation team. The implementation of this service went smoothly and as planned. Based on the results of the pretest and posttest, an increase in the knowledge and skills of participants was obtained. Based on interviews, it was found that service participants received positive benefits from the service carried out. Thus, concluded that this service was successfully carried out despite the limitations in its implementation. This community service advice re-conducting basic life support training for hotel staff so that the knowledge and skills received are beneficial in the long term. Basic life support, Emergency, Health tourism, Hotel, Tourist destination Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija being, encompassing protection from criminal activities and other potential dangers. Every individual requires protection from environmental threats, accidents, or illnesses (Ruminem, 2021). being, encompassing protection from criminal activities and other potential dangers. Every individual requires protection from environmental threats, accidents, or illnesses (Ruminem, 2021). Various tourist activities entail risks of accidents ranging from minor to severe. The available tourist activities range from sports, meditation, shopping, culinary tours, museum visits, amusement parks, zoos, to adrenaline-charged activities such as rafting, hill trekking, waterfall visits, swings, and more (“Top 12 aktivitas wisata di Ubud”, 2019). These tourist activities pose risks of diseases, accidents, and emergencies. Therefore, the readiness of resources in handling emergency situations for tourists is crucial, ensuring that tourists encountering emergency situations are properly and effectively managed.i One of the preparedness measures required is proficiency in Basic Life Support (BLS). BLS is the initial effort to restore respiratory and/or circulatory functions in individuals experiencing respiratory arrest and/or cardiac arrest due to accidents or certain diseases (Aditianingsih, 2022). Several simple skills need to be mastered to assist in maintaining the life of someone experiencing respiratory or cardiac arrest by clearing the airway, providing breathing assistance, and aiding in blood flow to vital areas of the victim’s body. Conversely, tourism workers lack readiness in responding to emergencies. Typically, if a tourist experiences health problems such as a heart attack, accident, or drowning, they are immediately taken to the nearest health service.fi Bali Municipality Police Department (Polda Bali) recorded an average of 6 traffic accidents involving foreign tourists in Bali in 2023. This figure is relatively higher compared to the number of traffic accidents involving foreign tourists in 2022, which amounted to 68 incidents. The traffic accidents were spread across several regions. Gianyar Regency reported the highest number of accidents involving tourists, with 23 incidents, followed by Denpasar City with 18 cases, and Buleleng and Klungkung Regencies each with 3 cases (Sugiharto, 2023). The distance between hotels and clinics or community health centers ranges from 1-2 kilometers. However, it is rare to find healthcare professionals, either doctors or nurses, on duty at hotels. Most hotels collaborate with nearby clinics to address health issues for hotel staff and tourists. Ubud Raya Resort is one of the hotels located on Jalan Raya Sayan, Banjar Sindu Sayan, Ubud, Bali, Indonesia. The staff at Ubud Raya Resort also lack readiness in handling emergencies. 2. METHODS The service dedication activity is conducted through training sessions to enhance the knowledge and skills regarding emergencies for hotel staff. This service dedication activity took place at Ubud Raya Resort, Gianyar, Bali in July 2023. The activity commenced with the determination of the timing for the service dedication. Based on the availability and agreement between the service team and the hotel management, the activity was scheduled for Friday, July 14, 2023, from 09:00 to 13:00. Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija Based on interviews with hotel management, an average of 3 emergency cases involving tourists occurs per month. Common emergency incidents include slipping and cramps while swimming. Sick tourists are taken to the hotel’s collaborating clinic without providing first aid for accidents because hotel staff lack skills in handling emergency cases. To date, no training activities for everyday emergency handling skills have been conducted. Based on this, a service dedication activity through empowering hotel staff in emergency handling is organized with the aim of improving BLS skills for hotel staff and enhancing service quality for tourists. Improving the skills and service quality of hotel staff in maintaining health can increase the sense of security and comfort for tourists staying at the hotel. 1. INTRODUCTION Ubud is one of the tourist destinations located in Gianyar Regency, known for its thriving arts and culture scene in Bali. Through this allure, Ubud manages to attract tourists to visit and stay. The number of tourist visits to Ubud has been reported to increase each year (Warta Bali, 2022). The increase in the number of visits necessitates hotel staff readiness in providing security and comfort assurance for tourists. The need for security and comfort is considered one of the fundamental human needs according to Maslow’s theory. Comfort is a state where the basic human needs for tranquility, relief, and transcendence are fulfilled. Meanwhile, security is the need for protection for human physical well- Corresponding Author: Luh Seri Ani: Tel. +62 361 222510 │ E-mail: seriani@unud.ac.id Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija Practices Practice in performing basic life support on victims was provided to all service participants for approximately 100 minutes. The activity began with the screening of an illustrative video depicting victims experiencing emergencies. This was followed by a direct demonstration of the proper and correct basic life support techniques. Each participant was given the opportunity to practice basic life support techniques with the assistance of instructional aids. Training continued until all participants were assessed as capable of performing basic life support using the correct techniques Presentation and Question-and-Answer Session The presentation and question-and-answer session lasted for 1 hour. The training material on basic life support provided insights into the potential risks of emergencies for tourists and covered basic life support techniques. The training material was delivered by a nurse from the STIKES Bina Usada Bali institution. The provision of basic life support material began with a pre-test for all participants. The event continued with the delivery of basic life support material followed by a question-and-answer session. A post-test was then administered to all participants to assess the improvement in their knowledge of basic life support. ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 Evaluation Design Evaluation of the implementation of the emergency training program for hotel staff using three metrics of achievement from the training activities includes success from the execution of the activities, from the participants, and from the organizers of the activities. The measure of success from the execution of the activities involves assessing the adequacy of the timing and the number of meetings that were planned beforehand. The measure of success from the participants involves the scores obtained from the pretest and post-test of the presentation and practice of basic life support activities. The measure of success from the organizers is assessed by the benefits derived by the participants from the activities. Activity Methods The activity that was attended by 15 hotel staff was carried out using two methods, namely presentation and question and answer as well as practice of providing basic life support. \| 57 \| Results In Table 1, it was found that most of the training participants were aged 19-24 years (53.3%), had a high school/vocational education level (53.3%) and had worked for > 5 years (60%). In Table 1, it was found that most of the training participants were aged 19-24 years (53.3%), had a high school/vocational education level (53.3%) and had worked for > 5 years (60%). Table 1. Characteristics of participants Characteristics n % Age 19-24 8 53,3 25-45 6 40,0 >45 1 6,7 Education level Senior/Vocational High School (SMA/SMK) 8 53,3 Diploma 7 46,7 Work experience <5 years 6 40 >5 years 9 60 Table 1. Characteristics of participants \| 58 \| Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija Presentation Training activities are filled with lectures and demonstrations. In Figure 1, it is shown that almost all participants seemed enthusiastic about listening to the material presented by the lecturer, namely an emergency nurse specialist from STIKES Bina Usada Bali. Participants’ knowledge about potential emergency risks for tourists and basic life support was assessed to have increased. The assessment of participants’ increased knowledge is assumed to be based on the participants’ ability to answer questions correctly after being given a lecture. Figure 1. The enthusiasm of the participants Figure 1. The enthusiasm of the participants Figure 1. The enthusiasm of the participants Figure 1. The enthusiasm of the participants Practice F Practices g y pp y all participants were shown the correct procedures when helping people experiencing heart failure. Furthermore, hotel staff participating in the training were given the opportunity to practice basic life support procedures with the help of phantoms. The trainer supervises and guides the participants until they are able to perform well and correctly. In the picture, it can be seen that the participants are able to carry out the correct method of basic life support procedures. Figure 2 shows a demonstration activity of basic life support skills. In this demonstration activity, all participants were shown the correct procedures when helping people experiencing heart failure. Furthermore, hotel staff participating in the training were given the opportunity to practice basic life support procedures with the help of phantoms. The trainer supervises and guides the participants until they are able to perform well and correctly. In the picture, it can be seen that the participants are able to carry out the correct method of basic life support procedures. Practices Figure 2 shows a demonstration activity of basic life support skills. In this demonstration activity, all participants were shown the correct procedures when helping people experiencing heart failure. Furthermore, hotel staff participating in the training were given the opportunity to practice basic life support procedures with the help of phantoms. The trainer supervises and guides the participants until they are able to perform well and correctly. In the picture, it can be seen that the participants are able to carry out the correct method of basic life support procedures Figure 2. Demonstration of basic life support by trainers and participants practicing the procedures Closing The service activity closed with an evaluation activity on the implementation of service for hotel Figure 2. Demonstration of basic life support by trainers and participants practicing the procedures Figure 2. Demonstration of basic life support by trainers and participants practicing the procedures osing Figure 2. Demonstration of basic life support by trainers and participants practicing the procedures Figure 2. Demonstration of basic life support by trainers and participants practicing the procedure staff. partic Clos Closing participants knowledge and skills regarding basic life support. Several participants were randomly selected to be interviewed about staff perceptions of the benefits of implementing this service activity. The service activity ended with the event of giving certificates and mementos from the implementing team to the service participants, as in Figure 3. The service activity closed with an evaluation activity on the implementation of service for hotel staff. Evaluation is carried out by filling out a posttest questionnaire to determine the increase in participants' knowledge and skills regarding basic life support. Several participants were randomly selected to be interviewed about staff perceptions of the benefits of implementing this service activity. The service activity ended with the event of giving certificates and mementos from the implementing team to the service participants, as in Figure 3. The service activity closed with an evaluation activity on the implementation of service for hotel staff. Evaluation is carried out by filling out a posttest questionnaire to determine the increase in participants’ knowledge and skills regarding basic life support. Several participants were randomly selected to be interviewed about staff perceptions of the benefits of implementing this service activity. The service activity ended with the event of giving certificates and mementos from the implementing team to the service participants, as in Figure 3. \| 59 \| ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 nterviewed about staff perceptions of the benefits of implementing this vity ended with the event of giving certificates and mementos from the i ti i t i Fi 3 Figure 3. Handing over basic life support training certificates Figure 3. Handing over basic life support training certificates Figure 3. Handing over basic life support training certificates Figure 3. Handing over basic life support training certificates Activity Materia The mate Activity Materials The material used in the dedication for hotel staff is in line with the teaching materials from the Travel Medicine course. Travel medicine, also known as travel health or emporiatrics, is a branch or specialization of medicine that specifically studies diseases and health conditions resulting from travel The material used in the dedication for hotel staff is in line with the teaching materials from the Travel Medicine course. Travel medicine, also known as travel health or emporiatrics, is a branch or specialization of medicine that specifically studies diseases and health conditions resulting from travel and their management efforts (Wirawan & Made, 2016). Various health problems are often experienced by tourists during their travels. Therefore, preventive measures are needed for tourists (Wirawan et al., 2020). The material for the dedication on basic life support practice is adjusted according to the basic life support protocol prepared by the Ministry of Health of the Republic of Indonesia. The basic life support training material is also tailored to the needs of participants who are classified as non-healthcare professionals. Therefore, the depth of the material, the number of meetings, and the training delivery methods are adjusted for the general public. The implementation of community dedication activities is divided into 5 meetings, as shown in Table 2. Table 2. Schedule 1st Meeting Activities - Introduction between the service team and service participants - Explanation of the reasons and benefits of service activities - Explanation of the methods of service activities - Explanation of the evaluation of service activities - Pretest Goals - Introduction to the service team and introducing the reasons, benefits and methods of implementing the service program. Discussion The training on basic life support is typically provided to medical professionals. However, emergency victims are often encountered in various locations far from healthcare facilities. The success of emergency patient management is determined by the quality of the first aid received before receiving medical treatment. Proficiency in providing basic life support increases the success rate of managing patients with cardiac arrest. Therefore, the ability to provide assistance in cases of respiratory and cardiac arrest needs to be imparted not only to healthcare professionals but also to other professions. One profession that needs to acquire knowledge about basic life support is hotel staff. The skills required by hotel staff for basic life support include the ability to perform cardiopulmonary resuscitation (CPR), which involves chest compressions and airway management to maintain blood circulation and oxygenation in patients experiencing a heart attack. Another essential skill for hotel staff is understanding wound care and how to manage bleeding and other injuries (My CPR Now, 2024; Sing, 2023).fi Basic life support training is essential for hotel staff as it enables them to provide first aid in emergency situations. This training aims to enhance employees’ skills in delivering first aid and basic life support. Basic life support training encompasses various skills, such as cardiopulmonary resuscitation, wound management, and other emergency actions. Basic life support training can be conducted in various settings, including hotels, and involves learning methods such as theory, discussion, and practical sessions. Therefore, basic life support training is highly relevant and crucial for enhancing the readiness of hotel staff in dealing with emergencies and providing safe service to their guests (Mayfield, 2021). Based on research conducted at a hotel, it was found that basic life support training improves the knowledge and preparedness of hotel staff in providing first aid for emergencies. Approximately 60% of hotel staff were found to have good knowledge of basic life support after receiving training (Prihastini et al., 2023). Contrary results were found among healthcare workers. Although the degree of training and proficiency in basic life support differs between healthcare workers and hotel staff, the knowledge of hotel staff was found to be higher than that of healthcare workers. A study found that only 12% of healthcare workers had adequate knowledge of basic life support (Chaudhary et al., 2023). Activity Materia The mate Activity Materials Schedule \| 60 \| Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija Activity Materia The mate Activity Materials - Introduce the activity and evaluation methods used in implementing this service program - To find out the initial knowledge of service participants, so that the presenters can adjust the material provided 2nd Meeting Activities - Providing material on the topic of potential emergency risks for tourists Goals - To be able to provide knowledge to participants regarding the risks of emergencies for tourists 3rd Meeting Activities - Explanation of the meaning, objectives, procedures and evaluation of providing basic life support Goals - To provide knowledge about basic life support for tourists 4th Meeting Activities - Practice of providing basic life support by service participants Goals - To provide experience and make service participants able to carry out basic life support correctly 5th Meeting Activities - Post test Goals - To determine changes in the level of knowledge and abilities of participants in carrying out basic life support correctly Table 2. Schedule 1st Meeting Activities - Introduction between the service team and service participants - Explanation of the reasons and benefits of service activities - Explanation of the methods of service activities - Explanation of the evaluation of service activities - Pretest Goals - Introduction to the service team and introducing the reasons, benefits and methods of implementing the service program. - Introduce the activity and evaluation methods used in implementing this service program - To find out the initial knowledge of service participants, so that the presenters can adjust the material provided 2nd Meeting Activities - Providing material on the topic of potential emergency risks for tourists Goals - To be able to provide knowledge to participants regarding the risks of emergencies for tourists 3rd Meeting Activities - Explanation of the meaning, objectives, procedures and evaluation of providing basic life support Goals - To provide knowledge about basic life support for tourists 4th Meeting Activities - Practice of providing basic life support by service participants Goals - To provide experience and make service participants able to carry out basic life support correctly 5th Meeting Activities - Post test Goals - To determine changes in the level of knowledge and abilities of participants in carrying out basic life support correctly Table 2. Discussion Most healthcare workers, especially nurses, require improvement in their knowledge of basic life support (Adal & Emishaw, 2023).f The emergency training activities for hotel staff that have been conducted are considered successful. Based on the evaluation results, the success of the implementation of the activities was attributed to the appropriateness of the timing and the number of meetings planned beforehand. This success was influenced by the support from the hotel staff and management themselves. Despite the increasing hotel visits and the busy schedules of hotel staff, the training activities proceeded smoothly. Additionally, the expertise and delivery methods of the service team were considered engaging, resulting in enthusiastic participation from the service participants from the first meeting to the end. Interviews also revealed that training participants perceived the benefits of this basic life support training. The skills acquired can be applied to guests staying at the hotel where they work. Previously, if guests experienced health problems or emergencies, the hotel staff only contacted the clinic for assistance without providing first aid to the victims.f Based on the pretest and post-test evaluations, it was found that the knowledge of hotel staff is assumed to increase after receiving information about the potential risks of emergencies for tourists. The average pretest knowledge score was 37.3±1.4, which increased to 78.9±16.2 in the post-test. Similarly, the ability of participants to practice basic life support improved. The average pretest score for basic life support skills among hotel staff was 28.3±2.2, which increased to 82.4±21.6 in the post-test. This success \| 61 \| ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 can be attributed to the methods used in providing basic life support training to hotel staff. The method used was a combination of presentations and direct practice of basic life support, ensuring that the techniques were performed correctly and effectively. An approach combining mini-lectures and practical sessions is an effective method for delivering education and basic life support training to the general public, thereby enhancing their ability to provide first aid in emergency situations (Ghozali et al., 2023).f can be attributed to the methods used in providing basic life support training to hotel staff. Discussion The method used was a combination of presentations and direct practice of basic life support, ensuring that the techniques were performed correctly and effectively. An approach combining mini-lectures and practical sessions is an effective method for delivering education and basic life support training to the general public, thereby enhancing their ability to provide first aid in emergency situations (Ghozali et al., 2023).f Although the knowledge and skills of hotel staff appear to have improved, there are still some points of knowledge and skills that need to be enhanced. Some questions about basic life support were found to have low scores after lectures on basic life support. These questions pertained to the correct location for performing chest compressions, the frequency and speed of chest compressions, and the depth of pressure during chest compressions. These scores were 43.7, 37.5, and 31.2, respectively. Similarly, low scores were found for practical skills in basic life support. The lowest score was found in the skill of checking the pulse to ensure the absence of a pulse, with a score of 42.8. The low scores of knowledge and skills obtained by hotel staff may be because the basic life support training was conducted for the first time. Therefore, basic life support training is recommended to be conducted regularly every six months, with a focus on knowledge and skills points that are still categorized as less proficient. This activity also has limitations that may hinder the success of the Community Service program. The time interval between the pretest and post-test evaluations of participants’ knowledge and skills is relatively short, so this assessment may not reflect participants’ knowledge and skills over the long term. Another limitation is the use of instructional aids in training, which may differ from the actual situation when basic life support is applied directly to victims. Therefore, the limitations of this service need to be considered to ensure the success of the program being conducted (Lahaya et al., 2021). 4. CONCLUSION AND RECOMMENDATIONS The service activity for hotel staff was successfully conducted. The success of implementing this activity was assessed based on the adequacy of the timing and the number of meetings planned beforehand. Success was also observed in the improvement of participants’ knowledge and skills after receiving basic life support training. However, some points of knowledge and skills that need to be enhanced were still identified. The factors supporting the success of this dedication include support from the hotel staff and management, as well as the expertise and delivery methods of the service team, which were deemed engaging, resulting in enthusiastic participation from the participants from the first meeting to the end. On the other hand, factors hindering this dedication activity include the relatively short time interval between the pretest and post-test evaluations of participants’ knowledge and skills, which may not fully reflect participants’ knowledge and skills over the long term. Additionally, the use of instructional aids in training may create different situations and conditions compared to when basic life support is applied directly to victims. Based on the evaluation results of the training, it was found that there were still low scores of knowledges and skills on basic life support, so it is recommended to provide basic life assistance training on a regular basis once in six months with emphasis on the point-point of the knowledge and skill assessed as under-mastered. In addition, in order to overcome the limitations in the use of security tools, it is recommended that service activities for hotel staff be carried out with simulation methods involving health personnel as well as supporting advice to provide a more realistic situation so that it can provide experience for the hotel staff in the implementation of basic life support. \| 62 \| Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija Training hotel staff in early emergency management to enhance tourist comfort at Ubud Raya Resort area Luh Seri Ani, Made Agus Hendrayana, Ni Komang Matalia Gandari, Ida Ayu Agung Laksmi, I Made Cahyadi Agastiya, Govinda Vittala, I Gusti Ngurah Juniartha, Ida Bagus Nyoman Putra Dwija ACKNOWLEDGEMENTS Acknowledgment to the management of the Ubud Raya Resort-Bali Hotel who has become a partner in this service activity. Acknowledgment was also given to the Travel Medicine Unit of FK Unud, for financing this service activity. REFERENCES Adal, O., & Emishaw, S. (2023). Knowledge and attitude of healthcare workers toward advanced cardiac life support in Felege Hiwot Referral Hospital, Bahir Dar, Ethiopia, 2022. SAGE Open Medicine, 11. https://doi.org/10.1177/20503121221150101 Aditianingsih, D. (2022, August 9). Bantuan hidup dasar (basic life support and first aid training). Kemenkes: Direktorat Jenderal Pelayanan Kesehatan. https://yankes.kemkes.go.id/view_ artikel/1241/bantuan-hidup-dasar-basic-life-support-and-first-aid-training Bali Tripon. (2022, March 18). Top 12 aktivitas wisata di Ubud yang populer dan wajib dicoba. Bali Tripon. Retrieved from: https://www.balitripon.com/aktivitas-wisata-di-ubud/ Chaudhary, G. P., Sah, K., Malla, J., Das, N., Chaudhary, S., Chaudhary, I., & Pandey, J. (2023). Knowledge regarding Basic Life Support among Health Care Workers of the Hospital of Nepal. Journal of Healthcare Engineering, 2023. https://doi.org/10.1155/2023/9936114 Ghozali, M. T., Nugraheni, T. P., & Halimatussa’diyah, S. (2023). Pelatihan Dasar Manajemen Bantuan Hidup Dasar (BHD) Karang Taruna Dusun Sribit Dan Sekarsuli, Kapanewon Berbah, Sleman, Yogyakarta. Jurnal Surya Masyarakat, 5(2), 244-249. https://doi.org/10.26714/jsm.5.2.2023.244-249 Lahaya, I. A., Rahman, S. F., & Bate, C. I. G. (2021). Perencanaan dan Penganggaran Badan Usaha milik Desa (BUMDes) Karya Mandiri Desa Sepakat Kutai Kartanegara [Community Service Report]. Universitas Mulawarman Samarinda. Mayfield, G. (2021, July 4). Workplace CPR training helps hotel employee save a guest’s life \| BLS, PALS, ACLS and First Aid. Health Force Training Center. https://www.healthforcetrainingcenter. com/post/workplace-cpr-training-helps-hotel-employee-save-a-guest-s-life-bls-pals-acls- and-first-aid My CPR Now. (n.d.). First aid training for the hospitality industry. My CPR Now. Retrieved from: https://cprcertificationnow.com/blogs/mycpr-now-blog/first-aid-training-for-the-hospitality- industry-a-must-have-skill Prihastini, K. A., Nugraha, I. M. A., & Putra, I. G. N. R. (2023). Pengaruh pelatihan Bantuan Hidup Dasar (BHD) terhadap pengetahuan Karyawan Hotel XX sebagai upaya kesiapsiagaan dalam kasus kegawatdaruratan. Proceedings Seminar Psikologi Bencana, 9-15. Ruminem, R. (2021). Konsep kebutuhan rasa aman dan nyaman [Teaching Materials]. Universitas Mulawarman Samarinda. http://repository.unmul.ac.id/handle/123456789/36880 Sing, M. (2023, March 10). Is important to have a basic first aid training for hotel and school staff? LinkedIn. https://www.linkedin.com/pulse/important-have-basic-first-aid-training-hotel- school-staff-mihir-sing Sugiharto, J. (2023, January 20). Banyak turis asing kecelakaan di Bali, ini datanya. Tempo. https:// otomotif.tempo.co/read/1681861/banyak-turis-asing-kecelakaan-di-bali-ini-datanya \| 63 \| ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 ABDIMAS: Jurnal Pengabdian Masyarakat Universitas Merdeka Malang Volume 9, No 1, February 2024: 56-64 Warta Bali. (2022, April 17). Kunjungan wisatawan ke Ubud meningkat signifikan. Warta Bali Online. https://wartabalionline.com/2022/04/17/kunjungan-wisatawan-ke-ubud-meningkat- signifikan/ Warta Bali. (2022, April 17). Kunjungan wisatawan ke Ubud meningkat signifikan. Warta Bali Online. https://wartabalionline.com/2022/04/17/kunjungan-wisatawan-ke-ubud-meningkat- signifikan/ Wirawan, A., & Made, I. (2016). Kesehatan pariwisata: Aspek kesehatan masyarakat di daerah tujuan wisata. Archive of Community Health, 3(1), 9-14. Wirawan, A., & Made, I. (2016). Kesehatan pariwisata: Aspek kesehatan masyarakat di daerah tujuan wisata. Archive of Community Health, 3(1), 9-14. Wirawan, I. M. A., Putri, W. C. W. S., Kurniasari, N. M. D., Mulyawan, K. H., Hendrayana, M. A., & Suharlim, C. (2020). Geo-mapping of hazards, risks, and travel health services in Bali: Results from the first stage of the integrated travel health surveillance and information system at destination (TravHeSID) project. Travel Medicine and Infectious Disease, 37. https://doi.org/10.1016/j. tmaid.2020.101698 \| 64 \|
https://openalex.org/W4285306231	https://www.e3s-conferences.org/10.1051/e3sconf/202235201077/pdf	English	null	Numerical simulation of polymer flooding in water flooding reservoir	E3S web of conferences	2,022	cc-by	1,997	Numerical simulation of polymer flooding in water flooding reservoir Xiaofei Sun, Qingquan Zhang, Zhiyi Teng, Xueqian Wang, and Jiaming Cai etroleum Engineering, China University of Petroleum (East China), Qingdao, China Abstract. Different influencing factors in the process of polymer flooding have different degrees of influence on the development effect, so it is necessary to carry out the dynamic prediction of polymer flooding before implementing the development scheme. In this study, the reservoir model of C Block is established and the production performance is fitted. The fitted reservoir model is used to study the influence of polymer concentration, injection time, injection rate multiple and injection timing on the development effect of C Block. The results show that the cumulative oil production increases gradually with the increase of polymer concentration, and combined with the principle of economic development, there is an optimal polymer concentration in C Block, which is 0.15 wt%; With the increasing of injection time and injection rate multiple, the cumulative oil production and incremental oil production (compared with water flooding) show an increasing trend; For injection timing, the earlier polymer injection is more conducive to obtaining higher cumulative oil production and oil increment. The cumulative oil production under the optimal development scheme of C block is 23.8494×104 m3, which is 1.5042×104 m3 higher than that of water flooding. 2.1 Establishment of geological model Based on seismic, logging and reservoir test data, the geological model (grid and reservoir properties) established by petrel software was imported into CMG- SRATS software. The reservoir structure and properties are shown in Fig.1. The model has 47×25×25 (29375) grids. grids. (a) Structural model (b) Permeability (c) Porosity (d) Oil saturation Figure 1. Numerical simulation model of C block. g p y [ ] The C Block has experienced the stages of natural energy development, initial water injection and subdivided water injection development. In 2017, the production of injection-production adjustment reached the peak. It is urgent to carry out the research on polymer flooding scheme to improve oil production. Therefore, in order to scientifically develop reservoirs, it is necessary to evaluate the stimulation effect of the scheme before the implementation scheme [5]. In this paper, the CMG- SRATS software is used to study the development effect of different influencing factors of polymer flooding. Finally, the development effect under the optimal scheme is predicted, so as to provide theoretical basis and guidance for the implementation of polymer flooding in future. (d) Oil saturation Figure 1. Numerical simulation model of C block. Corresponding author: 1209475702@qq.com © 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/). 2. Reservoir model At present, most oil fields in eastern China have entered the late stage of high water cut development, and the existing water flooding cannot excavate more remaining oil [1]. So improving oil recovery at a small economic cost is the main task at present. In China, polymer flooding is one of the most mature and widely used EOR methods in tertiary oil recovery technology [2]. Polymer flooding is to inject polymer solution into the oil reservoir and increase recovery by changing mobility ratio and increasing sweep efficiency [3, 4]. https://doi.org/10.1051/e3sconf/202235201077 https://doi.org/10.1051/e3sconf/202235201077 E3S Web of Conferences 352, 01077 (2022) ESAT 2022 2.2 Establishment of fluid model The results of history matching are shown in Fig.2. It can be seen from Fig.2 that the fitting accuracy of actual production data and model data is high, which provides a reliable reservoir model for subsequent program prediction research. Table 2. Development schemes under different polymer concentration. Scheme number Injection timing year Polymer concentration wt.% Injection time year Injection rate multiple 1 1 0(water flooding) 5 1 2 1 0.01 5 1 3 1 0.05 5 1 4 1 0.1 5 1 5 1 0.15 5 1 6 1 0.2 5 1 Figure 3. Simulation results under different polymer concentration. Table 2. Development schemes under different polymer concentration. Figure 2. Historical matching results of oil production and water cut in the whole area. Figure 3. Simulation results under different polymer concentration. Figure 2. Historical matching results of oil production and water cut in the whole area. Figure 3. Simulation results under different polymer concentration. 3.2 Effect of injection time Combined with the field operation parameters of C Block, the development schemes under different influence parameters of polymer flooding are formulated, and the influences of polymer concentration, injection time, injection rate multiple and polymer injection timing on the development effect are studied. Table 3 and Fig.4 show the development schemes and simulation results of polymer flooding under different injection time. It can be seen from Fig.4 that with the increase of polymer injection time, the cumulative oil production increased from 22.5028×104 m3 to 23.1396×104 m3. This is because the amount of polymer increases as the injection time increases. The crude oil in the pores is continuously affected and produced under the action of more polymers, thereby increasing the cumulative oil production and incremental oil production. 2.2 Establishment of fluid model In order to fully simulate the mechanism of enhanced oil recovery by polymer flooding, three components are defined in the fluid model of CMG, including oil, water and polymer. The fluid model not only defines the basic properties of oil and water, but also considers some special properties of polymer, such as viscosity, residual E3S Web of Conferences 352, 01077 (2022) ESAT 2022 https://doi.org/10.1051/e3sconf/202235201077 resistance factor and so on. Table 1 shows the rock and fluid properties in the model. resistance factor and so on. Table 1 shows the rock and fluid properties in the model. Table 1. Rock and fluid properties in the model Reservoir Val ue oil Val ue Polymer Val ue Depth, m 21 70 Density, g/cm3 0.9 6 Density, g/cm3 1.0 08 Pressure, MPa 21. 7 Viscosity, mPa·s 8.5 Viscosity, mPa·s 20 9.3 Temperat ure, °C 80 Volume factor 1.0 35 Residual resistance factor 2 concentration, the cumulative oil production and incremental oil production (compared with water flooding) increase gradually. This is because with the increase of polymer concentration, the entanglement intensity of the molecular chain increases due to the characteristic that the single molecular chain of the polymer is easy to tangle with each other. Therefore, the increase of the traction radius of the polymer molecule causes the residual oil at both ends of the pores to be dragged out, thereby increasing the volume sweep coefficient. However, when the polymer concentration increased from 0.15 % to 0.2 %, the cumulative oil production only changed 0.055×104 m3. This is due to the fact that the polymer injected in the early stage can be adsorbed and mechanically detained on the reservoir rock, resulting in the weakening of the polymer flooding ability and mobility of subsequent injection. In addition, according to the principle of economic development, the optimal polymer concentration is 0.15 wt %. 2.3 Establishment of production dynamic model Production dynamic model is the dynamic presentation of reservoir development process and production situation. Based on the production parameters of C Block, the production dynamic model is established. In order to ensure that the established reservoir model can better reflect the actual reservoir conditions and complete the study of polymer flooding development effect, the cumulative oil production and water cut are taken as the fitting target, and the history matching is carried out by adjusting the relative permeability curve and other reservoir parameters. Table 5. Optimal parameters of target block. Injection timing year Polymer concentration wt.% Injection time year Injection rate multiple 1 0.15 5 1.4 4. Conclusion Based on the relatively fine geological model and history matching, the well-fitting reservoir model is used to complete the research on the influence of different polymer flooding parameters in C Block. The development effect of the optimal development scheme is predicted, and the relevant conclusions are obtained as follows: Figure 5. Simulation results at different injection rate multiples. 3.1 Effect of polymer concentration Table 2 and Fig.3 show the development schemes and simulation results under different polymer concentration. It can be seen from Fig.3 that with the increase of polymer 2 2 E3S Web of Conferences 352, 01077 (2022) ESAT 2022 https://doi.org/10.1051/e3sconf/202235201077 Table 4. Development schemes under different injection timing. Scheme number Injection timing year Polymer concentration wt.% Injection time year Injection rate multiple 1 1 0.2 5 1 2 2 0.2 5 1 3 3 0.2 5 1 4 4 0.2 5 1 5 5 0.2 5 1 Figure 6. Simulation results under different polymer injection timing. Table 3. Development schemes of polymer flooding under different injection time. Scheme number Injection timing year Polymer concentration wt.% Injection time year Injection rate multiple 1 1 0.2 1 1 2 1 0.2 2 1 3 1 0.2 3 1 4 1 0.2 4 1 5 1 0.2 5 1 Figure 4. Simulation results under different injection time. Table 4. Development schemes under different injection timing. Table 3. Development schemes of polymer flooding under different injection time. Figure 4. Simulation results under different injection time. Figure 6. Simulation results under different polymer injection timing. 3.5 Prediction of optimal development scheme 3.5 Prediction of optimal development scheme Combined with the influence of different polymer flooding parameters and the principle of economic development, the optimal development scheme is shown in table 5. The cumulative oil production under this scheme is 23.8494×104 m3, which is 1.5042 ×104 m3 higher than that of water flooding. Fig.5 shows the simulation results of polymer flooding under different injection rate multiples (0.6~1.4). It can be seen from Fig.5 that with the increase of injection rate multiple, both cumulative oil production and incremental oil production show an increasing trend. Figure 5. Simulation results at different injection rate multiples. Table 5. Optimal parameters of target block. Injection timing year Polymer concentration wt.% Injection time year Injection rate multiple 1 0.15 5 1.4 Table 5. Optimal parameters of target block. 3.4 Effect of polymer injection timing Table 4 and Fig.6 show the development plan and simulation results under different polymer injection timing. It can be seen from Fig.6 that the earlier polymer injection is more conducive to obtaining higher cumulative oil production and oil increment. When the injection starts in the first year, the cumulative oil production can reach 23.196×104m3, which is 0.7944×104m3 higher than that of water flooding. This is because the early injection can give full play to the role of polymer and increase the amount of crude oil affected. （1）The cumulative oil production increases gradually with the increase of polymer concentration, and the optimal polymer concentration is 0.15 wt% in the target area; With the increase of injection time and injection rate multiple, the cumulative oil production gradually increases; The earlier polymer injection is more conducive to obtaining higher cumulative oil production and incremental oil production. 3 https://doi.org/10.1051/e3sconf/202235201077 E3S Web of Conferences 352, 01077 (2022) ESAT 2022 （2）The cumulative oil production under the optimal development scheme is 23.8494×104m3, which is 1.5042×104m3 higher than that of water flooding. References 1. C. Sun, J. Hou, G.M. Pan, et al. Optimized polymer enhanced foam flooding for ordinary heavy oil reservoir after cross-linked polymer flooding, J. Sci. Journal of Petroleum Exploration & Production Technology, China University of Petroleum (East China), 6 (2016):777-785. 2. H. Yu, Y. Wang, W. Ji , et al. A Laboratory Study of the Microgel Used for Polymer Flooding, J. Sci. Petroleum Science and Technology, China University of Petroleum (East China), 29(2011):715- 727. 3. Z.H. Chen, X.T. Zhao, Z.B. Wang, et al. A comparative study of inorganic alkaline/polymer flooding and organic alkaline/polymer flooding for enhanced heavy oil recovery, J. Sci. Colloids and Surfaces A: Physicochemical and Engineering Aspects, China University of Petroleum (East China), 469(2015):150-157. 4. A.M. Alsofi, M.J. Blunt. Polymer flooding design and optimization under economic uncertainty, J. Sci. Journal of Petroleum Science & Engineering, Imperial College London, 124(2014):46-59. 5. M. Algharaib, A.A. lajmi, R. Gharbi. Improving polymer flood performance in high salinity reservoirs, J. Sci. Journal of Petroleum Science & Engineering, Kuwait University, 115(2014):17-23. 4
https://openalex.org/W2101444245	https://www.bio-conferences.org/articles/bioconf/pdf/2011/01/bioconf_skills_00029.pdf	English	null	Design Guidelines for the Development of Virtual Reality and Augmented Reality Training Systems for Maintenance and Assembly Tasks	Bio web of conferences/BIO web of conferences	2,011	cc-by	2,790	Abstract The current work describes design guidelines for the development of Virtual Reality (VR) and Augmented Reality (AR) platforms to train technicians on maintenance and assembly tasks of industrial machineries. The main skill involved in this kind of tasks is the procedural skill. Based on past literature and studies conducted within the SKILLS project, several main design guidelines were formulated. First, observational learning integrated properly within the training protocol increases training efficiency. Second, training protocols combining physical and cognitive fidelity enhances procedural skills acquisition. Third, guidance aids should be provided in a proper and controlled way. And last, enriched information about the task helps trainees to develop a useful mental model of the task. These recommendations were implemented in both VR and AR training platforms. Design Guidelines for the Development of Virtual Reality and Augmented Reality Training Systems for Maintenance and Assembly Tasks Nirit Gavish * Teresa Gutierrez † Sabine Webel ‡ Jorge Rodriguez‡ Franco Tecchia** ()Technion – Israel Institute of Technology, Israel (†)TECNALIA, Spain (‡)Fraunhofer IGD, Germany (‡)CEIT – University of Navarra, Spain (*)PERCRO - Scuola Superiore Sant'Anna, Italy E-mail: niritg@tx.technion.ac.il, teresa.gutierrez@tecnalia.com, sabine.webel@igd.fraunhofer.de, jrodriguez@ceit.es, franco@sssup.it fundamentally a combination of two elements: the comprehension of the basic working principles of the machines to be maintained, and, more important, the learning of complex procedures (sequence of steps) to be performed in order to assemble or replace some parts of the machine. Hence, the main skill involved in a maintenance and assembly task is the procedural skill. Procedural skills reflect the ability of the operator to obtain a good mental representation of how to perform each of the steps of a task and the correct order to perform them, which is reflected in their hierarchic organization [1]. They are based on both procedural memory and on a good and correct mental model of the machine, its components and required tools. Until now, coherent recommendations for designing VR and AR systems for procedural skills training in a general context, and for IMA activities training specifically, are still missing. BIO Web of Conferences 1, 00029 (2011) DOI: 10.1051/bioconf/20110100029 © Owned by the authors, published by EDP Sciences, 2011 BIO Web of Conferences 1, 00029 (2011) DOI: 10.1051/bioconf/20110100029 © Owned by the authors, published by EDP Sciences, 2011 1 SKILLS project (IST-2006-035005) 3.1 Observational learning The common approach to training with VR training systems is the enactive approach, which stresses the importance of physical action within the environment to improve perception and enhance learning. However, under the theory of embodied cognition, verbal or visual stimuli are neurologically coupled with – and can prime – related motor activities. Hence, observational learning might replace active learning during some part of the training process. Two studies were conducted to address the use of observational learning in VR training systems. Figure 1. IMA-VR Training Platform: the trainee interacts and manipulates the virtual scene combining haptic, audio and visual feedback. The platform can be used with different types of haptic devices such as desktop workspace (right) and large workspace (left). g g y The first study made use of a 75-steps Lego assembly task, where the trainees had to learn how to assemble a model helicopter [2]. Training was performed using a 3D haptic virtual reality system. Two conditions were compared: an active condition, in which trainees had to both identify each target brick and assemble it correctly in the Lego model; and a partly observational learning condition, in which trainees were required only to select the correct brick, which would then be automatically positioned by the system. Results showed that training time was reduced substantially with the incorporation of an observational learning phase, while final performance with the real Lego task was similar for both conditions. Thus, observational learning enhances training efficiency if it is integrated properly within Virtual Reality-based training. The second study [3] demonstrated that preliminary observational learning, in which the training starts with a demonstration, and the trainee performs the task only after acquiring a basic knowledge of the necessary steps, can shorten training time. Figure 1. IMA-VR Training Platform: the trainee interacts and manipulates the virtual scene combining haptic, audio and visual feedback. The platform can be used with different types of haptic devices such as desktop workspace (right) and large workspace (left). In the IMA-AR platform, the trainee interacts with the real machine and tools using a tablet PC that acts as interactive see-through device. For this purpose, a camera is attached to the tablet PC. A moveable mount allows for depositing the tablet PC and still using it as see-through device. Thus, the trainee has both hands free for physically performing the training task. 3.1 Observational learning Furthermore, the trainee is equipped with a vibrotactile bracelet that is used for providing additional information through haptic feedback. See Figure 2. Figure 2. The trainee interacts with the IMA-AR platform using a tablet PC that acts as see-through device. Additional haptic feedback is presented to the trainee via a vibrotactile bracelet. The interactive training application is running on the tablet PC. Based on these studies, preliminary observational learning strategy was implemented in the IMA-VR platform. In this strategy, instead of having an active interaction with the virtual scenario, trainees just watch the system which provides visual information about how to undertake the task in order to help trainees to develop a mental model of the assembly/disassembly process. This strategy is being used at the beginning of the training process. Figure 2. The trainee interacts with the IMA-AR platform using a tablet PC that acts as see-through device. Additional haptic feedback is presented to the trainee via a vibrotactile bracelet. The interactive training application is running on the tablet PC. 3. Design guidelines: research and implication in the training platforms During the SKILLS project, several studies have been conducted in order to develop design guidelines for the VR and AR IMA training platforms. The fundamental research enabled us to provide applicative recommendations, which were applied in the platforms. Four main domains of research will be 1. Introduction Within the SKILLS project1, two different platforms were developed for the training of IMA tasks, one based on Virtual Reality technologies (IMA-VR) and the other one based on Augmented Reality (IMA-AR). Using the IMA-VR system, the trainee learns the task within a virtual environment. In the IMA-AR system the trainee learns the task through assembly operations, The current work describes design guidelines for the development of Virtual Reality (VR) and Augmented Reality (AR) platforms to train technicians on maintenance and assembly tasks of industrial machineries. These machineries usually need complex and lengthy maintenance procedures to be performed periodically by teams of highly trained technicians. Industrial maintenance and assembly (IMA) training is This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License 3.0, which permits unrestricted use, distribution, and reproduction in any noncommercial medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License 3.0, which permits unrestricted use, distribution, and reproduction in any noncommercial medium, provided the original work is properly cited. Article available at http://www.bio-conferences.org or http://dx.doi.org/10.1051/bioconf/20110100029 BIO Web of Conferences which are linked to the real machines by superimposing virtual objects on the live camera image, and using the real instruments for interaction. discussed: observational learning, cognitive fidelity versus physical fidelity, guidance aids and enriched information. In the IMA-VR platform, the trainees interact with the virtual machine and tools in a multimodal way combining haptic, audio and visual feedback, see Figure 1. This platform provides different multimodal aids, not available in the real world, that help and guide the trainees during the training process. It also allows remote supervision/training through an on-line interaction between trainer and trainee. 3.3 Guidance aids During the execution of a task, the trainees will sometimes need to receive direct information about how to proceed with the current step: guidance aids. However, the addition of guidance aids can have adverse effects on performance, because it encourages shallow performance strategies and little exploration. To examine this potential training trap, two experimental studies were conducted. A study was conducted to compare real-world training and two alternative virtual trainers, one emphasizing the physical fidelity and the other the cognitive fidelity of the task [4]. Participants were randomly assigned to one of four training groups in the 75-steps Lego assembly task: Virtual-Physical Fidelity, Cognitive Fidelity, Real-World and Control. A post- training test to assess the development of procedural skills was conducted. Results demonstrated the Cognitive Fidelity training was inferior in terms of test time compared with the Real-World training. In contrast, only the Real-World and the Cognitive Fidelity groups required significantly less time than the Control group for error correction. Conclusions are that the two training methods have complementary advantages, hence combining physical fidelity and cognitive training methods enhances procedural skills acquisition. In the first study [5], each trainer instructed trainees on how to perform a 3D virtual puzzle in two conditions: Vocal guidance, where only vocal instructions were possible, and vocal guidance with mouse pointing, where the trainer could also use a mouse to point out the target positions on the trainee’s screen. The results showed that while the use of the mouse pointer reduced trainees’ mental load during training, it also drastically lowered performance level on a non-supervised test. These results suggest that the addition of guidance information should be considered very carefully. However, specific training protocols can improve the use of guidance tools. This was the goal of the second study. This study made use of the 75-steps Lego assembly task and a 3D haptic virtual reality system. Two conditions were compared: building the virtual Lego model with the help of a printed instructions book (with step-by-step diagrams of the 75 stages); and building the virtual Lego with the help of direct aids provided by the virtual platform (change the color of the target brick and then show a copy in its the final position) but only on participants’ demand. Both groups performed similarly in the real Lego test. The International Conference SKILLS 2011 cognitively represented within the simulator. The physical fidelity approach claims that the simulator should replicate the real-world task to the greatest degree possible. In contrast, the cognitive fidelity approach states that the simulator should engage the trainee in the type of cognitive activities involved in the real-world task, without needing to duplicate the physical elements of the task. 3.2 Procedural skills are acquired through repeated exposure to a certain task. Hence, it is important to determine whether this task should be physically or 00029-p.2 The International Conference SKILLS 2011 4. Conclusions Several past studies have demonstrated that when learners of a new procedural task are provided with enriched information about the task, in addition to the "how-to-do-it" knowledge, their performance becomes more accurate, faster and more flexible. This information enhances the building of the trainee’s mental model about the task [6], and, as a result, improves performance. Taking into account that industrial maintenance tasks are based on complex procedures, it was proposed to divide the whole task into a set of logic sub-tasks. Providing enriched information about these sub-tasks could help trainees to develop an appropriate and more accurate mental model of the whole task. Based on past literature and studies conducted within the SKILLS project, several main design guidelines were formulated for the development of VR and AR training platforms in the IMA domain. The guidelines are: 1) observational learning integrated properly within the training protocol increases training efficiency; 2) training protocols combining physical and cognitive fidelity enhance procedural skills acquisition; 3) guidance aids should be provided in a proper and controlled way; and 4) enriched information about the task helps trainees to develop a useful mental model of the task. The recommendations were implemented in both IMA-VR and IMA-AR platforms and tested in a skills transfer evaluation performed by technicians. A study was conducted to explore the efficiency of providing information in the step or sub-task level in an industrial task. In this study, the trainees used a haptic virtual environment to learn how to assemble part of an actuator. The task was composed of 23 steps grouped in five sub-tasks. Two conditions were compared: assembling the actuator receiving information in the step level or in the sub-task level. On the following day, participants had to assemble the real actuator. Results showed that although performance time was similar in both groups, the number of non-solved errors was larger in the group trained with step level information. The conclusions are that the use of enriched information should be encouraged in order to help the trainees develop a better mental model of the task. 3.3 Guidance aids Results showed that the use of guidance aids in a proper and controlled way, as providing aids only on trainees’ demand, does not impede task learning. These outcomes were implemented in both platforms. In addition to the high physical fidelity training, the IMA-VR platform also provides cognitive training. The cognitive training is carried out by providing information about the current step not directly on the manipulated objects, but on a second display next to the virtual machine in which the system shows all the pieces and tools involved in the current step in their final position and a textual aid which describes the step and lists the names of the necessary pieces/tools (see Figure 3, left). This implementation allows cognitive training, since the trainees need to cognitively translate the display and the textual information to the actions that must be undertaken with the manipulated objects. Similarly, in the IMA-AR platform the information about the step and the required tools is presented near the real object instead of using traditional Augmented Reality overlays as 3D animations (see Figure 3, right). Implementation of these outcomes in the platforms design is to provide guidance aids in a limited and controlled way, only when the trainee requests it. In the IMA-VR platform, the requested guidance aids are provided by means of visual aids (highlighting the target tool icon, changing the color of the target piece or displaying a copy of the target piece/tools in its final position) and haptic aids (exerting an attraction force towards the target piece/position). In the IMA-AR platform, although a superimposed 3D-animation of a pulsing circle showing the part of the machine which is relevant for the performance of the current step is permanently presented, it provides only a hint about the required action, and the trainee needs to actively request additional information. Figure 3. Information about the current step in the IMA-VR platform (left) and in the IMA-AR Platform (right). Figure 3. Information about the current step in the IMA-VR platform (left) and in the IMA-AR Platform (right). 00029-p.3 BIO Web of Conferences 5. Acknowledgments The activities described in this paper were carried out with the financial assistance of the EU which co- funded the project SKILLS (IST FP6 ICT-IP-035005- 2006) the “Ministerio de Ciencia e Innovación” (Spain). Authors wishes to thank especially to all the scientists/technicians who helped in the development of the IMA training systems from DLR, FhG, CEIT, PERCRO, SIDEL, TECHNION and TECNALIA. References [1] J.R Anderson. Acquisition of cognitive skill. Psychological Review, 89, 369-406, 1982. Based on these outcomes both platforms provide the trainees enriched information by means of information in the sub-task level and information about the progress in the task. In the IMA-VR platform, enriched information in the sub-task level is provided in a second display in which the system shows the pieces involved in all the steps of the current sub-task in their final position (see Figure 4, left). Similarly, in the IMA-AR platform enriched information is provided with animated image of the current sub-task along with a progress bar (see Figure 4, right). [2] N. Yuviler-Gavish, J. Rodríguez, T. Gutiérrez, E. Sánchez, and S. Casado. (Revised and re-submitted). Observational learning versus enactive approach in virtual reality training. Human Factors. [3] N. Yuviler-Gavish, and M. Shelef (Submitted). Evaluating two modes of observational learning in cognitive-spatial task training. Applied Cognitive Psychology. [4] I. Hochmitz, I., & N. Yuviler-Gavish (Accepted). Physical fidelity versus cognitive fidelity training in procedural skills acquisition. Human Factors. Figure 4. Enriched Information at the sub-task level in the IMA-VR platform (left) and in the IMA-AR Platform (right). [5] N. Yuviler-Gavish, E. Yechiam, and A. Kallai. Learning in multimodal training: Visual guidance can be both appealing and disadvantageous in spatial tasks. International Journal of Human-Computer Studies, 69, 113-122, 2011. [6] N.A. Taagten, D. Huss, D. Dickison, and J.R. Anserdon, The acquisition of robust and flexible cognitive skills. Journal of Experimental Psychology: General, 137, 548-565, 2008. Figure 4. Enriched Information at the sub-task level in the IMA-VR platform (left) and in the IMA-AR Platform (right). 00029-p.4
https://openalex.org/W4206602327	https://www.scielo.br/j/pci/a/T795Mfh4PLsBdxWvbMVRswj/?lang=pt&format=pdf	Portuguese	null	Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à luz da Arquivologia	Perspectivas em Ciência da Informação	2,021	cc-by	8,587	Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 92 Pedro Vinicius Asterito Lapera I http://orcid.org/0000-0002-7547-1566 I Universidade Federal Fluminense, RJ, Brasil. Docente no Programa de Pós-Graduação em Cinema e Audiovisual. http://dx.doi.org/10.1590/1981-5344/4359 Este artigo apresenta uma análise do quadro de arranjo e dos instrumentos de pesquisa referentes ao fundo da Divisão de Censura de Diversões Públicas, custodiado no Arquivo Nacional. Partindo de uma revisão bibliográfica e da coleta de dados, pretendemos trabalhar as seguintes questões: a) de que modos o quadro de arranjo e os instrumentos de pesquisa orientam a busca por informações junto ao fundo DCDP? Secundariamente, como esses instrumentos de pesquisa estão integrados ao cenário arquivístico institucional e até mesmo nacional? Palavras-chave: Descrição arquivística; Censura; Ditadura; Arquivo Nacional. From twilight to reveal: a brief analysis of the arrangement and of the research instruments of Divisão de Censura de Diversões Públicas regarding Archivology I Universidade Federal Fluminense, RJ, Brasil. Docente no Programa de Pós-Graduação em Cinema e Audiovisual. I Universidade Federal Fluminense, RJ, Brasil. Docente no Programa de Pós-Graduação em Cinema e Audiovisual. Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à luz da Arquivologia Pedro Vinicius Asterito Lapera I http://orcid.org/0000-0002-7547-1566 1 A ela, referir-nos-emos como DCDP ao longo deste artigo a partir desse ponto. http://dx.doi.org/10.1590/1981-5344/4359 Este artigo apresenta uma análise do quadro de arranjo e dos instrumentos de pesquisa referentes ao fundo da Divisão de Censura de Diversões Públicas, custodiado no Arquivo Nacional. Partindo de uma revisão bibliográfica e da coleta de dados, pretendemos trabalhar as seguintes questões: a) de que modos o quadro de arranjo e os instrumentos de pesquisa orientam a busca por informações junto ao fundo DCDP? Secundariamente, como esses instrumentos de pesquisa estão integrados ao cenário arquivístico institucional e até mesmo nacional? Palavras-chave: Descrição arquivística; Censura; Ditadura; Arquivo Nacional. From twilight to reveal: a brief analysis of the arrangement and of the research instruments of Divisão de Censura de Diversões Públicas regarding Archivology erspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 92 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera This article presents an analysis of the arrangement and the research instruments related to the fund of the Divisão de Censura de Diversões Públicas, kept in Arquivo Nacional. Based on a bibliographic review and data collection, we intend to work on these questions: a) in what ways does the arrangement framework and research instruments guide the search for information from the DCDP fund? Secondly, how these instruments guide are integrated into their institucional and evennational context? Keywords: Archivaldescription; Censorship; Dictatorship; Arquivo Nacional. Keywords: Archivaldescription; Censorship; Dictatorship; Arquivo Nacional. 1 Introdução A censura foi uma função amplamente exercida pelo Estado brasileiro em seu período republicano. Inicialmente exercida pelas polícias estaduais (e, no caso do Rio de Janeiro, pela Polícia do Distrito Federal) nos primeiros anos da República, a censura ganhou um órgão específico no organograma do Estado brasileiro com a criação do Departamento de Propaganda e Difusão Cultural (DPDC), através do Decreto 24651/34, durante a Era Vargas (1930-1945). A centralização da atividade censória pensada pela ditadura civil- militar que se instalou após o Golpe de 1964 é fruto desse primeiro momento inaugural da ditadura varguista, lembrando que ela foi exercida mesmo durante o período democrático no intervalo entre esses regimes autoritários.A atuação da Divisão de Censura de Diversões Públicas1 configura mais um capítulo no histórico republicano do exercício dessa função.Ela foi o órgão que atuou diretamente na censura às obras de diferentes meios artísticos e de comunicação entre 1972 e 1988, sendo também responsável pela atuação cada vez mais enfática do órgão na repressão às vozes dissonantes do regime que atuavam nos meios de comunicação massiva e artísticos (Fico, 2001;Kushnir, 2004; Lapera, 2015; Ridenti, 2000; Simões, 1999). 93 Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera O Decreto 70665/722 inseria a DCDP na estrutura do Departamento de Polícia Federal (DPF) no inciso I do seu artigo 2º., situando-a na sua competência de exercer a censura de diversões públicas estabelecida no art 1º. do mesmo Decreto. Ainda havia o Conselho Superior de Censura, órgão colegiado de natureza consultiva e deliberativa do Ministério da Justiça, criado pelo Decreto 64416, de 19693, que possuía a atribuição de julgar todos os recursos impetrados contra as decisões da diretoria da Polícia Federal que, por sua vez, apreciavam os recursos contra as decisões da chefia da DCDP.Recordamos que ela foi oficialmente extinta com a promulgação da Constituição de 1988, uma vez que o então novo texto constitucional vedava ao Estado brasileiro a prática de censurar obras artísticas e meios de comunicação massivos. 2https://www2.camara.leg.br/legin/fed/decret/1970-1979/decreto-70665-2-junho-1972- 419313-publicacaooriginal-1-pe.html . Acesso em: em 27 jul. 2019. 3https://www2.camara.leg.br/legin/fed/decret/1960-1969/decreto-64416-28-abril-1969- 405956-publicacaooriginal-1-pe.html . Acesso em: 28 jul. 2019. 4 Ressaltamos que este artigo não se relaciona à tese já defendida, uma vez que se trata de um projeto de pesquisa posterior ao Doutorado. 5 Sigla de Sistema de Informações do Arquivo Nacional. Resumidamente, a base SIAN reúne documentos de diversos fundos custodiados pelo Arquivo Nacional. Ainda, apresenta esses fundos e suas informações, tais como instituição de origem, histórico, séries, subséries, descrições qualitativas a respeito dos documentos e dos fundos. Para acessá-la, basta realizar um breve cadastro por parte do/a pesquisador/a. Link: https://sian.an.gov.br/sianex/consulta/login.asp (Acesso em 25/11/2021). 1 Introdução Ao longo deste artigo, apresentaremos uma discussão sobre o organograma da DCDP, sobre a própria delimitação de seu fundo perante os fundos da Polícia Federal e do Ministério da Justiça, além de uma análise do quadro de arranjo do fundo DCDP e dos instrumentos de pesquisa elaborados pelo Arquivo Nacional – seção Brasília, instituição responsável pelo recolhimento da documentação permanente e pela sua custódia. As questões que trabalhamos ao longo de nosso trabalho são: a) de que modos o quadro de arranjo e os instrumentos de pesquisa orientam a busca por informações junto ao fundo da DCDP? Secundariamente, como esses instrumentos de pesquisa estão integrados ao cenário arquivístico institucional e até mesmo nacional?Enfatizamos que nossa pesquisa foi realizada através da coleta in loco e online de fontes e documentos junto ao Arquivo Nacional – seção Brasília e de uma breve revisão bibliográfica amparada em algumas discussões atuais na Arquivologia a respeito de questões como custódia, critérios de delimitação de fundos, de elaboração de quadros de arranjo e de elementos a serem incorporados na descrição arquivística. A coleta de fontes sobre o arquivo da DCDP vem ocorrendo em meio a uma pesquisa sobre a censura à atividade cinematográfica no Brasil, em andamento desde 2010 junto ao Arquivo Nacional. Por diversos caminhos, ao longo do Doutorado, percebemos que todos os filmes analisados se depararam em algum momento com a atividade censória e, em maior ou menor nível, tiveram sua circulação cerceada4. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 94 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Embora de modo intermitente, conseguimos reunir não apenas dados a partir das fontes coletadas especificamente a respeito do cinema no Brasil nos anos 1970 e 80, como também a respeito do próprio arquivo que custodia esses documentos analisados. Enquanto critério de seleção das fontes a serem analisadas, partimos do material disponibilizado ao longo das consultas efetuadas presencialmente no Arquivo Nacional para, em seguida, acrescentarmos novas fontes à medida que foram disponibilizadas. 1 Introdução Como usuário, acessamos os materiais que instrumentalizam a busca por documentos junto ao arquivo da DCDP ao longo desses anos e, em 2019, incorporamos as informações disponíveis no site do Arquivo Nacional a respeito desse arquivo à nossa exposição. Por fim, em 2021, resolvemos acrescentar também os registros na base SIAN5 do Arquivo Nacional ao escopo desta pesquisa. 6 Disponível em https://www2.camara.leg.br/legin/fed/declei/1970-1979/decreto-lei- 1077-26-janeiro-1970-355732-publicacaooriginal-1-pe.html. Acesso em: 23 jul. 2019. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional Antes de avaliarmos o quadro de arranjo e os instrumentos de pesquisa da DCDP, precisamos discutir a delimitação de um fundo. A DCDP era um órgão localizado dentro da estrutura da Polícia Federal que, por sua vez, estava submetida ao Ministério da Justiça. Tal subordinação foi amparada pelo Decreto-Lei 1077/70, que dispunha, em seu artigo 2º: “Caberá ao Ministério da Justiça, através do Departamento de Polícia Federal verificar, quando julgar necessário, antes da divulgação de livros e periódicos, a existência de matéria infringente da proibição enunciada no artigo anterior”6. Assim, a DCDP possuía uma atividade e uma competência legal definidas: exercer censura a diferentes meios atinentes às diversões públicas (teatro, cinema, música, televisão etc). Além disso, a chefia desta Divisão detinha um grau de autonomia relativo à Chefia da Polícia Federal, uma vez que suas decisões só poderiam ser contestadas por meio de recurso administrativo, que se inseria em um fluxo hierárquico definido. Por fim, possuía um organograma minimamente estável que Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 95 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera respondia à sua estrutura e dava conta das funções e atividades que o órgão exercia. respondia à sua estrutura e dava conta das funções e atividades que o órgão exercia. Desse modo, a DCDP atendia aos critérios elencados por Duchein (1982-1986, p. 19) na delimitação de um fundo próprio, na medida em que o órgão possuía uma existência jurídica imposta pelo Decreto-Lei citado, executou durante sua existência atividades estáveis em torno da função censória, tinha posição definida na hierarquia administrativa do Ministério da Justiça e chefia com autonomia relativa de atuação, além de um organograma conhecido. Considerando o fundo uma criação intelectual fruto de uma abstração, Cook discute as posições maximalistas e minimalistas na definição de um fundo. O autor analisou a importância de se delimitar os limites de um fundo (2017, p. 35-38) para a gestão dos arquivos correntes, intermediários e também os permanentes. Ainda, recordou a posição de alguns autores maximalistas (Jenkinson e seus seguidores), ao analisar que esses autores incluem todos os documentos produzidos por uma administração dentro de um todo orgânico (2017, p. 36). A isso, Cook (2017, p. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional 37) contrapõe a posição dos arquivistas norte-americanos (representados por Schellenberg), que se preocuparam em criar e adotar critérios de definição de fundos para melhor administrá- los em relação ao todo documental produzido pela Administração federal do governo norte-americano. No caso da DCDP, seu fundo foi individualizado no tratamento arquivístico elaborado pelo Arquivo Nacional em relação aos fundos dos entes administrativos aos quais o órgão se encontrava subordinado durante sua existência e atuação. Desse modo, ao se delimitar o fundo DCDP, adotou-se o critério minimalista, dissociando-o dos fundos da Polícia Federal e do Ministério da Justiça. No caso do fundo da DCDP, a opção pelo tratamento minimalista permitiu um destaque maior tanto do seu conjunto documental quanto da atividade principal exercida por ele, sem que se perdesse de vista o fato de que ele se insere em uma estrutura maior. Tal escolha revelou-se fundamental para divulgar aos usuários (sejam eles historiadores ou não) sua importância como vestígio de uma função – censura – que sustentava o regime que o originou, jogando luz sobre os processos administrativos e os documentos produzidos por eles. Ainda, podemos considerar que a aplicação do critério maximalista na delimitação de um fundo seria bastante problemática em se tratando dos documentos referentes à Administração pública brasileira durante a ditadura civil-militar. Além de serem notórios os relatos em torno da perda de arquivos inteiros e de eliminação arbitrária de documentos pelos agentes da ditadura na ocultação de seus crimes (Figueiredo, 2015), também recordamos que o Ministério da Justiça centralizava parte 96 Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera considerável do aparato repressor do regime civil-militar (Polícia Federal, Serviço Nacional de Informação – SNI, além da própria DCDP). considerável do aparato repressor do regime civil-militar (Polícia Federal, Serviço Nacional de Informação – SNI, além da própria DCDP). Desse modo, não há a garantia de que as perdas documentais ocorreram exclusivamente através da avaliação arquivística e a consequente eliminação de parte da massa documental, na medida em que a adoção do critério maximalista exige a visão completa do fluxo documental arquivos correntes, intermediários e permanentes na Administração pública (Cook, 2007, p. 36) e tal fato torna-se impossível no contexto descrito. 7 “To declare a document authentic means to say that it is precisely as it was when first transmitted or set aside for preservation, and that its reliability, or the trustworthiness it had at that moment, has been maintained intact” (Tradução fo autor). 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional Sobre a custódia do arquivo da DCDP pelo Arquivo Nacional, é preciso situá-la dentro do exposto por Duranti a respeito da ligação entre ela e a autenticidade dos documentos: declarar um documento autêntico significa dizer que ele se encontra do mesmo jeito que foi transmitido primeiramente ou colocado para preservação e que sua confiabilidade ou a veracidade relacionada àquele momento foi mantida intacta.7 (DURANTI, 2007, p. 454) Ainda, a autora relaciona a custódia dos arquivos ao processo de formação dos Estados nacionais a partir do século XVIII em razão dos valores jurídico-administrativo e histórico dos documentos (DURANTI, 2007, p. 455-457). Desse modo, podemos localizar esses documentos em meio à relação entre a custódia estatal, a sua autenticidade e a sua necessidade de preservação, vista como fundamental para a construção do conhecimento histórico sobre eventos, sujeitos, instituições e processos no passado. De acordo com Kushnir (2004, p. 208), o organograma da DCDP era estruturado a partir do seu Gabinete da Direção, que ocupava o topo da escala hierárquica. Dele, partiam todos os níveis inferiores, o que demonstra uma centralização interna do próprio órgão. Em um nível hierárquico inferior, há duas seções (Orientação e Coordenação e Controle) e um arquivo, além do Serviço de Censura que, por sua vez, em um nível hierárquico inferior, se desdobra em censura a cinema, a televisão e rádio, a teatro e congêneres, além das seções de expediente e de projeção. 97 Perspectivas em Ciência da Informação, v.26, número 4, p. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional 92-112, dez/2021 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Retomaremos a esse organograma na discussão sobre o quadro de arranjo elaborado pelo Arquivo Nacional, que reproduzimos no quadro abaixo8: arranjo elaborado pelo Arquivo Nacional, que reproduzimos no quadro abaixo8: Quadro 1 Seção Série Subsérie Administração Geral (15 caixas) Controle de documentos - termos de eliminação - CDO - 1 caixa Correspondência Oficial - COF Ofícios de comunicação e solicitação - CSO - 5 caixas Informações Sigilosas - ISI - 1 caixa Manifestações da Sociedade Civil - MSC - 6 caixas Relatórios de Atividades - RAT - 2 caixas Censura Prévia - CPR (3187 caixas) Cinema - CIN Programação Cinematográfica - PCN - 9 caixas Filmes - FIL - 862 caixas Publicações - PUB - 28 caixas Publicidade - PBL - 11 caixas Rádio - RAD Programas de Rádio - PGI - 8 caixas Programação de Emissoras de Rádio - PRI - 3 caixas Radionovelas - RDN - 66 caixas Teatro - TEA Peças teatrais - PTE - 764 caixas Programação de Teatro - 2 caixas Televisão - TVE Programas de Televisão - PGV - 96 caixas Programação de Emissoras de Televisão - PTV - 6 caixas Seriados - SER - 413 caixas Telenovelas - TLN - 158 caixas 8 O quadro foi elaborado de acordo com o quadro de arranjo de autoria da técnica em arquivo Camilla França publicado no Diário Oficial da União em 6 de maio de 2009 e todas as informações reproduzidas nesta tabela constam no referido quadro. Além das seções apresentadas, há 17 caixas avulsas. 98 Perspectivas em Ciência da Informação, v.26, número 4, p. 9http://dibrarq.arquivonacional.gov.br/index.php/divisao-de-censura-de- diversoes-publicas-4. Acesso em: 27 jul. 2019. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional 92-112, dez/2021 99 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Pedro Vinicius Asterito Lapera permite entender o arquivo como um todo em que cada peça está relacionada em virtude da procedência e do trâmite. Algo bem diferente de uma bem ordenada coleção de documentos, reunidos por interesse de pesquisa, por capricho ou por necessidade de ordenar peças procedentes de várias origens‟ (Cortés Alonso, 1981). Entra aqui, mais uma vez, a questão da organicidade dos documentos de arquivo. Se estes resultam de diferentes ramos das funções e das competências de um órgão público, as sequências de documentos não têm sentido fora dessa sucessão, assim como não podem estar relacionados a outros tipos de competências. A organicidade é, portanto, a „relação entre a individualidade do documento e o conjunto no qual ele se situa geneticamente, sendo precisamente a base da noção de fundo de arquivo‟ (Guaye, 1984, v. 34, p. 15-23). Esse axioma deve estar sempre presente, quase que implacavelmente, nas tarefas de organização interna de fundos (BELLOTTO, 2004, p. 152). Bellotto avalia que o objetivo do arranjo é a “ordenação dos conjuntos documentais remanescentes das eliminações (...), obedecendo a critérios que respeitem o caráter orgânico dos conjuntos, interna e externamente” (BELLOTTO, 2004, p. 136). Ao refletir sobre a ligação entre a predominância da forma administrativa no arranjo e a recuperação da informação para os usuários, a autora pondera que a importância do fundo não reside exclusivamente no conteúdo dos documentos, mas também nas relações que guardam entre eles e, principalmente, entre eles e as funções e atividades desempenhadas por um órgão (BELLOTTO, 2004, p. 139-140). Cruzando os dados do organograma com os do quadro de arranjo, é possível verificar algumas semelhanças entre a estrutura do órgão e o modo como os documentos foram organizados. Por exemplo, o serviço de Censura Prévia e as seções de Orientação e de Coordenação e Controle presentes no organograma encontram ressonância nas seções homônimas no quadro de arranjo. Além disso, a seção Censura Prévia no quadro de arranjo reúne em suas séries algumas das divisões dentro do setor que a gerou.Isso significa que o quadro de arranjo adotou o critério estrutural em sua elaboração, ao menos parcialmente. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional 92-112, dez/2021 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Música - MUI Letras Musicais - LMU - 736 caixas Programação de Música - PMU - 14 caixas Coordenação e Controle - CCO (89 caixas) Autorização Especial - AES - 16 caixas Direito Autoral - DAU - 5 caixas Fiscalização - FIS - 19 caixas Registros de Firmas e Publicações - RFP Empresas cinematográficas e agências de publicidade - ECA - 16 caixas Estabelecimento Comercial - ECO - 3 caixas Publicações - PUB - 30 caixas Orientação - ORI (16 caixas) Cursos - CUR - 1 caixa Normatização - NOR - 5 caixas Recursos - RER - 10 caixas Fonte: produção do autor. Pedro Vinicius Asterito Lapera Fonte: produção do autor. É importante ressaltar que este quadro de arranjo se refere aos documentos escritos que se encontram fisicamente guardados na seção Brasília do Arquivo Nacional. Também há documentos audiovisuais do mesmo fundo da DCDP guardados no Arquivo Nacional, na seção do Rio de Janeiro, cujos itens documentais são compostos por 354 filmes cinematográficos e por 16 fitas videomagnéticas9, porém enfatizamos que a parte referente a documentos audiovisuais do fundo da DCDP não é o objeto desta pesquisa. Sobre os critérios a serem considerados na elaboração de um quadro de arranjo, existem o tipológico, o estrutural e o funcional. O primeiro se ateria aos tipos documentais presentes no arquivo. Por sua vez, o segundo levaria em consideração principalmente a estrutura da empresa/instituição originária do fundo. Por fim, o terceiro critério – que é o mais recomendado e utilizado no campo da Arquivologia –é pautado pelas funções e atividades exercidas por um órgão/instituição e como os documentos são relacionados a elas (BELLOTTO, 2004, p. 147-160). De acordo com Bellotto: Se, para a fixação do fundo, do grupo ou seção e do subgrupo ou subseção, o fundamental é o órgão produtor (o “autor”), para a série ou subsérie, o que vigora são a função e o tipo documental. No que diz respeito aos documentos, é com esses elementos que „se reconstroem o organograma e as atividades que os originam, o que Perspectivas em Ciência da Informação, v.26, número 4, p. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional Entretanto, a disposição das seções no quadro de arranjo também responde às funções desempenhadas pela DCDP ao longo do seu período ativo, uma vez que cabia a ela realizar a censura prévia das diversões públicas e também exercer alguns mecanismos de controle e fiscalização e, ainda, na formação e atualização nas práticas dos censores. Assim, podemos classificar o quadro de arranjo da DCDP como estrutural- Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 100 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera funcional, em linhas gerais, sendo o critério tipológico adotado no caso mais raramente. funcional, em linhas gerais, sendo o critério tipológico adotado no caso mais raramente. Ainda sobre o quadro de arranjo da DCDP,verificamos que este se estrutura em torno de 4 seções, a saber: Administração Geral; Censura Prévia; Coordenação e Controle; e Orientação. A respeito dessa divisão em um quadro de arranjo, Bellotto prescreveu: “tanto o fundo quanto suas primeiras divisões – grupos ou seções e subgrupos ou subseções (se houver) – são, na verdade, nomes, no arquivo, aglutinações de documentos” (2004, p. 152). Na seção Administração Geral, há um conjunto de documentos referentes às atividades administrativas da DCDP, tais como: correspondências com outros órgãos de dentro e mesmo externos à estrutura do Ministério da Justiça, cartas escritas por cidadãos apoiando ou questionando a atuação da DCDP, pedindo maior ou menor rigor na avaliação de obras, troca de informações sigilosas envolvendo obras avaliadas pela DCDP, personalidades dos diferentes campos da arte e da cultura brasileira dos anos 1960 e 70 e a atuação de funcionários do próprio órgão na repressão da ditadura contra os agentes da cultura brasileira. Também estão guardados os relatórios de atividades dos diferentes setores da DCDP desde sua implementação até sua extinção com a Constituição de 1988. Nessa seção, duas séries foram nomeadas a partir de tipos documentais (Correspondência Oficial e Relatório de Atividades) e não a partir de funções, como no restante do quadro de arranjo da DCDP, havendo uma mistura de critérios na nomeação de séries. Em princípio isso contraria a teoria arquivística, que preconiza a adoção de um critério na nomeação de grupos de mesmo nível (seção, série, subsérie) (BELLOTTO, 2004, p. 10 Agradecemos a/ao parecerista por essa observação, que incorporamos à nossa análise. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional 150-160)10 e, em um primeiro momento, poderia dificultar a visualização por parte do usuário dos documentos elencados nessas séries. De fato, tais séries poderiam ter sido nomeadas de outro modo para indicar com mais precisão as funções desempenhadas por esses documentos em relação à totalidade do fundo. Entretanto, recordamos que justamente essas séries foram alvos de uma descrição arquivística bastante meticulosa, a ser ainda abordada neste artigo, contornando a questão da consulta por parte do usuário externo com mais informações sobre esses conjuntos de documentos. Por sua vez, a seção Censura Prévia constitui o “núcleo duro” da documentação do fundo DCDP, justamente por se remeter à principal 10 Agradecemos a/ao parecerista por essa observação, que incorporamos à nossa análise. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 101 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera atividade-fim do órgão. Nela, estão reunidas em 7 séries que se desdobram em 13 subséries os documentos referentes à censura prévia das obras de diferentes campos da cultura: música, teatro, cinema, rádio e televisão, o que certamente justifica o maior acúmulo documental ter ocorrido justamente nela (algo comprovado pelas 3187 caixas arquivadas). À À primeira vista, os nomes de algumas séries dessa seção – Cinema, Televisão, Publicidade, por exemplo – podem parecer um recorte por assunto e não funcional, na medida em que estes se confundem com temas a serem potencialmente pesquisados na documentação do fundo11. A respeito disso, precisamos sublinhar a ligação entre a atividade de censura prévia feita pela DCDP e a função em cada caso, a saber: censura ao cinema, à televisão, à publicidade, para continuarmos nos exemplos.Na seção Coordenação e Controle, encontram-se os documentos referentes às atividades de controle no campo da cultura exercidas pela DCDP, tais como recolhimento de taxas de direitos autorais de obras dos diversos meios, registros de empresas cinematográficas e de agências publicitárias (que precisavam disso para funcionarem). Além disso, há documentos a respeito da fiscalização exercida pela DCDP no âmbito das diversões públicas, tais como autos de infração, alvarás de funcionamento, multas. Nessa seção, também é possível ter uma impressão de que assuntos foram privilegiados em detrimento de funções, sobretudo a série “Direito Autoral”. 11 Agradecemos à/ao parecerista por mais essa observação feita sobre os nomes das séries e subséries. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional No entanto, recordamos que a série citada responde diretamente a uma atividade de controle (tal como expresso no nome da seção). Na seção Orientação, há uma documentação acumulada referente aos cursos para os próprios funcionários da DCDP que se remetiam à atividade censória, comentários e explicações sobre a legislação aplicada pelo órgão e os recursos endereçados por particulares a ele. Completando o fundo, ainda há dezessete caixas avulsas que incluem alguns documentos referentes a cinema, tais como Festival de Cinema de Gramado e instrumentos de busca referentes a filmes, além de outros assuntos como livros e material apreendidos. Deste modo, realizou-se a operação do arranjo em seu sentido material e intelectual, tal como preconizado por Bellotto (2004, p. 139), uma vez que à organização física dos documentos no fundo e em suas seções, séries e subséries, há a correspondência com a função desses documentos no conjunto do arquivo, o que evidencia o respeito ao princípio da organicidade. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 102 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Pedro Vinicius Asterito Lapera j p q Censura de Diversões Públicas à Luz da Arquivologia Na delimitação do fundo DCDP, observamos as consequências apontadas por Duchein: Separa as divisões ou secções internas de um organismo que não respondam às condições requisitadas para produzir um Fundos de arquivo; Todo organismo possuindo uma existência jurídica e um nível de competências própria produz um Fundos de arquivo, mesmo se está subordinado a outro organismo de nível mais elevado; Os órgãos locais dependentes de um organismo central produzem Fundos de arquivos que lhes são próprios; Por fim, parece inevitável introduzir em Arquivologia uma noção nova: aquela de hierarquia de Fundos dos organismos produtores, provocando a subordinação de certos Fundos em relação a outros (DUCHEIN, 1982-1986, p. 21). É possível inferir que o fundo da DCDP estaria hierarquicamente submetido aos fundos da Polícia Federal e do Ministério da Justiça, na medida em que aquele se constituiu em relação aos últimos. Ao avaliarmos o tratamento concedido pelo Arquivo Nacional ao fundo DCDP, constatamos algumas interrelações com os fundos da Polícia Federal e do Ministério da Justiça em alguns elementos da descrição dos itens documentais e no histórico do fundo apresentado pela instituição. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional Além do quadro de arranjo, existem vários instrumentos de pesquisa referentes às subséries em que os documentos estão alocados. Por ocasião da análise desses instrumentos, reproduziremos um pequeno trecho da primeira página, pois se tratam de documentos com muitas páginas, cujas reproduções integrais tornariam nosso trabalho inviável. Sublinhamos que a consulta a esses instrumentos de pesquisa pode ser feita no site do Arquivo Nacional através do SIAN e localmente na seção Brasília do Arquivo Nacional. A descrição como função arquivística está diretamente relacionada ao acesso por parte dos usuários. Bellotto infere que “o processo de descrição consiste na elaboração de instrumentos de pesquisa que possibilitem a identificação, o rastreamento, a localização e a utilização de dados” (2004, p. 179). Assim sendo, os instrumentos de pesquisa serviriam aos gestores dos arquivos e aos usuários na consulta a arquivos permanentes, uma vez que estes permitiriam recuperar as informações em diferentes níveis, de acordo com os interesses de cada consulente. Bellotto apresenta a definição de instrumento de pesquisa como “obras de referência que identificam, resumem e localizam, em diferentes graus e amplitudes, os Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 103 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera fundos, as séries documentais e/ou unidades documentais existentes em um arquivo permanente” (2004, p. 180), enfatizando seu papel na comunicação com o usuário. Sobre a descrição, Yeo (2017, p. 142-148) recupera alguns argumentos críticos à descrição retrospectiva, isto é, à descrição realizada somente após os documentos serem considerados permanentes e recolhidos à instituição de guarda. O autor apontou a necessidade de se atrelar a descrição ao ciclo vital dos documentos através da inserção de metadados no momento de sua criação, destacando que a crítica à descrição retrospectiva ocorre porque ela “exige recursos a que os arquivistas não têm acesso; causa enormes gargalos no processamento; é incapaz de registrar importantes informações contextuais” (2017, p. 142), dentre outros. Yeo também recordou seus defensores que, segundo ele, “identificam um papel específico para a descrição retrospectiva na averiguação da autenticidade ou no fornecimento de uma perspectiva contextual mais cuidadosa” (2017, p. 143). Em seguida, ponderou que as informações referentes à descrição podem ser inseridas ao longo do ciclo vital dos documentos (2017, p. 143). 12http://arquivonacional.gov.br/br/consulta-ao-acervo/instrumentos-de- pesquisa.html. Acesso em: 26 jul. 2019. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional No caso do fundo da DCDP, é possível inferir que a descrição retrospectiva se mostrou a única opção possível a ser seguida, visto que não havia o debate sobre metadados no momento da criação desses documentos nem condições tecnológicas para isso. Assim, tal exigência revela-se um anacronismo histórico na avaliação das práticas arquivísticas. E como a produção de metadados se trata de uma prática relativamente recente no âmbito da Arquivologia nos países do Primeiro Mundo e muito recente no caso brasileiro, esta irá demorar a ter alguma ressonância no tratamento de documentos considerados permanentes recolhidos a instituições de guarda. Sobre os diferentes tipos de instrumentos de pesquisa, Bellotto (2004) apontou que estes serviriam para apresentar a instituição e os arquivos guardados por ela em diferentes níveis. Bellotto (2004, p. 180) apresenta os guias como um panorama de todos os arquivos guardados por uma instituição, enquanto os inventários e catálogos seriam mais específicos na delimitação do universo informacional descrito. Sobre os guias, Bellotto destaca a popularidade destes perante o público, visto que adotam uma linguagem menos técnica e apresentam de modo bastante panorâmico os arquivos (2004, p. 191). Ressaltamos que não iremos nos debruçar sobre o guia de arquivos ofertado pelo Arquivo Nacional em seu website12, uma vez que o objetivo de apresentar os Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 104 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera instrumentos de pesquisa neste capítulo refere-se ao aspecto qualitativo de nossa pesquisa no fundo da DCDP. instrumentos de pesquisa neste capítulo refere-se ao aspecto qualitativo de nossa pesquisa no fundo da DCDP. Já o inventário é definido pela autora como “o instrumento de pesquisa que descreve conjuntos documentais ou partes do fundo. É um instrumento do tipo parcial, trazendo descrição sumária e não analítica, esta, própria do catálogo” (BELLOTTO, 2004, p. 197). Por sua vez, o catálogo serviria para descrever os documentos de uma ou várias séries unitariamente (2004, p. 2 Análise do quadro de arranjo e dos instrumentos de pesquisa da DCDP/Arquivo Nacional 202). A esses dois últimos tipos de instrumentos de pesquisa, dirigimos nossa análise. Iniciamos com o instrumento de pesquisa referente à seção Administração Geral Consistindo em um catálogo de 39 páginas que contém os itens documentais referentes a essa seção, é possível verificar que há uma descrição qualitativa em torno do tipo do documento (e, em alguns casos, a descrição é mais minuciosa quanto ao conteúdo do documento), a notação do mesmo no arquivo, a data, a caixa onde se encontra e o número de páginas que possui. Trata-se do maior nível de descrição encontrado no tratamento do fundo DCDP que não será repetido em outras séries e subséries, provavelmente em razão de ser um conjunto documental com menos itens que as séries representativas das atividades-fim do órgão. Ainda, é possível apontar que esta série é relevante para a compreensão do fundo DCDP em sua integralidade, uma vez que reúne documentos que se ligam a outras séries e também outros documentos a respeito da relação entre o órgão, a Polícia Federal e o Ministério da Justiça, superiores hierarquicamente a ela. Desse modo, a escolha pela descrição em um nível mais detalhado mostra-se correta, pois ajuda o usuário externo na compreensão de como o fundo foi organizado e se encontra disposto. Por sua vez, o instrumento de pesquisa da subsérie filmes, da série Cinema, apresenta somente o título de cada obra avaliada pela DCDP, o diretor, o ano em que a obra foi submetida à apreciação e a caixa guardada, não havendo nenhuma descrição qualitativa em torno do Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 105 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera conteúdo dos processos administrativos, conforme podemos conferir abaixo: conteúdo dos processos administrativos, conforme podemos conferir abaixo: conteúdo dos processos administrativos, conforme podemos conferir abaixo: Através desse instrumento, que também é um catálogo de 651 páginas, é possível verificar que a ordem de guarda dos processos administrativos referentes às obras cinematográficas deu-se de modo aleatório, sem respeitar nenhum tipo de critério cronológico, alfabético ou mesmo de entrada dos filmes para a avaliação da DCDP. Este padrão repete-se nos instrumentos de pesquisa – basicamente, catálogos – das subséries de peças teatrais (com 461 páginas), programas de rádio (com 2 páginas) e de letras musicais (com 455 páginas). Uma organização bastante similar é seguida no instrumento de pesquisa da subsérie programação, um catálogo com 5 páginas, da mesma série Cinema: Aqui, também há o número da caixa, a data do documento e uma breve descrição da sua tipologia. Apenas ressaltamos a diferença de que este instrumento segue uma ordem cronológica da produção documental, organizando os documentos entre 1978 e 1988. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 106 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Pedro Vinicius Asterito Lapera arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia No caso dos instrumentos de pesquisa referentes à série televisão, as informações são ainda mais escassas. No caso da subsérie telenovela, um catálogo com 36 páginas, reproduzimos este trecho: No caso dos instrumentos de pesquisa referentes à série televisão, as informações são ainda mais escassas. No caso da subsérie telenovela, um catálogo com 36 páginas, reproduzimos este trecho: Podemos verificar que há menção somente ao título, ao ano de apreciação pela DCDP e à caixa, não havendo menção a seus autores, diretores, gênero, faixa de horário nem emissora que veiculou a telenovela. E o mesmo acontece no instrumento de pesquisa referente a outros programas de TV (talk shows, seriados, programas de humor, desenhos animados, programas jornalísticos, infantis, etc.), também um catálogo com 73 páginas. 13 É importante ressaltar que o Arquivo Nacional oferta aos usuários a possibilidade de acessar os processos administrativos do fundo DCDP – parcial ou totalmente – através de requisição, presencialmente ou por e-mail, serviço ao qual este pesquisador recorreu em algumas vezes, tendo sido atendido com a devida presteza. conteúdo dos processos administrativos, conforme podemos conferir abaixo: Em comum, as seções Censura Prévia, Coordenação e Controle e Orientação apresentam uma descrição com elementos bem básicos referentes aos processos administrativos de avaliação das obras submetidas à DCDP (série Censura Prévia) ou mesmo se atendo à função exercida pelas seções do órgão extinto. A título de exemplo, não há referência aos documentos contidos nesses processos, tais como pareceres, ofícios, cartas, recursos, documentos referentes ao trâmite processual, além dos materiais das obras avaliadas – roteiros, textos de peças, scripts de telenovelas, sinopses, fichas técnicas, partituras, letras de música, dentre outros13. Possivelmente, isso ocorreu em razão de o volume de documentos dessas séries ser muito maior, o que impediu uma descrição retrospectiva mais minuciosa. Finalmente, aproveitamos para apresentar também a descrição do fundo DCDP disponível na base SIAN do Arquivo Nacional. O referido fundo e cada uma de suas seções possuem uma descrição geral de acordo 13 É importante ressaltar que o Arquivo Nacional oferta aos usuários a possibilidade de acessar os processos administrativos do fundo DCDP – parcial ou totalmente – através de requisição, presencialmente ou por e-mail, serviço ao qual este pesquisador recorreu em algumas vezes, tendo sido atendido com a devida presteza. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 107 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera com a norma ISAD(G), que preconiza a elaboração de campos como áreas de identificação, de contextualização, de conteúdo e de estrutura, de condições de acesso e de uso, de fontes relacionadas e de notas e de controle (Bellotto, 2004, p. 182-183), dentro do seu objetivo de “estabelecer diretrizes gerais para a preparação de descrições arquivísticas, podendo ser usada juntamente com as normativas nacionais dos vários países-membros do CIA [Conselho Internacional de Arquivos]” (BELLOTTO, 2004, p. 182). com a norma ISAD(G), que preconiza a elaboração de campos como áreas de identificação, de contextualização, de conteúdo e de estrutura, de condições de acesso e de uso, de fontes relacionadas e de notas e de controle (Bellotto, 2004, p. 182-183), dentro do seu objetivo de “estabelecer diretrizes gerais para a preparação de descrições arquivísticas, podendo ser usada juntamente com as normativas nacionais dos vários países-membros do CIA [Conselho Internacional de Arquivos]” (BELLOTTO, 2004, p. 182). 14 Acesso em 27 jul. 2021. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 15 Em razão da extensão do artigo, não reproduzimos mais imagens extraídas da base SIAN sobre a descrição do fundo DCDP nem de suas seções. Por ocasião do acesso em 27/9/2021, foi possível verificar o preenchimento dos campos requeridos pela norma ISAD(G) quanto à descrição, sem repetições desnecessárias. Destacamos também que a parte dos documentos audiovisuais do fundo DCDP também se encontra descrita na mesma base. conteúdo dos processos administrativos, conforme podemos conferir abaixo: Reproduzimos abaixo duas imagens extraídas da base SIAN14 referentes à descrição do fundo DCDP, mais precisamente quanto à sua identificação e contextualização. 108 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Pedro Vinicius Asterito Lapera j p q Censura de Diversões Públicas à Luz da Arquivologia É possível deduzir dessa descrição a preocupação da instituição quanto à recuperação da história do fundo DCDP tanto em relação à sua função principal (censura) quanto à sua inserção na estrutura jurídico- administrativa do Estado brasileiro (órgãos a que a DCDP era subordinada, transformações anteriores à existência do órgão quanto à atividade de censura), além de outros dados referentes ao período englobado, gênero da documentação (textual) e metragem, cumprindo as normas ISAD(G) e NOBRADE quanto à descrição arquivística15. Ainda, é possível fazer considerações sobre as dificuldades operacionais de se descrever qualitativamente o fundo da DCDP, tais como a necessidade de a instituição de guarda ter um ritmo de trabalho que permita acolher e tratar vários fundos de diferentes órgãos federais, a restrição de tempo e de profissionais para a realização da descrição arquivística. Mesmo assim, os instrumentos de pesquisa mostram-se adequados à recuperação da informação custodiada pelo fundo analisado ao longo deste artigo. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 109 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Pedro Vinicius Asterito Lapera j p q Censura de Diversões Públicas à Luz da Arquivologia 3 Considerações finais 92-112, dez/2021 Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia Pedro Vinicius Asterito Lapera em sua totalidade. Ainda, a adaptação na descrição da seção referente a Censura Prévia mostrou-se eficaz na recuperação das informações, na medida em que é possível localizar os processos referentes às obras musicais, cinematográficas, teatrais, televisivas etc. a partir de seus nomes. em sua totalidade. Ainda, a adaptação na descrição da seção referente a Censura Prévia mostrou-se eficaz na recuperação das informações, na medida em que é possível localizar os processos referentes às obras musicais, cinematográficas, teatrais, televisivas etc. a partir de seus nomes. Finalmente, precisamos reconhecer que a retomada da discussão sobre censura é sempre algo bem doloroso. Implica destacar as perdas e as baixas provocadas por um autoritarismo que impunha sua vontade de modo ilegítimo. Pelas ironias de um tempo histórico bastante conturbado, este trabalho começou com o objetivo de recuperar alguns pontos do debate sobre arquivos e censura e, a partir dele, a análise dos instrumentos de pesquisa do arquivo da DCDP, dentro do esforço de situar essa atividade censória como uma marca de origem do nosso regime republicano. Infelizmente, o autoritarismo presente durante toda a vida da nossa república (com “r” minúsculo, visto que ela historicamente representou muito poucos) – em alguns momentos de modo mais recalcado – retornou à esfera pública com tanta força que revela a atualidade de nossa empreitada. 3 Considerações finais Tentamos pensar o fundo da DCDP diante dos desafios do cenário arquivístico brasileiro, visando reposicionar algumas questões teóricas da Arquivologia à luz das suas complexidades e dificuldades. Ainda, pretendemos contribuir para um conhecimento arquivístico que incorpore as limitações materiais, históricas, estruturais, culturais e de gestão dos arquivos e dos documentos. Isto não significa abrir mão dos debates sobre melhorias nos diversos âmbitos da área, mas reconhecer que o cenário adverso representa um desafio a concepções ideais de arquivo e de tratamento documental e, simultaneamente, a possibilidade de construir outros caminhos teóricos que valorizem o enfrentamento das adversidades pelos arquivistas no seu cotidiano profissional. Ressaltamos a necessidade de estabelecer um diálogo horizontal com o debate arquivístico de outros países. Reconhecer os ganhos desse debate implica posicionar autores, teorias e textos em realidades arquivísticas historicamente condicionadas e não como pensadores a serem acriticamente reproduzidos como portadores de uma “visão ideal” de Arquivologia. E até que ponto reside a importância do estabelecimento de normas gerais no tratamento arquivístico? Se em alguns cenários institucionais, é difícil estabelecer uma correspondência entre essas normas e as práticas de tratamento documental, isso não pode ser negligenciado como um dado limitador da generalização pretendida. Fazer isso é penalizar ainda mais os sujeitos imersos em contextos arquivísticos cotidianamente sacrificados com cortes orçamentários, instáveis política e administrativamente e menos reconhecidos socialmente, a saber: fora dos países do Primeiro Mundo. A análise dos instrumentos de pesquisa também precisa se mostrar sensível a esses fatores e reconhecer as adaptações e os esforços feitos pelos arquivistas para disponibilizar essas informações aos usuários externos de modo mais rápido e eficaz. Neste artigo, tivemos a intenção de analisar desses instrumentos de pesquisa criados pelo Arquivo Nacional para que o usuário externo pudesse acessar as informações contidas nos documentos custodiados pelo fundo da DCDP e também as informações sobre a própria história do fundo e de sua instituição geradora. Tal objetivo não teria sido possível sem o trabalho dos funcionários do Arquivo Nacional que, diante dos limites impostos ao fazer arquivístico, conseguiram criar esses instrumentos. Em linhas gerais, agiram acertadamente ao delimitar o fundo da DCDP e, dentro do volume documental apresentado por ele, ao eleger a seção Administração Geral com o nível mais detalhado de descrição, pelo fato de ali estarem os documentos que evidenciam a estrutura do fundo 110 Perspectivas em Ciência da Informação, v.26, número 4, p. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 Referências BELLOTTO, H. L. Arquivos permanentes: tratamento documental. Rio de Janeiro: FGV, 2004. BELLOTTO, H. L. Arquivo: estudos e reflexões. Belo Horizonte: UFMG, 2014. COOK, T. O conceito de fundo arquivístico: teoria, descrição e proveniência na era pós-custodial. Rio de Janeiro: Arquivo Nacional, 2017. DUCHEIN, M. O respeito aos fundos em Arquivística: princípios teóricos e problemas práticos. In: Rio de Janeiro, Arquivo e Administração, v.10-14, n. 2, 1982-1986. DURANTI, L. Archives as a place. Archives & Social Studies: a Journal of Interdisciplinary Research, v. 1, March 2007, p. 445-466. FICO, C. Reinventando o otimismo: ditadura, propaganda e imaginário social no Brasil. Rio de Janeiro: FGV, 2001. FIGUEIREDO, L. Lugar nenhum: militares e civis na ocultação dos documentos da ditadura. São Paulo: Cia. das Letras, 20015. KUSHNIR, B. Cães de guarda: jornalistas e censores, do AI-5 à Constituição de 88. São Paulo: Boitempo, 2004. Perspectivas em Ciência da Informação, v.26, número 4, p. 92-112, dez/2021 111 Pedro Vinicius Asterito Lapera Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Do ocaso à revelação: uma breve análise do quadro de arranjo e dos instrumentos de pesquisa da Divisão de Censura de Diversões Públicas à Luz da Arquivologia j p q Censura de Diversões Públicas à Luz da Arquivologia j p q Censura de Diversões Públicas à Luz da Arquivologia LAPERA, P. V. A. Entre brechas, cortes e rasuras: relações étnico-raciais e censura cinematográfica na ditadura militar. Famecos, Porto Alegre, v.22, n. 2, abr/jun 2015, p. 82-98. LAPERA, P. V. A. Entre brechas, cortes e rasuras: relações étnico-raciais e censura cinematográfica na ditadura militar. Famecos, Porto Alegre, v.22, n. 2, abr/jun 2015, p. 82-98. RIDENTI, M. Em busca do povo brasileiro: artistas da revolução, do CPC à era da TV. Rio de Janeiro, São Paulo: Record, 2000. SIMÕES, I. Roteiro da intolerância: a censura cinematográfica no Brasil. São Paulo: SENAC, 1999. YEO, G. Debates em torno da descrição.In: EASTWOOD, T.; MACNEIL, H. Correntes atuais do pensamento arquivístico. Belo Horizonte: Ed. UFMG, 2018 p 135 169 RIDENTI, M. Em busca do povo brasileiro: artistas da revolução, do CPC à era da TV. Rio de Janeiro, São Paulo: Record, 2000. RIDENTI, M. Em busca do povo brasileiro: artistas da revolução, do CPC à era da TV. Referências Rio de Janeiro, São Paulo: Record, 2000. SIMÕES, I. Roteiro da intolerância: a censura cinematográfica no Brasil. São Paulo: SENAC, 1999. SIMÕES, I. Roteiro da intolerância: a censura cinematográfica no Brasil. São Paulo: SENAC, 1999. YEO, G. Debates em torno da descrição.In: EASTWOOD, T.; MACNEIL, H. Correntes atuais do pensamento arquivístico. Belo Horizonte: Ed. UFMG, 2018, p. 135-169. YEO, G. 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https://openalex.org/W4249990614	https://ojs.unud.ac.id/index.php/beta/article/download/42466/26057	Indonesian	null	Analisis Teknis Penggunaan Sumber Daya Air Tanah Untuk Irigasi Tanaman Padi di Kabupaten Jembrana	BETA (Biosistem dan Teknik Pertanian)	2,018	cc-by	5,282	JURNAL BETA (BIOSISTEM DAN TEKNIK PERTANIAN Program Studi Teknik Pertanian, Fakultas Teknologi Pertanian Universitas Udayana http://ojs.unud.ac.id/index.php/beta Volume 6, Nomor 2, September 2018 JURNAL BETA (BIOSISTEM DAN TEKNIK PERTANIAN Program Studi Teknik Pertanian, Fakultas Teknologi Pertanian Universitas Udayana http://ojs.unud.ac.id/index.php/beta Volume 6, Nomor 2, September 2018 Analysis Technical of the Use of Ground Water Resources for Rice Plantation Irrigation in Jembrana Regency Angga Hendrayana S1, Sumiyati1, Ida Ayu Gede Bintang Madrini1 1 Program Studi Teknik Pertanian, Fakultas Teknologi Pertanian, Unud Email : anggahendrayana@gmail.com Abstrak Air tanah merupakan salah satu sumber daya air yang dapat digunakan untuk memenuhi kebutuhan irigasi. Dengan keterbatasan ketersediaan air, maka diupayakan dengan memanfaatkan air tanah dengan menggunakan sumur pompa. Penggunaan sumur pompa belum maksimal dalam memenuhi kebutuhan air irigasi maka diperlukan penelitian lebih lanjut. Tujuan dari penelitian ini adalah : (1) Mengetahui teknis penggunaan sumber daya air tanah untuk irigasi menggunakan sumur pompa, dan (2) Menganalisis kemampuan sumur pompa pada subak sampel di Kabupaten Jembrana terhadap kebutuhan air irigasi. Penelitian ini menggunakan analisis deskriptif melalui metode survei, pengambilan data menggunakan data primer dan sekunder. Analisis debit tersedia diperoleh melalui metode survei langsung kelapangan dan analisis kebutuhan air irigasi dilakukan dengan bantuan software CROPWAT 8.0. Berdasarkan analisis neraca air irigasi dilakukan pengurangan antara kebutuhan air irigasi dengan ketersediaan air irigasi terhadap 3 sampel subak. Pada Subak Sari Merta terjadi kekurangan air pada selama 2 periode yaitu periode Juni I sebesar 171.900 l/hari, Januari I 149.700 l/hari. Pada Subak Brawantangi terjadi kekurangan air sebanyak 4 periode yaitu periode Agustus I sebesar 123.900 l/hari, Agustus II 99.200 l/hari, Maret I 98.000 l/hari, Maret II 78.200. Dan pada Subak Tegal Badeng terjadi kekurangan air sebanyak 8 periode yaitu periode Juni II sebesar 64.600 l/hari, Juni III 178.500 l/hari, Agustus III 35.600 l/hari, September I 35.100 l/hari, September II 38.400 l/hari, September III 32.900 l/hari, November I 48.400 l/hari, November III 149.700 l/hari. Defisit air terjadi karena pada periode bulan tersebut dilakukan pengolahan tanah sehingga kebutuhan air irigasi cenderung tinggi. Untuk dapat memenuhi kebutuhan air irigasi yang diperlukan untuk pengolahan tanah, pada Subak Sari merta dilakukan sistem giliran memakai air irigasi supaya aktifitas pertanian dapat berjalan baik. Kata Kunci: Sumur Pompa, Air Tanah, Kebutuhan Air, Ketersediaan Air, Irigasi. Abstract The Ground water is one of water resources that can be used to fullfill the needs of irrigation. With the limited availability of water, it is attempted by utilizing ground water using pump wells. The use of pump wells has not been maximal in fulfilling the irrigation water requirement so further research is needed. The objectives of this research are: (1) To know the technical use of groundwater resources for irrigation using pump wells, and (2) to analyze the ability of pump wells in subak samples in Jembrana Regency to irrigation water needs. This research uses descriptive analysis through survey method, taking data using primary and secondary data. Available discharge analysis is obtained through direct survey method of spaciousness and analysis of irrigation water needs carried out with the help of CROPWAT 8.0 software. Based on the irrigation water balance analysis, there is a reduction between the irrigation water demand and the availability of irrigation water to 3 subak samples. In Subak Sari Merta it is known that there is water shortage 2 periods that is the period of June I equal to 171.900 l / day, January I 149.700 l / day. Subak Brawantangi is known that there is water shortage as much as 4 periods that is the period of August I equal to 123.900 l / day , August II 99.200 l / day, March I 98,200 l / day, March II 78.200 l / day. Subak Tegal Badeng is known that there is water shortage for 8 periods that is June II period is 64.000 l / day, June III 178.500 l / day, August III 35.600, September I 35.100 l/day, September II 38.400 l/day, September III 32.900 l/ day, November I 48,400 l / day, November III 149.700 l / day. Water deficit occurs because in the period of the month is done so that the processing of irrigation water needs tend to be high. To be able to meet the needs of irrigation water needed for the processing of the soil, the Subak Sari is done by the turn system using irrigation water so that agricultural activities can run well. 98 Keywords: Pump Well, Ground Water, Water Requirement, Water Availability, Irrigation. Keywords: Pump Well, Ground Water, Water Requirement, Water Availability, Irrigation. PENDAHULUAN Untuk memenuhi kebutuhan air baku pada daerah yang mengalami kekurangan air dalam rangka memenuhi kebutuhan masyarakat, mendorong pertumbuhan pertanian, perkebunan diperlukan pembangunan/pembuatan sumur bor termasuk jaringannya oleh pemerintah. PENDAHULUAN Budidaya tanaman padi tidak bergantung pada musim, dimana padi dapat ditanam di musim kemarau atau hujan. Pada musim kemarau produksi dapat meningkat apabila air selalu tersedia. Namun di daerah Kebupaten Jembrana pada musim kemarau banyak daerah yang tidak tercukupi oleh suplai dari irigasi permukaan, maka diperlukan pembangunan sumber daya air tanah. Budidaya tanaman padi tidak bergantung pada musim, dimana padi dapat ditanam di musim kemarau atau hujan. Pada musim kemarau produksi dapat meningkat apabila air selalu tersedia. Namun di daerah Kebupaten Jembrana pada musim kemarau banyak daerah yang tidak tercukupi oleh suplai dari irigasi permukaan, maka diperlukan pembangunan sumber daya air tanah. Kebutuhan air baku dari tahun ketahun terus meningkat seiring dengan pertumbuhan penduduk, ekonomi, pertanian, perkebunan dan peternakan yang semakin banyak membutuhkan air baku. Sedangkan ketersediaan air permukaan semakin menurun akibat dari penggundulan hutan, alih fungsi lahan, pemanasan global dan lain-lain. Alternatif untuk memenuhi kebutuhan air bagi daerah yang mengalami kekeringan dapat dilakukan dengan memanfaatkan sumber air dalam tanah (Soemarwoto,1991). Terdapat 128 sumur di Kabupaten Jembrana yang telah dibangun oleh Pendayagunaan Air Tanah Pelaksanaan Jaringan Pemanfaatan Air Balai Wilayah Sungai (BWS) Bali – Penida dan dioperasikan 92 buah, paling banyak jika dibandingkan daerah lainnya. Sumur pompa yang ada di Kabupaten Jembrana dimaksudkan untuk membantu petani lokal yang tinggal atau mempunyai areal pertanian di daerah yang tidak tercakup dalam sistem irigasi permukaan atau tidak dapat bercocok tanam sepanjang tahun. Air tanah merupakan salah satu sumber daya air, selain air sungai dan air hujan.Air tanah juga memiliki peranan penting terutama menjaga keseimbangan dan ketersediaan air, dalam hal ini air tanah memberikan kontribusi yang besar bagi peningkatan produksi pertanian, terutama pada musim kemarau. Maka pengembangan air tanah tetap menjadi salah satu solusi untuk membantu para petani lokal mewujudkan pertanian sepanjang tahun dengan memanfaatkan sumber daya air tanah untuk irigasi (Asdak,2002). Penggunaan sumur pompa dalam menjaga umur pakai pompa dalam penggunaannya dibatasi waktu kerja mesin, akibatnya petani pada proses olah tanah musim tanam masih bergilir penggunaanya. Selain itu, pemberian air pada lahan petani masih menggunakan teknik perkiraan. Sehingga memungkin lahan petani mendapatkan kelebihan air irgasi. Kemudian kelebihan tersebut akan terbuang dengan percuma. Dari permasalahan diatas maka diperlukan studi yang mengkaji mengenai ”Analisis Teknis Penggunaan Sumber Daya Air Tanah Untuk Irigasi Tanaman Padi di Kabupaten Jembrana” Potensi air dalam tanah untuk kebutuhan hidup dan bahkan dapat dikembangkan untuk memenuhi kebutuhan pertanian, perkebunan dan peternakan yang merupakan mata pencaharian sebagian masyarakat di Bali.. METODE j g y p Kabupaten Jembrana memiliki potensi air yang meliputi air tanah, air permukaan dan mata air. Diantara sumber daya air tersebut, di Kabupaten Jembrana potensi sumber daya air yang paling melimpah adalah air tanah. Potensi air tanah di Kabupaten Jembrana adalah sebesar 10,85 m3/dt (Anonim, 2014).Dengan keterbatasan ketersediaan air, maka diupayakan dengan memanfaatkan air tanah dengan cara membuat sumur pompa. Pemanfaatan air tanah dapat digunakan sebagai air irigasi pada daerah kekurangan air, dimana air permukaan tidak memadai atau tidak ada sama sekali sedangkan daerah tersebut memiliki potensi pertanian. Salah satu tanaman yang memiliki potensi dalam bidang pertanian adalah tanaman padi. Tanaman padi merupakan tanaman yang memerlukan air untuk masa pertumbuhannya. Tempat dan Waktu Penelitian Tempat dan waktu penelitian ini dilaksanakan pada 3 subak sampel yang ada di daerah Kabupaten Jembran, yaitu Subak Sari Merta, Subak Brawantangi, Subak Tegal Badeng. Waktu pelaksanaan penelitian ini yakni dilaksanakan mulai dari bulan September sampai dengan bulan November 2017. HASIL DAN PEMBAHASAN c. Jadwal tanam, setelah didapatkan evapotraspirasi potensial dan curah hujan efektif selanjunya dimasukan data jadwal tanam pada 3 sampel Subak, yaitu Subak Sari Merta, Subak Brawantangi, Subak Tegal badeng. Data tanaman meliputi sifat dari tanaman yang tergantung dari koefisien tanaman (Kc) dan lama pertumbuhan yang tergantung dari jenis tanaman dan fase pertumbuhan. Dari data tanaman ini dapat dihitung kebutuhan air untuk tanaman, dengan menambahkan data tanggal tanam, maka kebutuhan air irigasi untuk tanaman dapat ditentukan. Analisis Kecukupan Sumur Pompa Analisis Kecukupan Sumur Pompa Analisis kecukupuan sumur pompa menggunakan hasil perhitungan dari neraca air irigasi. Hasil dari neraca air, apabila ketersediaan memenuhi kebutuhan air irigasi maka kebutuhan air irigasi tersebut tercukupi. Namun ketersediaan air kurang untuk memenuhi kebutuhan air irigasi maka diperlukan perhitungan untuk mengetahui seberapa besar kekurangan air yang dibutuhkan di subak tersebut serta berapa diperlukan tambahan pompa untuk memenuhi kekurangan air irigasi tersebut. g g a. Evapotraspirasi Potensial (Eto), dalam perhitungan evapotranspirasi potensial menggunakan persamaan Penman dengan bantuan sofware CROPWAT 8.0, menggunakan data klimatologi yang terdiri dari : temperatur, kelembaban, presentase lama penyinaran matahari dan kecepatan angin. Data klimatologi yang dimaksudkan yaitu data rata-rata lima tahun terakhir dari tahun 2012 sampai dengan tahun 2016. Data iklim yang diambil dari dari Balai Besar Meteorologi Klimatologi dan Geofisika Stasiun Klimatologi Jembrana-Bali. g g a. Evapotraspirasi Potensial (Eto), dalam perhitungan evapotranspirasi potensial menggunakan persamaan Penman dengan bantuan sofware CROPWAT 8.0, menggunakan data klimatologi yang terdiri dari : temperatur, kelembaban, presentase lama penyinaran matahari dan kecepatan angin. Data klimatologi yang dimaksudkan yaitu data rata-rata lima tahun terakhir dari tahun 2012 sampai dengan tahun 2016. Data iklim yang diambil dari dari Balai Besar Meteorologi Klimatologi dan Geofisika Stasiun Klimatologi Jembrana-Bali. g g a. Evapotraspirasi Potensial (Eto), dalam perhitungan evapotranspirasi potensial menggunakan persamaan Penman dengan bantuan sofware CROPWAT 8.0, menggunakan data klimatologi yang terdiri dari : temperatur, kelembaban, presentase lama penyinaran matahari dan kecepatan angin. Data klimatologi yang dimaksudkan yaitu data rata-rata lima tahun terakhir dari tahun 2012 sampai dengan tahun 2016. Data iklim yang diambil dari dari Balai Besar Meteorologi Klimatologi dan Geofisika Stasiun Klimatologi Jembrana-Bali. Metode Analisis Dengan penelitian ini digunakan metode analisis deskriptif untuk mengetahui a) teknis penggunaan sumber daya air tanah, b) kecukupan pompa untuk irigasi. b. Curah hujan efektif, perhitungan curah hujan efektif dilakukan dengan bantuan sofware CROPWAT 8.0 yang menggunakan rumus Depandable rain (FAO/AGLW formula). Kebutuhan Air Irigasi Kebutuhan air sawah untuk padi ditentukan oleh faktor-faktor sebagai berikut: penyiapan lahan, penggunaan konsumtif, perkolasi,dan curah hujan efektif (Purwanto dkk, 2006). Perhitungan kebutuhan air irigasi dilakukan dengan bantuan sofware CROPWAT 8.0, tahapan dan cara kerja yang dilaksanakan untuk menentukan kebutuhan air irigasi adalah sebagai berikut. Jenis Data dan Variabel Penelitian Jenis Data dan Variabel Penelitian Data yang didapatkan oleh peneliti pada saat pelaksanaan penelitian menggunakaan 2 jenis data, yaitu data primer dan sekunder. Dalam penelitian ini ada beberapa variabel yang diamati yaitu: Spesifikasi Wilayah, Ketersediaan Air/Kemampuan Sumur Pompa, Kebutuhan Air Irigasi, Neraca Air Irigasi dan Analisis Kecukupan Pompa 99 saling mempengaruhi satu sama lain dalam pertumbuhan tanaman. Ketersediaan Air dan Kemampuan Pompa Ketersediaan Air dan Kemampuan Pompa Ketersediaan Air dan Kemampuan Pompa Untuk mengetahui ketersediaan air di ketiga subak tersebut. Metode yang digunakan pada tahapan ini adalah metode wawancara dan pengamatan secara langsung. Dengan metode tersebut akan diketahui data-data yang diperlukan. Data-data yang diperlukan dalam tahapan ini adalah jumlah pompa, jenis pompa, daya pompa, dan debit yang dapat dihasilkan pompa. e. Kebutuhan Air Irigasi, setelah didapatkan evapotraspirasi potensial, curah hujan efektif dan jadwal tanam selanjutnya dapat ditentukan kebutuhan air irigasi yang sesuai dengan jadwal tanam yang diterapkan pada 3 sampel subak. e. Kebutuhan Air Irigasi, setelah didapatkan evapotraspirasi potensial, curah hujan efektif dan jadwal tanam selanjutnya dapat ditentukan kebutuhan air irigasi yang sesuai dengan jadwal tanam yang diterapkan pada 3 sampel subak. Neraca air irigasi Neraca air irigasi dapat dihitungan dengan persamaan berikut: NAI = KA – KAI Dimana : NAI = neraca air irigasi KA = ketersediaan air KAI = kebutuhan air irigasi Spesifikasi Wilayah Setiap wilayah memiliki spesifikasi yang berbeda- beda karena dipengaruhi oleh iklim dan keadaan geologi. Di daerah Bali memiliki potensi air tanah yang bisa memenuhi kekurangan kebutuhan sumber daya air khususnya Kabupaten Jembrana. Air tanah banyak digunakan pada daerah persediaan air sangat terbatas dan tidak merata. Penelitian dilakukan di 3 sampel subak di Kabupaten Jembrana, yaitu Subak Sari Merta, Subak Tegal Badeng, dan Subak Brawantangi Dengan luas lahan Subak Sari Merta 32 ha, Subak Brawantangi 125 ha, Subak Tegal Badeng 165 ha. Subak Sari Merta yaitu memiliki 32 anggota dengan 5 kelompok, subak Brawantangi memiliki 186 anggota dengan 11 d. Jenis tanah, tanah sebagai media tumbuh tanaman yang memiliki sifat dan karakteristik yang dapat dilihat dari sifat fisik, kimiawi, maupun bioloogisnya dimana ketiganya berintegrasi dan 100 Perhitungan Evapotranspirasi Potensial Bulan Temp (OC) Hum (%) Wind (km/ hari) Sun (jam) Eto (mm/ day) Januari 27.1 83 58 4.4 3.57 Februari 26.7 84 34 4.7 3.59 Maret 27.1 83 48 5.4 3.72 April 27.0 84 48 5.4 3.45 Mei 26.5 85 38 5.9 3.18 Juni 25.9 84 48 5.7 2.93 Juli 25.0 83 67 5.7 2.96 Agustus 24.7 82 72 6.3 3.33 September 25.5 80 80 7.0 3.89 Oktober 26.9 80 72 6.9 4.18 November 27.7 81 62 5.8 4.00 Desember 27.3 84 48 4.1 3.46 Average 26.4 83 56 5.6 3,52 kelompok, dan Subak Tegal Badeng memiliki 130 anggota dengan 8 kelompok. memiliki 130 Perhitungan Evapotranspirasi Potensial Perhitungan Evapotranspirasi Potensial Teknis Penggunaan Sumber Daya Air Tanah Teknis Penggunaan Sumber Daya Air Tanah gg y Penggunaan sumber daya air tanah diperlukan upaya pengambilan / pengangkatan air dalam tanah kepermukaan tanah. Pengangkatan air menggunakan bantuan pompa. Pompa air digerakan dengan motor penggerak berbahan bakar solar, bensin, listrik atau tenaga angin. Dari beberapa sumur pompa kebanyakan semuanya menggunakan mesin diesel sebagai penggerak. Hal ini karena penggunaan genset/ mesin diesel lebih menguntungkan dibandingkan menggunakan energi listrik (PLN) karena petani tidak perlu mengeluarkan biaya rutin bulanan untuk membayar beban gardu listrik meskipun mesin tidak beroperasi. Tahapan selanjutnnya setelah didapatkan hasil dari evapotranspirasi potensial selanjutnya dilakukan perhitungan untuk menentukan besarnya curah hujan efektif yang dapat dimanfaatkan oleh tanaman. Perhitungan curah hujan efektif dilakukan dengan menggunakan rumus Dependable rain (FAO/AGLW formula) dengan bantuan software CROPWAT 8.0. Dalam perhitungan curah hujan efektif, data yang digunakan adalah data rata-rata lima tahun terakhir dari tahun 2012 sampai dengan tahun 2016 yang bersumber dari stasiun hujan yang berlokasi di Kabupaten Jembrana, dapat dilihat pada Tabel 2. p p Untuk mengalirkan air dari pompa ke lahan petani, maka perlu dibangun jaringan irigasi air tanah (JIAT). JIAT sumur pompa adalah suatu sistem penyediaaan air irigasi yang bersumber dari air tanah, diekploitasi melalui pembangunan sumur bor dengan kedalaman tertentu, kemudian airnya didistribusikan melalui jaringan perpipaan dengan bantuan pompa submersible. Operasi JIAT Sumur Pompa merupakan tata cara pengoprasian pompa untuk mengalirkan air dari sumber air tanah sumur bor ke petak-petak tanaman melalui jaringan perpipaan. Setelah itu air ditampung dalam box pembagi untuk disalurkan ke petak-petak sawah sesuai dengan luasan tiap arealnya melalui saluran terbuka/ salurak kecil. Sehingga air irigasi dapat dimanfaatkan pada petak pemanfaatan atau petak tanaman. Tabel 2 Perhitungan Curah Hujan Efektif Bulan CHtota l(mm/bulan) CHefektif (mm/bulan) Januari 262.5 186.0 Februari 413.7 307.0 Maret 170.2 112.2 April 129.5 79.6 Mei 129.6 79.7 Juni 94.5 51.6 Juli 96.7 53.4 Agustus 42.1 15.3 September 33.8 10.3 Oktober 105.9 60.7 November 128.7 79.0 Desember 304.3 219.4 Total 1911.5 1254.0 Tabel 2 Perhitungan Curah Hujan Efektif Kebutuhan Air Irigasi. Data jadwal tanam padi yang diterapkan pada ke 3 Subak sampel dapat dilihat pada masa tanam subak Sari Merta yang dimulai pada tanggal 10 Juni dan panen tanggal 07 Oktober, Subak Brawantangi yang dimulai pada tanggal 14 Agustus dan panen tanggal 11 Desember, Subak Tegal Badeng yang dimulai pada tanggal 01 Juli dan panen tanggal 28 Oktober sehingga umur padi dari mulai penanaman bibit padi sampai dengan panen adalah 120 hari. Setalah penentuan jadwal tanam dilakukan penentuan koefisien tanaman (Kc). Keterangan: Penentuan Kc dilakukan diaplikasi dengan menginput data jadwal tanam dan jenis tanaman. Setelah penentuan Kc, dilakukan penentuan jenis tanah yang terdapat pada 3 subak sampel adalah jenis tanah yang bertekstur lempung (loam). Tahapan selanjutnya setelah dilakukan perhitungan evapotranspirasi potensial, curah hujan efektif, jadwal tanam dan jenis tanah adalah ditentukan kebutuhan air irigasi yang sesuai dengan jadwal tanam yang diterapkan pada 3 Subak yaitu Subak Sari Merta, Subak Brawantangi, Subak Tegal Badeng. Hasil analisis dapat dilihat pada Gambar 1. Analisis kebutuhan air irigasi merupakan salah satu tahap penting yang diperlukan dalam perencanaan dan pengelolaan sistem irigasi. Kebutuhan air tanaman didefinisikan sebagai jumlah air yang dibutuhkan oleh tanaman pada suatu periode untuk dapat tumbuh dan produksi secara normal. Kebutuhan air nyata untuk areal usaha pertanian meliputi evapotranspirasi potensial (ETo), sejumlah air yang dibutuhkan untuk pengoperasian secara khusus seperti penyiapan lahan dan penggantian air, serta kehilangan selama pemakaian (Sudjarwadi, 1990). Tahapan selanjutnya setelah dilakukan perhitungan evapotranspirasi potensial dan curah hujan efektif selanjutkan akan ditentukan data musim tanam padi pada ke 3 sampel subak. Setelah itu dilakukan input jenis tanaman untuk menentukan fase dari jadwal tanam. Data jadwal tanam padi yang diterapkan pada ke 3 Subak sampel dapat dilihat pada masa tanam subak Sari Merta yang dimulai pada tanggal 10 Juni dan panen tanggal 07 Oktober, Subak Brawantangi yang dimulai pada tanggal 14 Agustus dan panen tanggal 11 Desember, Subak Tegal Badeng yang dimulai pada tanggal 01 Juli dan panen tanggal 28 Oktober sehingga umur padi dari mulai penanaman bibit padi sampai dengan panen adalah 120 hari. Setalah penentuan jadwal tanam dilakukan penentuan koefisien tanaman (Kc). Keterangan: Penentuan Kc dilakukan diaplikasi dengan menginput data jadwal tanam dan jenis tanaman. Gambar 1. Kebutuhan Air Irigasi. Grafik kebutuhan air irigasi ketiga subak sampel p Tabel di atas menunjukan jadwal tanam I bahwa kebutuhan air irigasi pada 3 sampel subak saat dilakukan pengolahan tanah yang membutuhkan air irigasi cukup banyak, pada Subak Sari Merta yaitu periode Juni I sebesar 17,19 mm/hari. Sedangkan pada fase pertumbuhan, rata-rata kebutuhan air irigasi berkisar antara 6,13 mm/hari sampai dengan 3,55 mm/hari. Kebutuhan air irigasi yang terkecil yaitu periode Oktober I sebesar 0,94 mm/hari. Pada periode Oktober I memerlukan sedikit air karena tanaman padi sudah mencapai fase generatif di mana tanaman padi sudah berumur 120 hari. Pada Subak Brawantangi yaitu periode Agustus I sebesar 12,39 mm/hari. Sedangkan pada fase pertumbuhan, rata-rata kebutuhan air irigasi berkisar antara 5,86 mm/hari sampai dengan 1,88 mm/hari. Kebutuhan air irigasi yang terkecil yaitu periode Desember II sebesar 0,3 mm/hari. Pada periode Desember II memerlukan sedikit air karena tanaman padi sudah mencapai fase generatif di mana tanaman padi sudah berumur 120 hari. Pada subak Tegal Badeng yaitu periode Juni III sebesar 17,85 mm/hari. Sedangkan pada fase pertumbuhan, rata-rata kebutuhan air irigasi berkisar antara 6,46 mm/hari sampai dengan 2,4 mm/hari s. Kebutuhan air irigasi yang terkecil yaitu periode Oktober III sebesar 0,56 mm/hari. Pada periode Oktober III memerlukan sedikit air karena tanaman padi sudah mencapai fase generatif di mana tanaman padi sudah berumur 120. g p j j Setelah penentuan Kc, dilakukan penentuan jenis tanah yang terdapat pada 3 subak sampel adalah jenis tanah yang bertekstur lempung (loam). Tahapan selanjutnya setelah dilakukan perhitungan evapotranspirasi potensial, curah hujan efektif, jadwal tanam dan jenis tanah adalah ditentukan kebutuhan air irigasi yang sesuai dengan jadwal tanam yang diterapkan pada 3 Subak yaitu Subak Sari Merta, Subak Brawantangi, Subak Tegal Badeng. Hasil analisis dapat dilihat pada Gambar 1. Analisis kebutuhan air irigasi merupakan salah satu tahap penting yang diperlukan dalam perencanaan dan pengelolaan sistem irigasi. Kebutuhan air tanaman didefinisikan sebagai jumlah air yang dibutuhkan oleh tanaman pada suatu periode untuk dapat tumbuh dan produksi secara normal. Kebutuhan air nyata untuk areal usaha pertanian meliputi evapotranspirasi potensial (ETo), sejumlah air yang dibutuhkan untuk pengoperasian secara khusus seperti penyiapan lahan dan penggantian air, serta kehilangan selama pemakaian (Sudjarwadi, 1990). Kebutuhan Air Irigasi. Kebutuhan air irigasi dihitung berdasarkan data klimatologi yang digunakan yaitu data rata-rata lima tahun terakhir dari tahun 2012 sampai dengan tahun 2016 yang diambil dari Balai Besar Meteorologi Klimatologi dan Geofisika Stasiun Klimatologi Jembrana-Bali. Proses perhitungan yang dilakukan dengan menghitung evapotranspirasi potensial dengan menggunakan persamaan Penman dengan bantuan software CROPWAT 8.0 dapat diperoleh data ETo seperti tabel 1. Tabel 1 menunjukan hasil dari perhitungan yang dilakukan untuk menentukan evapotranspirasi potensial dapat diketahui evapotranspirasi potensial yang cukup tinggi terdapat pada bulan Oktober yaitu 4,18 mm/hari.Sedangkan evapotranspirasi potensial yang terkecil yaitu bulan Juni sebesar 2,93 mm/hari. Tabel 1 Data curah hujan efektif yang diambil dari stasiun hujan di Kabupaten Jembrana, dapat diketahui bahwa pada bulan Februari terjadi curah hujan yang cukup tinggi yaitu 307.0 mm/hari, sedangkan pada bulan September terjadi curah hujan yang kecil yaitu 10.3 mm/hari. Pada musim hujan sebagian Tabel 1 101 Gambar 1. Grafik kebutuhan air irigasi ketiga subak sampel besar kebutuhan air dipenuhi oleh hujan, sementara dalam musim kemarau kebutuhan air irigasi yang tidak dapat dipenuhi oleh air hujan dapat dipenuhi oleh air irigasi. Besarnya jumlah air yang bersumber dari curah hujan sulit diperkirakan, karena curah hujan sangat bervariasi setiap tahunnya. Curah hujan yang jatuh tidak semua digunakan oleh tanaman. Sebagian hujan hilang karena limpasan permukaan (run off) atau karena perkolasi. Jumlah curah hujan yang jatuh dan efisien untuk pertumbuhan tanaman tergantung pada intensitas hujan, topografi lahan, sistem penanaman dan fase pertumbuhan (Sari, 2004). g y j y g dari curah hujan sulit diperkirakan, karena curah hujan sangat bervariasi setiap tahunnya. Curah hujan yang jatuh tidak semua digunakan oleh tanaman. Sebagian hujan hilang karena limpasan permukaan (run off) atau karena perkolasi. Jumlah curah hujan yang jatuh dan efisien untuk pertumbuhan tanaman tergantung pada intensitas hujan, topografi lahan, sistem penanaman dan fase pertumbuhan (Sari, 2004). Tahapan selanjutnya setelah dilakukan perhitungan evapotranspirasi potensial dan curah hujan efektif selanjutkan akan ditentukan data musim tanam padi pada ke 3 sampel subak. Setelah itu dilakukan input jenis tanaman untuk menentukan fase dari jadwal tanam. Ketersediaan Air Pada Subak Brawantangi debit pompa yang dihasilkan sebesar 1,76 l/dt/ha. Debit pompa tersebut dapat pula dianalisi untuk mendapatkan volume yang dihasilkan. Volume dihasilkan adalah 76.032 l/ha/hari. Pada Subak Tegal Badeng pada debit pompa yang dihasilkan sebesar 0,72 l/dt/ha. Debit pompa tersebut dapat pula dianalisi untuk mendapatkan volume yang dihasilkan. Volume dihasilkan adalah 31.104 l/ha/hari. Kemampuan Pompa a. Subak Sari Merta Debit pompa yang dimiliki subak Sari Merta yaitu 10 l/d, pompa yang dimiliki sebanyak 5 pompa. Dengan luas lahan 32 ha, jadi dalam satu pompa dapat mengairi sekitar 6 sampai 7 ha lahan subak Kemampuan Sumur Pompa Kemampuan Sumur Pompa Pembuatan jaringan irigasi air tanah dimaksudkan untuk memenuhi kebutuhan air irigasi yang mengalami kekurangan, sehingga mampu menjamin ketersediaan air baik pada musim penghujan maupun kemarau. Pemanfaatan air tanah untuk 102 Ketersediaan Air Ketersediaan air adalah air yang tersedia untuk mengairi atau memenuhi kebutuhan irigasi pada lahan pertanian. Besarnya ketersediaan air pada suatu wilayah (subak) didapat dengan menghitung debit air pada pompa. Diperoleh data ketersediaan air irigasi pompa sebagai berikut pada Tabel 5. Jaringan irigasi air tanah direncanakan dengan sistem saluran tertutup atau perpipaan dan sistem jaringan tertutup didalam tanah. Dengan tiap-tiap outletnya menggunakan box pembagi untuk disalurkan kesawah sesuai dengan luasan tiap arealnya melalui saluran terbuka. Tabel 5. Ketersediaan Air Subak Kemam puan Pompa (l/det) Jumlah Pompa Luas Laha n (ha) Ketersediaan Air debit(l/ d/ha) Vol (l/ha//h ari) Sari Merta 10 5 35 1,42 61.344 Brawa ntangi 20 11 125 1,76 76.032 Tegal Baden g 15 8 165 0,72 31.104 Pada Subak Sari Merta debit pompa yang dihasilkan sebesar 1,42 l/dt/ha. Debit pompa tersebut dapat pula dianalisi untuk mendapatkan volume yang dihasilkan. Volume dihasilkan adalah 61.344 l/ha/hari. Pada Subak Brawantangi debit pompa yang dihasilkan sebesar 1,76 l/dt/ha. Debit pompa tersebut dapat pula dianalisi untuk mendapatkan volume yang dihasilkan. Volume dihasilkan adalah 76.032 l/ha/hari. Pada Subak Tegal Badeng pada debit pompa yang dihasilkan sebesar 0,72 l/dt/ha. Debit pompa tersebut dapat pula dianalisi untuk mendapatkan volume yang dihasilkan. Volume dihasilkan adalah 31.104 l/ha/hari. Tabel 5. Ketersediaan Air Subak Kemam puan Pompa (l/det) Jumlah Pompa Luas Laha n (ha) Ketersediaan Air debit(l/ d/ha) Vol (l/ha//h ari) Sari Merta 10 5 35 1,42 61.344 Brawa ntangi 20 11 125 1,76 76.032 Tegal Baden g 15 8 165 0,72 31.104 y Kemampuan dan jumlah sumur pompa di Subak Sari Merta, Subak Brawantangi, Subak Tegal Badeng berbeda-beda tergantung luas wilayah dari lahan subak petani. Dalam hal ini dibutuhkan spesifikasi pompa disetiap subak sampel. Spesifikasi Pompa Tabel 4. Spesifikasi pompa pada 3 Subak Sampel Nama Subak Pompa Mesin Genset Jenis/Ti pe Mere k M od el Je ni s Mer ek Powe r (KV A) Mo del Sb. Sari Merta Submer sible pump Grou ndfl ow Sp 45 -4 D is el Fg Wil son 27 P27 PI Sb. Brawa ntangi Submer sible pump Grou ndfl ow Sp 60 -7 D is el Per kins 40 P40 P3 Sb Tegal Baden g Submer sible pump Grou ndfl ow Sp 45 -4 D is el Fg Wil son 33 P33 -1 Pada Subak Sari Merta debit pompa yang dihasilkan sebesar 1,42 l/dt/ha. Debit pompa tersebut dapat pula dianalisi untuk mendapatkan volume yang dihasilkan. Volume dihasilkan adalah 61.344 l/ha/hari. Neraca Air N i Neraca air dimaksudkan merupakan perhitungan jumlah masukan (inflow) dan keluaran (outflow) dalam tinjauan periode waktu tertentu pada suatu sub-sistem hidrologi (Harto, 2000). Secara kuantitatif, neraca air menggambarkan bahwa selama periode tertentu masukan air total sama dengan keluaran air total ditambah dengan perubahan air cadangan. Perhitungan neraca air akan menentukan terhadap pola tanam akhir yang akan dipakai untuk jaringan irigasi (Juhana,dkk. 2015). Apabila debit tersedia pada saluran inlet melimpah, maka luas daerah irigasi akan terpenuhi kebutuhan airnya, apabila debit pada saluran inlet terjadi kekurangan air irigasi, terdapat tiga pilihan yang harus dipertimbangkan yaitu luas daerah irigasi dikurangi, melukan modifikasi dalam pola tanam, rotasi atau golongan. Neraca air irigasi dilakukan dengan mengurangi ketersediaan air irigasi dengan b. Subak Brawantangi Debit pompa yang dimiliki subak Brawantangi yaitu 20 l/d, pompa yang dimiliki sebanyak 11 pompa. Dengan luas lahan 125 ha, jadi dalam satu pompa dapat mengairi sekitar 20 sampai 25 ha lahan subak. c. Subak Tegal Badeng Debit pompa yang dimiliki subak Tegal Badeng yaitu 15 l/d, pompa yang dimiliki sebanyak 8 pompa. Dengan luas lahan 165 ha, jadi dalam satu pompa dapat mengairi sekitar 20 sampai 25 ha lahan petani. 103 Gambar 4. Grafik Neraca Air Irigasi di Subak Tegal Badeng Gambar 4. Grafik Neraca Air Irigasi di Subak Tegal Badeng kebutuhan air irigasi pada 3 sampel subak yaitu Subak Sari Merta, Subak Brawantangi dan Subak Tegal Badeng. Grafik neraca air irigasi dari pengurangan antara kebutuhan air irigasi dengan ketersediaan air irigasi pada 3 sampel subak dapat dilihat pada Gambar 2, 3 dan 4 sebagai berikut : kebutuhan air irigasi pada 3 sampel subak yaitu Subak Sari Merta, Subak Brawantangi dan Subak Tegal Badeng. Grafik neraca air irigasi dari pengurangan antara kebutuhan air irigasi dengan ketersediaan air irigasi pada 3 sampel subak dapat dilihat pada Gambar 2, 3 dan 4 sebagai berikut : Dari hasil grafik potensial diatas menunjukan kebutuhan air irigasi dan ketersediaan air pada sampel subak. Garis putus-putus menandakan ketersediaan air pada sampel subak dan garis lurus menandakan kebutuhan air irigasi pada sampel subak. Grafik diatas adalah perbandingan antara debit pompa dengan besarnya kebutuhan air irigasi. Pada Gambar 10,11,12 dapat dilihat jika pada Subak Sari Merta terjadi kekurangan air pada selama 2 periode yaitu periode Juni I sebesar 171.900 l/hari, Januari I 149.700 l/hari. Selain kekurangan air, di Subak Sari Merta juga terjadi kelebihan air. KESIMPULAN DAN SARAN Juhana, E.A., Permana, S. Farida, I. 2015. Analisis Kebutuhan Air Irigasi Pada Daerah Irigasi Bangbayang UPTD SDAP Leles Dinas Sumber Daya Air dan Pertambangan Kabupaten Garut. Sekolah Tinggi Teknologi Garut. Garut. ISSN : 2302-7312 Vol. 13 No. 1. Neraca Air N i Pada Gambar 10 dapat dilihat jika Subak Brawantangi terjadi kekurangan air sebanyak 4 periode yaitu periode Agustus I sebesar 123.900 l/hari, Agustus II 99.200 l/hari, Maret I 98.000 l/hari, Maret II 78.200. Dan pada Gambar 11 dapat dilihat pada Subak Tegal Badeng terjadi kekurangan air sebanyak 8 periode yaitu periode Juni II sebesar 64.600 l/hari, Juni III 178.500 l/hari, Agustus III 35.600 l/hari, September I 35.100 l/hari, September II 38.400 l/hari, September III 32.900 l/hari, November I 48.400 l/hari, November III 149.700 l/hari. Gambar 2. Grafik Neraca Air Irigasi di Subak Sari Merta. Gambar 2. Grafik Neraca Air Irigasi di Subak Sari Merta. Gambar 2. Grafik Neraca Air Irigasi di Subak Sari Merta. Gambar 3. Grafik Neraca Air Irigasi di Subak Brawantangi , Analisis dari ketersediaan air menggunakan pompa di Subak Sari Merta, Subak Brawantangi, Subak Tegal badeng dapat terpenuhi. Defisit air yang terjadi seperti pada Gambar 10, 11 dan 12 karena pada periode tersebut sedang dilakukan pengolahan tanah sehingga kebutuhan air irigasi tinggi. Untuk dapat memenuhi kebutuhan air irigasi yang diperlukan untuk pengolahan tanah, di Subak Sari Merta, Subak Brawantangi dan Subak Tegal Badeng dilakukan sistem pergiliran air. Pergiliran air yang dimaksud adalah pergiliran jadwal olah tanah yang menggunakan air, sehingga olah tanah yang dilakukan oleh petani pada subak tersebut tidak serempak. Tujuan dari penerapan pergiliran olah tanah ini adalah agar petani bisa bergiliran menggunakan air untuk pengolahan tanah. Misalnya di bagian hulu terlebih dahulu mengolah tanah, setelah itu baru dibagian hilir dilakukan pengolahan tanah. Selain itu untuk mengatasi kekurangan air pada ketiga subak khususnya subak Tegal Badeng perlu dilakukan sistem irigasi metode SRI (System of Rice Intensification) untuk menghemat air irigasi yang tersedia. Hal ini karena pada sistem SRI air yang diberikan selama pertumbuhan dari mulai tanam sampai panen dibatasi maksimal 2 cm. Pertumbuhan padi paling baik saat air yang diberikan secara macak-macak dengan ketinggian Gambar 3. Grafik Neraca Air Irigasi di Subak Brawantangi Gambar 3. Grafik Neraca Air Irigasi di Subak 104 sekitar 5 mm dan ada periode pengeringan sampai tanah retak (irigasi terputus) (Anggraini, 2013). Harto, S.B. 2000. Hidrologi Teori, Masalah dan Penyelesaian. Nafiri Offset. Yogyakarta. Saran Adapun saran yang dapat diberikan hasil penelitian yang telah dilakukan adalah untuk mengatasi kekurangan air pada ketiga subak khususnya subak Tegal Badeng perlu dilakukan sistem irigasi metode SRI (System of Rice Intensification). Selain itu perlu juga dilakukan penelitian pada Subak yang lain sehingga bisa diketahui apakah penggunaan sumber daya air tanahnya sudah bisa memenuhi kebutuhan air irigasinya atau belum.. Kesimpulan Teknis penggunaan sumur daya air tanah dimulai dari penggangkatan air menggunakan pompa lalu dialirkan melalui pipa sampai ke bak pembagi dan selanjutnya di alirkan ke petak-petak sawah Teknis penggunaan sumur pompa pada masing- masing subak rata-rata dapat digunakan maksimal selama 12 jam. Debit pompa setiap subak berbeda- beda, Subak Sari Merta mampu mendistribusikan air sebanyak 10 l/d, Subak Brawantangi 20 l/d, Subak Tegal Badeng 15 l/d. Purwanto, dan Jazaul Ikhsan. Analisis Kebutuhan Air Irigasi Pada Daerah Irigasi Bendung Mrican1. Jurnal Semesta Teknika 9.1 (2006): 83-93. Pada Subak Sari Merta terjadi kekurangan air pada selama 2 periode yaitu periode Juni I dan Januari I. Pada Subak Brawantangi terjadi kekurangan air sebanyak 4 periode yaitu periode Agustus I, Agustus II, Maret I, Maret II. Dan pada Subak Tegal Badeng terjadi kekurangan air sebanyak 8 periode yaitu periode Juni II, Juni III, Agustus III, September I, September II, September III, November I, November III. Defisit air terjadi karena pada periode bulan tersebut dilakukan pengolahan tanah sehingga kebutuhan air irigasi cenderung tinggi. Sari, N, Y, 2004. Optimasi Pola Tanam Bedasarkan Ketersediaan Debit Air Irigasi di Daerah Irigasi Situbala Kabupaten Bogor, Jawa Barat (Skripsi). Jurusan Teknik Pertanian. Fakultas Teknologi Pertanian. IPB. Bogor. Soemarwoto, Otto.1991. Ekologi, lingkungan hidup dan pembangunan. Djambatan. Bandung. Sudjarwadi, 1990. Teori dan Praktek Irigasi. Pusat Antar Universitas Ilmu Teknik. UGM. Yogyakarta. DAFTAR PUSTAKA Anggraini, F., Suryanto, A., & Aini, N. (2013). Sistem tanam dan umur bibit pada tanaman padi sawah (Oryza sativa L.) varietas Inpari 13. Jurnal Produksi Tanaman, 1(2). Anonim. 2014. Direktorat Jendral Sumber Daya Air Kementrian Pekerjaan Umum dan Perumahan Rakyat. Balai Wilayah Sungai Bali-Penida. Provinsi Bali Anonim. 2014. Direktorat Jendral Sumber Daya Air Kementrian Pekerjaan Umum dan Perumahan Rakyat. Balai Wilayah Sungai Bali-Penida. Provinsi Bali Asdac, C. 2002. Hidrologi dan Pengelolaan Daerah Aliran Sungai. Gadjah Mada University Press. Yogyakarta. 105
https://openalex.org/W2899452182	https://europepmc.org/articles/pmc6211681?pdf=render	English	null	Measuring health of highway network configuration against dynamic Origin-Destination demand network using weighted complex network analysis	PloS one	2,018	cc-by	9,819	RESEARCH ARTICLE OPEN ACCESS Citation: Tak S, Kim S, Byon Y-J, Lee D, Yeo H (2018) Measuring health of highway network configuration against dynamic Origin-Destination demand network using weighted complex network analysis. PLoS ONE 13(11): e0206538. https://doi. org/10.1371/journal.pone.0206538 Editor: Sergio Go´mez, Universitat Rovira i Virgili, SPAIN Copyright: © 2018 Tak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data used in this study are from the database of Korea highway system collected in 2015, which are public sources provided by the Korea Expressway Corporation. They are available at the URL: http://data.ex.co.kr/. Others can access the data in the same manner as the authors and the authors did not have any special access privileges. Abstract Ideal configuration or layout of highways should resemble the actual demands for the roads represented by Origin-Destination (OD) information. It would be beneficial if existing high- ways can be evaluated for their configurational fitness against the current demands, and newly planned highways can carefully be designed in terms of their layouts and topologies that would reflect the demands. Analysis techniques used for complex networks in the matured field of network theory can be applied for the highway layout health monitoring against the current OD information. This paper proposes a methodology of measuring the fitness of existing highways by comparing their structural configuration against conceptual OD networks using well-established techniques in network theory for complex networks. In the first phase, this paper conducts an empirical analysis and finds that both structural high- way network and OD network follow the “power law” distribution as they are weighted by capacity and traffic volume respectively. It is also found that the power law coefficient of the OD network dynamically changes throughout the day and week. In the second phase, a noble methodology of weighting and measuring the health, of structural highway networks against OD networks by means of comparing their power law coefficients is proposed. It is found that the proposed method is effective at detecting deviations from ideal structural con- figurations associated with actual demands. Measuring health of highway network configuration against dynamic Origin- Destination demand network using weighted complex network analysis Sehyun Tak1, Sunghoon KimID2, Young-Ji ByonID3, Donghoun Lee2, Hwasoo YeoID2* 1 Department of The Fourth Industrial Revolution and Transport, The Korea Transportation Institute (KOTI), Sejong, Republic of Korea, 2 Department of Civil and Environmental Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, 3 Department of Civil Infrastructure and Environmental Engineering, Khalifa University of Science and Technology, Abu Dhabi, UAE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * hwasoo@kaist.ac.kr Measuring health of highway network configuration against dynamic OD demand network centrality, betweenness centrality, straightness centrality, and clustering [1–6]. They analyse specific connection patterns of the road network to abstract the properties of a complicated transportation network structure because it is helpful for not only to understand the temporal and spatial changes of the transportation network but also to provide a guideline for road con- structions. To estimate the properties of large-scale transportation networks in an efficient way, many empirical studies have attempted to conduct a case study based on the railway, urban, aviation, and maritime transportation networks [7–11]. support program (IITP-2018-2016-0-00314) supervised by the IITP (Institute for Information & communications Technology Promotion) and the Ministry of Land, Infrastructure, and Transport (MOLIT, KOREA) [Project ID: 18TLRP-B146748- 01-000000, Project Name: Connected and Automated Public Transport Innovation (National R&D Project)]. Recently, the researches related to transportation networks have shifted their focus from topologic and geometric properties to large-scale statistical properties from a point of view of complex network theory [12]. In a complex network with the “power law” distributions, which is known as a scale-free network, the degree of a node indicates the importance and the node with a high degree is considered as critical [13,14]. By identifying critical components (nodes and links) in a network, the potential failure of a component can be prevented. Furthermore, if the distributions of a transportation network are identified beforehand, the evolutional direc- tion of transportation network can be predicted and the heterogeneity of transportation net- work can be estimated [2,15,16]. For these reasons, different kinds of transportation networks such as air network [17–19], railway network [20,21], public transportation network [22,23], and highway network [24] have been studied. In most of these studies, transportation net- works show small-world properties with different degree distributions for each network. For instance, for the airport networks in India and China, the degree distributions are found to fol- low the power law[17,18]. The degree distribution of Polish public transportation network and that of Indian railway network follow the exponential law [20,22]. The South Korean highway network has a heavy tail with relatively more homogeneous characteristics compared to other transportation networks such as public transportation network and airport transportation net- work [24]. Introduction Transportation networks, especially highway networks are considered as permanent invest- ments that are extremely expensive in building, maintaining and modifying them. Therefore, a careful and continuous monitoring of such crucial infrastructures is mandatory for the society. Many studies on transportation network focus on analysing the topological properties of a transportation network such as ringness, webness, circuitness, degree centrality, closeness Funding: This research was supported by the MSIT (Ministry of Science and ICT), Korea, under the ITRC (Information Technology Research Center) PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 1 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 The previous studies, which focus on the transportation networks based on statisti- cal properties from a point of view of complex network theory and graph theory, analyse the structure of transportation networks without any considerations on the traffic flow and traffic demand on the network, even though the traffic demand and network structure are mutually affected. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Traffic flow and traffic demand on a network shape the network structure in the long run, and instantaneous travel demands reflect the traffic flow. With this background, some previous studies evaluate the characteristics of transportation networks in accordance with the relation- ship between the desired properties of network structure perceived by road users and the prop- erties of transportation network [1,2,15,16,25–28]. They find the optimal network and measure the efficiency and continuity of transportation network based on the given traffic demand. In these studies, the annual traffic volume is used as the traffic demand on the net- works. However, the transportation network is also designed to solve temporal traffic prob- lems such as congestion, which recurrently occurs by the peak demand in a certain period. There is a need for an analysis to identify the relation between the short-term demand change and the properties of transportation network because congestion induced by the short-term peak demand significantly affects the performance of transportation network and the satisfac- tion of the transportation system users. Only a few studies have considered statistical properties of Origin-Destination(OD) demand networks based on actual travel patterns [29,30]. In general, traffic demands are not consistent throughout a day or months, and the congestion occurs due to temporal inconsis- tencies between the designed properties of physical transportation network and desired prop- erties of network structure perceived by road users. Therefore, it is important to study the effect of the temporal inconsistencies on performances of the transportation network. This paper proposes a noble, comprehensive and system-wide approach in evaluating the fitness or PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 2 / 21 Measuring health of highway network configuration against dynamic OD demand network health of existing highway network configurations. In the first phase, we explore the relation- ship between the underlying topological structure of highway systems and the dynamics of demand from a point of view of complex network theory. Analysis approach In order to assess the fitness of current physical transportation network against the conceptual road network perceived and used by the actual demand of people, structural highway network and hypothetical OD network are compared using the complex network theory. A road net- work can be modelled with different approaches by taking into account factors including exist- ing physical structures, number of lanes, actual usage, and traffic volume. In this paper, four different types of road networks are conceptualized of which two of them are inspired by the physical structure of the roads and the other two are hypothetical road networks based on actual trips taken by the demand in forms of O-D pairs. The structural highway network can be classified into two: conceptual road network (CN) and physical road network (PN). CN is the network that represents only the structural connec- tivity relationship among road links and nodes without the consideration of the number of lanes on each road link. On the other hand, PN considers the number of lanes on each road link in addition to the mere connectivity status of the physical network, which results in changes of degrees of the nodes in the network. CN can be seen as a non-weighted structural road network without consideration of link properties and PN is seen as a weighted structural road network with link properties considered. Second, the hypothetical OD network is also classified into two: movement network (MN) and volume-weighted network (VN). MN is the network that represents the actual travelled roads by the people from origins and destinations. In MN, each OD pair is generated if at least one person has travelled from the origin to the des- tination regardless of the traffic volume. On the other hand, VN considers the actual traffic volume on the generated OD pair. MN is seen as a non-weighted OD network and VN is seen as a weighted OD network. In order to reflect realistic physical road network and actual demands for assessing the current fitness of current infrastructure for the actual O-D trips, PN and VN are used to identify the contributing factors that affect the performance of a highway network. Throughout such procedure, we attempt to show three aspects of highway networks. First, this study will newly show the power law relation between the frequency of a degree and the degree of a node in the road network. In the second phase, we analyse the effect of this relationship on the network performance. This study aims to seek for possible solutions for controlling an entire highway network at the state-level and to give guidance for constructions of newly planned highways. We expect that the results of this study provide the foundation to improve the usage of a highway network. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Analysis approach The degree of node i is calculated as di ¼ PN j aijwij, where if nodes i and j are connected, aij = 1, and if not, aij = 0. N is the total number of nodes in a network and wij is the weighting factor between nodes i and j. Existing studies [31,32] on transportation networks show that the degree distribution of a road network appears as a bell shape. However, the weighted degree distribution of a road net- work actually follows the power law when the number of lanes in each road link is considered. Second, this study will analyse a hypothetical OD network based on real-world data and show the power law relation between the frequency of a degree and the degree of a node in the OD network. Unlike the existing studies on road networks, the studies on OD network are rare due to the difficulties in obtaining data for a large area. Therefore, the studies on the effect of dynamics of an OD network on the performance of existing structural road networks are also rare. Third, this study will propose an evaluation method for the performance of an entire PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 3 / 21 Measuring health of highway network configuration against dynamic OD demand network highway network. Existing researches have practiced mostly based on the assumption that the network performance is strongly related to the local traffic demand or local configuration of the network, and the performance level was generally represented by the summation of the median travel time or total travel time of the network. Therefore, most of the studies analysed the network based on the road link performance or effect of the link closure on the network performance. In this study, we present the characteristics of the OD network of a whole road network at the state-level by using a singular value, and then we evaluate the road network per- formance based on the measurements that represent the structural road network and hypo- thetical OD network. In order to derive the CN, PN, MN, and VN for the analysis, we use the databases of Korea highway system collected in 2015, which are public sources provided by Korea Expressway Corporation. Analysis approach To derive the degree distributions of the CN and PN, we use the network struc- tural database in 2015 that contains information on the number of lanes on each highway link, length of each link, and the locations of tollgates (TGs) and interchanges (ICs). The number of nodes in the Korea highway network is 614, and the degree of each node varies from 1 to 4. Both the non-weighted (CN) and weighted (PN) degree distributions of the structural highway network in Korea are derived using the degree values of each node and number of lanes on each road link. To derive the degree distributions of the MN and VN, we use the database of Toll Collection System (TCS), which contains information on the number of vehicles travelled for each OD pair collected hourly in 2013. Using this database, both the non-weighted degree distribution (MN) and the weighted degree distribution (VN) in Korea highway system are derived at every 4-hour interval. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Analysis of physical highway network Fig 1 shows the road network of Korean highway system, which is the structural network used in this study. As shown in the figure, the tollgates and interchanges on the road network are considered as the nodes. Tollgates (TGs) are the starting and ending points of each highway line. They are also the entering points to the highway or exiting points from the highway. Interchanges (ICs) are the intersecting points of two or more lines of the highway. In the road network of Korea highway, the lines connecting TGs and ICs are considered as the links. Based on given conditions, we propose to analyse the structural road network and draw the degree distribution in a log-log plane. At first, we draw un-weighted degree distribution of the road network of Korea highway. At this point, note that the general (un-weighted) degree distribution of a road network has some weakness in representing the nature of the road network. In the social network such as Facebook and Twitter, the link connection can easily be generated by just clicking a button on a computer and this action relatively does not cost much for connecting the link between two nodes. However, in a physical road network, constructing a new link between two nodes is associated with high construction cost. Particularly in a highway network system, the capacity of a road link can be increased by expanding the width instead of building a new link. The number of lanes in a link is increased by the width expansion. In order to reflect this nature in the degree distribution of road networks, we give weighting factors to a road link based on the number of lanes on the link. The number of lanes is considered to represent the road link capacity. In fact, if we were to consider the traffic flow theory more deeply, the capacity of each road lane may be a bit different. However, such deep consideration is not reflected because it does not match the scope of this study. Thus, homogeneous values are assumed for the capac- ity of all lanes in a link, and the number of lanes in a link represents the degree of the road capacity. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Analysis of physical highway network As an example, Hoedeok IC has the degree of three in un-weighted degree PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 4 / 21 Measuring health of highway network configuration against dynamic OD demand network Fig 1. Road network of Korea highway. (Drawn by authors using QGIS program, data source: Ministry of Land, Infrastructure and Transport (MOLIT), the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi org/10 1371/journal pone 0206538 g001 Fig 1. Road network of Korea highway. (Drawn by authors using QGIS program, data source: Ministry of Land, Infrastructure and Transport (MOLIT), the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). Fig 1. Road network of Korea highway. (Drawn by authors using QGIS program, data source: Ministry of Land, Infrastructure and Transport (MOLIT), the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g001 https://doi.org/10.1371/journal.pone.0206538.g001 distribution as shown on the upper right side of Fig 1. This is the example of the conceptual network (CN). In weighted degree distribution (bottom right corner of Fig 1), the degree of the node is 10, which is the summation of the number of lanes in all links that are connected to the node. This is the example of the physical network (PN). The un-weighted degree (CN) and the weighted degree (PN) distributions of the road net- work are to be drawn in a log-log plane. In this plot, two aspects are to be analysed. First, we show whether the degree distribution of the road network follows the normal distribution or the power law distribution. Second, if road network follows the power law distribution, we try to find that fd / d CPN, where fd is the frequency of a degree and d is proportional to the degree of a node to the power of a constant, CPN, in the road network. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 5 / 21 Measuring health of highway network configuration against dynamic OD demand network Fig 2. Degree distribution of road network in Korea highway. Analysis of physical highway network (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g002 Fig 2. Degree distribution of road network in Korea highway. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g002 Fig 2 shows the degree distributions of the structural Korea highway network. As shown in the figure, the general degree distribution of the structural road network (CN) and weighted degree distribution of structural road network (PN) show the linear relation in the log-log planes with -4.08 and -1.95 coefficient values, respectively. As mentioned above, the degree distribution of CN is not spread over a large range and shows the narrow range of values. It is difficult to say that the coefficient of the general degree distribution represents the nature of the road network reasonably well. The weighted degree distribution of PN shows a wider range of the values compared to CN, meaning that it deals with the various aspects of the net- work. So, for the network performance analysis, we use the coefficient of the degree distribu- tion of the PN as the representative value for the structural road network. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Analysis of Origin-Destination demand network Fig 3 shows the average daily traffic pattern of Korea highway, which is drawn based on the number of vehicles counted at each TG in 2015. This figure presents the patterns of traffic entering (attraction) and exiting (generation) the local areas at a glance. Fig 4(A) shows an example of the OD network of Korea highway system. In the OD network, each of the TGs is considered as a node, and the connections between TGs are considered as links when there is at least one traveller who moves from node i to node j. When there are no people moving from node i to node j, the link between i and j is not generated in the OD network. To analyse the OD network, we draw the directed graph as shown in Fig 4(A). Each node is a TG in the highway system and has in-degree and out-degree values. In-degree value is related to how many origins are directed to a destination, and out-degree value is related to how many destinations are directed from an origin. In this method, all links are considered to have the same weight regardless of the amount of people’s movements. This non-weighted OD network is called movement network (MN). Based on the degree distribution of the MN, we analyse the nature of the OD network. same weight regardless of the amount of people’s movements. This non-weighted OD network is called movement network (MN). Based on the degree distribution of the MN, we analyse the nature of the OD network. 6 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Measuring health of highway network configuration against dynamic OD demand network Fig 3. Average daily traffic pattern of Korea highway. (Drawn by authors using QGIS program, data source: MOLIT and Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g003 Fig 3. Average daily traffic pattern of Korea highway. (Drawn by authors using QGIS program, data source: MOLIT and Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g003 https://doi.org/10.1371/journal.pone.0206538.g003 In addition, each link in the OD networks has different demand value for movement. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Analysis of Origin-Destination demand network In order to reflect the demand of each link to the OD network, we assign weighting factors to each link based on the OD demand. The Fig 4(B) shows the example of the OD network of Korea highway system that is weighted by the number of travellers moving from node i and node j. In this study, this weighted OD network is called the volume-weighted network (VN), and the weighted degree distribution of the VN is to be also analysed in a log-log plane. Unlike the PN, the degree distribution of the VN changes continuously with time. For deal- ing with this nature of differences, we plan to estimate the coefficient of the distribution of OD network to find that fd / dCVNðtÞ, where fd is the frequency of a degree and d is proportional to the degree of a node to the power of constant CVN, at time t. Fig 5 shows the degree distributions of OD networks for the in-degree and out-degree MNs, where the density in the y-axis of Fig 5 indicates the average frequency density for each bin in the histogram of degree. As shown in the figure, both the in-degree and out-degree dis- tributions do not show the power law relation. The in-degree distribution shows a mixture of three normal distributions with {(μ, σ)\| (38, 12.447), (136, 13.168), (193, 15.265)}. The mixture model is verified based on the Kolmogorov-Smirnov (K-S) test, the corresponding D statistics and P-value are 0.00078 and 0.937, respectively. For the in-degree distribution, the degree val- ues show certain patterns by the size of the city that is near or within a certain node. As we can see in Fig 5(A), for the nodes near or within small-sized cities, approximately 38 origins are connected to each node. For the nodes near or within medium-sized cities, approximately 136 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 7 / 21 Measuring health of highway network configuration against dynamic OD demand network Fig 4. Example of Origin-Destination network. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). Fig 4. Example of Origin-Destination network. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Analysis of Origin-Destination demand network (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). Fig 4. Example of Origin-Destination network. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g004 8 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Measuring health of highway network configuration against dynamic OD demand network Fig 5. Degree distribution of Origin-Destination network. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g005 Fig 5. Degree distribution of Origin-Destination network. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g005 origins are connected to each node. For the nodes near or within big-sized cities, approxi- mately 193 origins are connected to each node. On the other hand, the out-degree distribution shows a mixture of two normal distributions with {(μ, σ)\| (41, 13.619), (159, 17.932)}. Similar to the in-degree distribution, the mixture model for the out-degree distribution is also verified based on the K-S test. The corresponding D statistics and P-value are 0.00112 and 0.798, respectively. As we can see in Fig 5(B), for the nodes near or within small-sized cities, approximately 41 destinations are connected from each node. For the nodes near or within big-sized cities, approximately 159 destinations are connected from each node. In the case of out-degree distribution, the degree distribution of the nodes near or within medium-sized cities and big-sized cities do not show a clear difference. It means that if a city becomes larger than a certain size, the nodes near the city have a higher tendency to be selected as the destination of trips started from various origins. These results of the MN show that, as a city gets bigger, the number of different OD pairs including the nodes near or within the city tends to increase. The degree distributions of VN show different results as shown in Fig 6. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Measuring health of highway network configuration against dynamic OD demand network some particular places. So, such a pattern is reflected in the degree distribution of the VN in Fig 6. The coefficient of VN also varies depending on the time of a day. Fig 6(A) and 6(B) show the distributions of afternoon and morning time during a weekday, respectively. The coeffi- cient in the afternoon is smaller than the coefficient in the morning, meaning that the travel demands in the afternoon tend to be more concentrated in specific places compared to the morning. It is because people show a greater tendency of traveling to specific places, and in this case, the places are the residential areas. In modern cities, the residential areas are usually placed together in specific areas. Hence, most people travel from each of their workplaces to the residential areas in the afternoon, and it causes the travel demand tending to be concen- trated in specific areas of the highway network. The time-dependent changes in the demand pattern also occur during the weekend, as shown in Fig 6(C) and 6(D). For more detailed analyses of trends on the regression coefficients of VNs with respect to day types, including weekday and weekend, several regression analyses are conducted based on the entire OD dataset in 2013. Fig 7 depicts the distributions of regression coefficients of VNs for each weekday and week- end, where the density in the y-axis of Fig 7 represents the average frequency density for each bin in the histogram of coefficient. As shown in Fig 7, the maximum density of regression coef- ficient value in the weekday is observed when the coefficient value is from -3.20 to -3.10, while Analysis of Origin-Destination demand network Note that the travel volumes of the OD pairs in the network represent VN. In order to collect the volume of an OD pair, multiple vehicle trips have to begin at the origin and they have to end at the destination. Therefore, VN represents the both in and out degrees of the vehicle trips within the highway system. The general trend of the degree distribution of VN follows the power law distribution. By comparing the distributions during weekdays and weekends, one can observe that the coefficient value of the weekend distribution shows less than that of the weekday distribution because the travel demands are more concentrated in specific nodes during the weekends. This difference is due to the different travel pattern between weekend and weekday. During weekdays, people use the highway system usually for the purpose of commuting and business. So, the travel demands are more likely distributed all over the PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 9 / 21 Measuring health of highway network configuration against dynamic OD demand network highway network rather than being concentrated in specific places. On the other hand, during weekends, people usually travel to similar destinations from the various origins. For example, during weekends, many people who live in various places travel to places where they can spend their leisure times. This pattern makes the travel demands be more concentrated in Fig 6. Example cases of the demand distribution of VN during weekday and weekend. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g006 Fig 6. Example cases of the demand distribution of VN during weekday and weekend. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g006 https://doi.org/10.1371/journal.pone.0206538.g006 highway network rather than being concentrated in specific places. On the other hand, during weekends, people usually travel to similar destinations from the various origins. For example, during weekends, many people who live in various places travel to places where they can spend their leisure times. This pattern makes the travel demands be more concentrated in PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 10 / 21 Fig 7. The distributions of regression coefficients of VNs for each weekday and weekend. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g007 Fig 7 The distributions of regression coefficients of VNs for each weekday and weekend (Drawn by authors using R program data source: Korea Fig 7. The distributions of regression coefficients of VNs for each weekday and weekend. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g007 https://doi.org/10.1371/journal.pone.0206538.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 11 / 21 Measuring health of highway network configuration against dynamic OD demand network the maximum density of regression coefficient value in the weekend is found when the coeffi- cient value ranges from -2.40 to -2.30. One can also observe that a greater density value is shown in the coefficient values ranging from -2.40 to -2.30, rather than those ranging from -3.20 to -3.10. These imply that the travel demands are more concentrated in certain OD pairs during the weekends compared to the weekdays’ pattern, which agrees with the research find- ing of the previous analysis, as shown in Fig 6. In other words, this result shows that the travel demand in the weekday has more evenly distributed OD pairs than in the weekend, while the travel demand in the weekend tends to be concentrated in specific places. Based on the observations in Fig 7, Fig 8 shows the weekday density trend according to the time evolution with a specific range of regression coefficient values, where the range of regres- sion coefficient values includes (a) from -2.80 to -2.70, (b) from -2.40 to -2.30, and (c) from -3.20 to -3.10. As seen in Fig 8, it can be found that there are several points in time for a dra- matic change of regression coefficient value. As shown in Fig 8(A), one can observe that the VN during the morning peak hour from 5 a.m. to 9 a.m. has the coefficient values ranging from -2.80 to -2.70. The coefficient value decreases to approximately -3.10 as the commuting demand decreases overall. After a while, the VN has a specific value of regression coefficient ranging from -2.40 to -2.30, as shown in Fig 8(B). We conjecture that the reason behind the concentration is due to the increased travel demands for logistics services. One can also find that the VN with the coefficient values ranging from -2.40 to -2.30 has the maximum density value around 3:00 a.m., while the VN with the coefficient values ranging from -3.20 to -3.10 shows the peak value around 12:00 a.m., as described in Fig 8(C). Such trends can be also observed in the weekend, as depicted in Fig 9. However, distinct from the observation in the weekday’s travel pattern, we find that there is a significant differ- ence in the density between the VN with the coefficient values ranging from -2.40 to -2.30 and the VN with the coefficient values ranging from -3.20 to -3.10. In other words, unlike the weekday’s travel pattern that people use the highway system usually for the purpose of com- muting and business, the OD travel demand is changed with the tendency toward dichotomization. As seen in Fig 10, we observe that the maximum P-values of the weekend and weekday are 1.99210−5 and 2.27510−5, respectively. It is also seen that the corresponding minimum values of adjusted R2 of the weekend and weekday are 0.75 and 0.791, respectively. Not only the maxi- mum P-values show much less than the significance level, but also the adjusted R2 values are high enough. This indicates that there is not sufficient evidence to reject the alternative hypothesis that the corresponding coefficient value is not equal to 0. Therefore, we can con- clude that the results of the analyses are statistically significant. Network performance analysis based on the weighted networks The performance of a highway network can be measured with various values such as travel time, frequency of congestion, and delay. In this study, we use the summation of the median travel time from origin i to destination j for measuring the performance of a highway network at time t as follows: Network Performance ðtÞ ¼ P ALL i;jTTmedian;i;jðtÞ ð1Þ ð1Þ ð1Þ Median travel time indirectly represents the congestion level of a highway network and shows the efficiency of the network for each given OD pair under a given total demand on the network. When a congestion occurs, which is caused when OD demand is larger than the road capacity, the median travel time becomes larger compared to the median travel time in free flow state. When OD demand is lower than the road capacity, the median travel time is the PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 12 / 21 Fig 8. The weekday density trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g008 Measuring health of highway network configuration against dynamic OD demand network Measuring health of highway network configuration against dynamic OD demand network Fig 8. The weekday density trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g008 ty trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, sway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would ) Fig 8. The weekday density trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g008 https://doi.org/10.1371/journal.pone.0206538.g008 same with the median travel time in a free flow state. Therefore, by summing the median travel times of all links, the performance of the entire network can be estimated. Network performance analysis based on the weighted networks We use the median travel time values provided in the database of Korea highway system for the performance eval- uation, which are computed by the Korea Expressway Corporation. The median travel time is computed based on the travel time values of multiple vehicles that have finished the trip same with the median travel time in a free flow state. Therefore, by summing the median travel times of all links, the performance of the entire network can be estimated. We use the median travel time values provided in the database of Korea highway system for the performance eval- uation, which are computed by the Korea Expressway Corporation. The median travel time is computed based on the travel time values of multiple vehicles that have finished the trip PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 13 / 21 Fig 9. The weekend density trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g009 Measuring health of highway network configuration against dynamic OD demand network Measuring health of highway network configuration against dynamic OD demand network Fig 9. The weekend density trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). htt //d i /10 1371/j l 0206538 009 Fig 9. The weekend density trend according to the time evolution with different regression coefficient values. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data) https://doi.org/10.1371/journal.pone.0206538.g009 https://doi.org/10.1371/journal.pone.0206538.g009 between origin i and destination j. The travel time of each vehicle is computed by calculating the difference between the inbound timestamp at origin i and outbound timestamp at destina- tion j. The timestamps are recorded at all tollgates by the highway TCS. between origin i and destination j. The travel time of each vehicle is computed by calculating the difference between the inbound timestamp at origin i and outbound timestamp at destina- tion j. Network performance analysis based on the weighted networks The timestamps are recorded at all tollgates by the highway TCS. With this performance measure, we plan to explore the causal factor degrading the highway performance among overall demands on the highway network and concentration of demands PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 14 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 Measuring health of highway network configuration against dynamic OD demand network Fig 10. The goodness-of-fit of each regression coefficient of VNs with respect to day type. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). Fig 10. The goodness-of-fit of each regression coefficient of VNs with respect to day type. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi.org/10.1371/journal.pone.0206538.g010 in a specific part of the network. At first, to verify the relationship between the total demand and network performance, we plan to analyse whether the following relation is satisfied or not. P All;i;jTTmedian;i;jðtÞ / P All;i;jDi;jðtÞ ð2Þ ð2Þ where Di,j is the collected number of people travelled from node i to j at time t. In many stud- ies, the assumption that the congestion is proportional to the total demand on a road network is general and many control methods try to mitigate the congestion by reducing and distribut- ing the total demand on a network. In this study, we plan to verify this relation also. Fig 11 shows the conceptual diagram of three different networks. In the figure, CCN, CPN, and CVN, represent the power law coefficient values of the conceptual network (CN), physical network (PN), and volume-weighted Network (VN) respectively. As shown in the figure, the difference between PN and VN can be estimated by the power law coefficient. This difference represents the imbalance of road configuration and road users’ desires to travel on the highway network. Considering the continuous changes in the demand, the coefficient difference is expressed as follows. DCVN;PNðtÞ ¼ CVNðtÞ CPN ð3Þ ð3Þ The difference between the degree distributions of a PN and VN can show the road perfor- mance. To figure out the effect of this difference on the road performance, we plan to explore the relationship between the power law coefficients of a network and the performance of a PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 15 / 21 Measuring health of highway network configuration against dynamic OD demand network Fig 11. Estimation method of coefficient difference between PN and VN. (Drawn by authors using R program). https://doi.org/10.1371/journal.pone.0206538.g011 Fig 11. Estimation method of coefficient difference between PN and VN. (Drawn by authors using R program). Fig 11. Estimation method of coefficient difference between PN and VN. (Drawn by authors using R program). https://doi.org/10.1371/journal.pone.0206538.g011 network based on whether the following relation is satisfied or not. network based on whether the following relation is satisfied or not. P All;i;jTTmedian;i;jðtÞ / DCVN;PNðtÞ ð4Þ P All;i;jTTmedian;i;jðtÞ / DCVN;PNðtÞ ð4Þ The power law coefficient of the network represents the dispersion of the degree. The low absolute value of the power law coefficient means that the demand is concentrated in a specific area of a network. Conversely, the high absolute value of power law coefficient means that the demand is almost uniformly distributed in the entire network. In this context, the difference of the power law coefficient between a PN and VN can show the difference of dispersion between the two networks, and such relation is set based on the assumption that congestions are caused by the difference between people’s desired movement patterns and the physical configuration of a road network. The total demand and coefficient difference show the different aspects of demand for the network. Total demand represents the entire quantity of travel desires on a network and the coefficient difference represents the difference between the characteristics of the traffic distri- butions in VN and PN. We also explore the following relation to consider these two different aspects of the road network. P All;i;jTTmedian;i;jðtÞ / DCVN;PNðtÞ P All;i;jDi;jðtÞ ð5Þ ð5Þ The coefficient of the VN dynamically changes and the difference of the coefficient between the VN and PN changes as well. Based on this phenomenon, we analyse the changes of the coefficient difference and total OD demand respect to the sum of the median travel time. The coefficient of the VN dynamically changes and the difference of the coefficient between the VN and PN changes as well. Based on this phenomenon, we analyse the changes of the coefficient difference and total OD demand respect to the sum of the median travel time. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 16 / 21 Measuring health of highway network configuration against dynamic OD demand network Fig 12. The effect on the road performance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi org/10 1371/journal pone 0206538 g012 Fig 12. The effect on the road performance. (Drawn by authors using R program, data source: Korea Expressway Corporation, the data are free source for everyone, the authors did not have any special privileges that others would have in obtaining the data). https://doi org/10 1371/journal pone 0206538 g012 https://doi.org/10.1371/journal.pone.0206538.g012 Fig 12(A) shows the relationship between the total demand and sum of median travel time. As we can see in the figure, when the total demand increases, the sum of median travel time also increases. This shows that the network performance is degraded as total demand increases. It is noticeable that there are two clusters in the relationships and each of them shows different increasing slope from each other. When total demand is small, the relationship is clearer with less sparse data points, whereas the relationship is sparser when the demand is big. Fig 12(B) shows the relationship between the difference of coefficient and the summation of the median travel time for each OD demand. As shown in the figure, the sum of the median travel time increases as the difference of coefficient increases. This means that the performance of a road network is affected by the coefficient difference as well. It can be noted that the data points showing the relationship are sparser compared to the relationship between the total demand and the sum of median travel time. Using only the coefficient difference may not be appropriate when evaluating the network performance. Fig 12(C) shows the product of coefficient difference and total demand respect to the sum of median travel time. As we can see in the figure, when we considered the two aspects together, it shows a much clearer relationship that is linear. In addition, we do not see the PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 17 / 21 Measuring health of highway network configuration against dynamic OD demand network Fig 13. The application of proposed relation. (Drawn by authors using R program). https://doi org/10 1371/journal pone 0206538 g013 Fig 13. The application of proposed relation. (Drawn by authors using R program). https://doi.org/10.1371/journal.pone.0206538.g013 Fig 13. The application of proposed relation. (Drawn by authors using R program). phenomenon of clustering in the relationship that is shown in Fig 12(A) anymore. Based on this result, it seems that using the product of the two aspects can more efficiently evaluate the performance of a road network. There are some outliers in the relationship, and it is assumed that these outliers occur due to the factors that are related to total demand, such as special cases like accidents and incidents. Still, these outliers have to be investigated in further studies. phenomenon of clustering in the relationship that is shown in Fig 12(A) anymore. Based on this result, it seems that using the product of the two aspects can more efficiently evaluate the performance of a road network. There are some outliers in the relationship, and it is assumed that these outliers occur due to the factors that are related to total demand, such as special cases like accidents and incidents. Still, these outliers have to be investigated in further studies. Fig 13 shows the possibility of applying the proposed evaluation measure to certain control methods for improving the network performance. When controlling the incoming vehicles in the TGs, we can lower the coefficient difference between the PN and VN by controlling the number of the incoming vehicles, as shown in Fig 13(A). The red dotted line represents the coefficient of the degree distribution of the PN, and the blue dotted line represents the coeffi- cient of the degree distribution of the VN. We can improve the entire network’s performance not only by considering the local traffic status but also by considering the pattern of the OD demands of the entire network system. At the same time, the proposed measure can give a more efficient direction when planning to construct a new road. Fig 13(B) shows the aggre- gated degree distribution of the VN at all periods. There is a difference between the coefficient values of the two different networks as we can see in the figure. During the construction plan- ning process, decisions can be made by checking if the coefficient difference would be lowered when the new road link is added to the existing network system. In this way, the performance of an entire road network can be improved efficiently. Conclusion In this paper, we analyse the structural road network and hypothetical Origin-Destination demand network of Korea highway system from a point of view of the complex network the- ory. It is found that both networks follow the “power law” distribution in the weighted condi- tions. Particularly, in the case of the OD demand network, the coefficient of node degree distribution dynamically changes according to the time and it can also represent different trav- elling behaviours during weekdays and weekends. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206538 November 1, 2018 18 / 21 Measuring health of highway network configuration against dynamic OD demand network By using the coefficient calculated from both weighted networks, we develop a network evaluation methodology. We analyse the effect of the total demand and coefficient difference of the structural network and OD demand network on the performance. The statistical analysis of the physical road network and demand network can be investigated with the difference between the designed properties of physical transportation network and desired properties of network structure perceived by road users. As we can see in the results, the total demand and coefficient difference show a positive linear relationship with respect to the network perfor- mance. Furthermore, a stronger linear relationship is observed when we consider the total demand and coefficient difference simultaneously. This means that not only the difference between the capacity of physical road network and the demand for road network but also the difference between the configuration of physical road network and distribution of origin-desti- nation demand have significant influences on network efficiencies. Based on these results, we can effectively evaluate the performance of an entire highway network with a simple calcula- tion method even in the environment in which the traffic demand continuously changes. The focus of this paper is not only to analyse the relationship between physical road net- work and Origin-Destination demand network but also to provide a basis for further applica- tions such as demand control for road network or planning of road network construction. It is shown that the proposed method is able to capture the network’s system-wide congestion caused by differences between characteristics of physical road networks and origin-destination demand networks. The proposed evaluation method can be used in two ways. First, it can give an efficient direction when modelling a control method to improve the performance of an entire road network. Conclusion Second, it can also give an efficient direction when planning a new road construction. The proposed method that uses the relationship between the structural road net- work and OD network can provide the foundation for road controls and related policies in terms of improving the performance of road network at the state level. Even if it is shown that the proposed method has a great advantage to evaluate the perfor- mance on the transportation network with a simple calculation and macroscopic perspective, the method is still required to be considered further for spatial information on the node distri- bution. Furthermore, since the ultimate goal of our proposed method is to provide a more effi- cient direction to the user, the effect of the proposed method on the urban structure and its future development may need to be further considered from the urbanization perspective. The recent researches such as [33,34] can be used for the network configuration setting by consid- ering the spatial features in the urban area. Acknowledgments This research was supported by the MSIT(Ministry of Science and ICT), Korea, under the ITRC(Information Technology Research Center) support program(IITP-2018-2016-0-00314) supervised by the IITP(Institute for Information & communications Technology Promotion) and the Ministry of Land, Infrastructure, and Transport (MOLIT, KOREA)[Project ID: 18TLRP-B146748-01-000000, Project Name: Connected and Automated Public Transport Innovation(National R&D Project)] References 1. Erath A, Lo¨chl M, Axhausen K. Graph-theoretical analysis of the Swiss road and railway networks over time. Networks Spat Econ. 2009; 9: 379–400. 2. Xie F, Levinson D. Measuring the structure of road networks. Geogr Anal. 2007; 39: 336–356. 3. Lin J, Ban Y. Complex network topology of transportation systems. Transp Rev. 2013; 33: 658–685. 4. Boccaletti S, Latora V, Moreno Y. Complex networks: Structure and dynamics. Phys Rep. 2006; 424: 175–308. 5. Latora V, Marchiori M. 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https://openalex.org/W3165972032	https://link.springer.com/content/pdf/10.1007/s40619-021-00893-6.pdf	Italian	null	Approccio terapeutico non chirurgico al paziente con oftalmopatia basedowiana	L'Endocrinologo	2,021	cc-by	1,794	L’Endocrinologo (2021) 22:103–105 https://doi.org/10.1007/s40619-021-00893-6 L’Endocrinologo (2021) 22:103–105 https://doi.org/10.1007/s40619-021-00893-6 TIROIDE TIROIDE R. Le Moli lemoli.rosario@tiscali.it Introduzione L’OB si può prevenire controllando alcuni fattori di rischio [1]: L’oftalmopatia basedowiana (OB) è una manifestazione ex- tratiroidea del morbo di Basedow (MB) determinata da au- toanticorpi contro il recettore dell’ormone tireotropo (TSH- rAb) espresso dai ﬁbroblasti dell’orbita. Interessa circa il 25% dei pazienti con MB ed è più frequente nelle donne (rapporto femmine/maschi di 5:1). Le forme lievi costitui- scono oltre l’85% dei casi, le forme moderato-gravi o gravi il 5–6% e l’1–2% dei casi, rispettivamente. Esistono le forme asimmetriche, unilaterali e la rara variante eutiroidea (preva- lenza di 0,02/10.000 casi). L’OB determina un cambiamento della ﬁsionomia e provoca alterazioni del visus che possono variare dalla diplopia ﬁno alla discromatopsia ed alla perdi- ta della visione. Queste manifestazioni cliniche determinano una riduzione signiﬁcativa della qualità della vita con effetti sul lavoro e sui rapporti sociali (Figg. 1–2) [1]. – il fumo è fattore di rischio di gravità, progressione e di non risposta alle terapie immunosoppressive; – il trattamento con 131I (RAI) e l’ipotiroidismo sono fattori di rischio per la presentazione e la progressione; – il diabete e la sindrome metabolica si associano alle forme più gravi [2]; asimmetriche, unilaterali e la rara variante eutiroidea (preva- lenza di 0,02/10.000 casi). L’OB determina un cambiamento della ﬁsionomia e provoca alterazioni del visus che possono – l’ipercolesterolemia incrementa il rischio di presentazio- ne e attività dell’OB. Le statine hanno un effetto protettivo indipendente [3]; p p variare dalla diplopia ﬁno alla discromatopsia ed alla perdi- ta della visione. Queste manifestazioni cliniche determinano una riduzione signiﬁcativa della qualità della vita con effetti sul lavoro e sui rapporti sociali (Figg. 1–2) [1]. – il sesso maschile e l’età >60 anni correlano con l’OB più grave; – il ritardo della diagnosi e della correzione dell’ipertiroi- dismo determina un’attivazione della risposta immuno- inﬁammatoria; – i TSHrAb correlano con la gravità dell’OB specie nei pa- zienti con anticorpi anti-tireoperossidasi (AbTPO) nega- tivi. Diagnosi La diagnosi di OB è deﬁnita dal coinvolgimento inﬁamma- torio bilaterale dei tessuti dell’orbita associato a diagnosi certa di MB. Può presentarsi entro 12 mesi o dopo diversi anni dall’insorgenza del MB, in pazienti eutiroidei o prima dell’ipertiroidismo, nel 10% dei casi in pazienti ipotiroidei. La diagnosi di OB è deﬁnita dal coinvolgimento inﬁamma- torio bilaterale dei tessuti dell’orbita associato a diagnosi certa di MB. Può presentarsi entro 12 mesi o dopo diversi anni dall’insorgenza del MB, in pazienti eutiroidei o prima dell’ipertiroidismo, nel 10% dei casi in pazienti ipotiroidei. Il 5–15% dei pazienti presentano il coinvolgimento di una sola orbita per cui è necessario ricorrere all’imaging radio- logico per effettuare una diagnosi differenziale. La diagnosi spesso è tardiva [1]. 1 U.O.C. di Endocrinologia, Dipartimento di Medicina Clinica e Sperimentale, Università di Catania, Catania, Italia Approccio terapeutico non chirurgico al paziente con oftalmopatia basedowiana Rosario Le Moli1 Accettato: 27 aprile 2020 / Pubblicato online: 2 giugno 2021 © The Author(s) 2021 Accettato: 27 aprile 2020 / Pubblicato online: 2 giugno 2021 © The Author(s) 2021 Forme attive moderato-gravi e gravi Richiedono una terapia immunosoppressiva. I corticosteroi- di per via parenterale costituiscono i farmaci di prima scel- ta. L’efﬁcacia e gli effetti collaterali sono proporzionali alla dose: è sconsigliata una dose cumulativa > 8 grammi [4]. La risposta clinica viene valutata alla terza settimana (forme gravi) ed alla sesta settimana di somministrazione mediante l’indice clinico composito proposto dall’European Group of Graves’ Ophthalmopathy (EUGOGO) [5]. I pazienti peggio- rati sospendono la terapia e, eventualmente, ricorrono alla chirurgia decompressiva. I pazienti stabili sono rivalutati al- la dodicesima settimana. Un’alternativa nei non responders può essere la ciclosporina associata ai corticosteroidi per os. Il Rituximab è un anticorpo monoclonale anti CD20 e può essere utilizzato nell’OB resistente ma esiste il rischio di ot- ticopatia [1]. La radioterapia retro-bulbare può essere asso- ciata alla terapia corticosteroidea specialmente nei pazienti con disturbi della motilità oculare. Fig. 2 Esempio di congiuntivite limbica L’attività e la QV devono essere valutate utilizzando [1]: – il clinical activity score (CAS) [1] che valuta il grado di inﬁammazione misurando i seguenti segni e sintomi cli- nici: dolore spontaneo o durante i movimenti del bulbo oculare, edema palpebrale, rossore delle palpebre, ros- sore delle congiuntive, chemosi delle congiuntive, iper- troﬁa della caruncola. Un CAS > 3 indica attività della malattia; – il clinical activity score (CAS) [1] che valuta il grado di inﬁammazione misurando i seguenti segni e sintomi cli- nici: dolore spontaneo o durante i movimenti del bulbo oculare, edema palpebrale, rossore delle palpebre, ros- sore delle congiuntive, chemosi delle congiuntive, iper- troﬁa della caruncola. Un CAS > 3 indica attività della malattia; Le dosi riportate in letteratura sono differenti. Controin- dicazione relativa è la presenza di diabete mellito o di reti- nopatia ipertensiva. Dati recenti hanno evidenziato che l’as- sociazione dei corticosteroidi per via parenterale con il mi- cofenolato è sicura ed efﬁcace nelle forme moderato-gravi attive [6]. Le terapie descritte sono comunque aspeciﬁche e spesso non risolutive; i corticosteroidi hanno scarso effetto sulla proptosi e sulla motilità oculare, per cui parecchi pa- zienti devono ricorrere alla chirurgia. Il Teprotumumab (T), un anticorpo monoclonale anti recettore dell’insulin growth factor-1 (IGF-1r) è stato approvato negli USA per la terapia dell’OB di grado moderato-grave. L’uso di T è razionale in quanto l’IGF-1r e il TSHr formano un complesso funzionale e interattivo che attivato dagli anticorpi e dalle citochine del MB determina proliferazione ﬁbroblastica e produzione di glicosamminoglicani nell’orbita. Terapia La gestione dell’OB comprende il controllo dell’ipertiroidi- smo e l’applicazione di programmi comportamentali e tera- peutici per i fumatori e per i pazienti con diabete e/o iper- colesterolemia. La scelta terapeutica dipende dell’attività e dalla gravità dell’OB. Lo specialista deve distinguere le for- me attive e progressive che richiedono una terapia immu- nosoppressiva e/o anti-inﬁammatoria dalle forme inattive e non progressive che non rispondono alla terapia medica. In ogni caso deve valutare la qualità di vita (QV) del paziente [1] Il 5–15% dei pazienti presentano il coinvolgimento di una sola orbita per cui è necessario ricorrere all’imaging radio- logico per effettuare una diagnosi differenziale. La diagnosi spesso è tardiva [1]. L’Endocrinologo (2021) 22:103–105 104 Fig. 1 Esempi di oftalmopatia basedowiana moderato-grave zione NOSPCES o EUGOGO, quest’ultima comprende la valutazione mediante GO-QoL. Fig. 1 Esempi di oftalmopatia basedowiana moderato-grave Fig. 2 Esempio di congiuntivite limbica zione NOSPCES o EUGOGO, quest’ultima comprende la valutazione mediante GO-QoL. zione NOSPCES o EUGOGO, quest’ultima comprende la valutazione mediante GO-QoL. Bibliograﬁa 1. Bartalena L, Baldeschi L, Boboridis K et al (2016) The 2016 Eu- ropean Thyroid Association/European Group on Graves’ Orbito- pathy Guidelines for the management of Graves’. Orbitopathy Eur Thyroid J 5:9–26 Forme attive moderato-gravi e gravi – la storia clinica e la determinazione dell’epoca d’insor- genza dei sintomi; – il controllo clinico periodico a intervalli non superio- ri ai 60 giorni che permette di valutare la progressione dell’OB; – il questionario Graves’ Orbitopathy – Quality of Life (GO-Qol) [1] che valuta l’impatto dell’OB sia sulle fun- zioni che sull’estetica del paziente. La gravità è valutata indipendentemente dall’attività ed è determinata dai cambiamenti dei tessuti muscolari e dal grasso retro-bulbare indotti dalla risposta immuno- inﬁammatoria. Le forme moderato-gravi sono caratterizzate da proptosi, cheratite da esposizione e/o disfunzione musco- lare, la presenza di otticopatia (Disthyroid Optic Neuropa- thy) e/o di danno corneale caratterizza le forme gravi. La gravità della OB viene determinata mediante la classiﬁca- I risultati clinici mostrano un effetto sulla proptosi com- parabile a quello chirurgico. Questo prova che l’IGF-1r è 105 L’Endocrinologo (2021) 22:103–105 speciﬁco nella patogenesi dell’OB [7]. Trial clinici sono in corso per valutare l’effetto degli analoghi della soma- tostatina (Pasireotide) e di un anticorpo monoclonale anti IgG. Open Access This article is licensed under a Creative Commons At- tribution 4.0 International License, which permits use, sharing, adap- tation, 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 chan- ges were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indica- ted 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/. Forme attive lievi Solitamente non determinano una riduzione della QV, sono auto-risolutive e non richiedono alcun trattamento. PREDI- GO è uno score che predice il rischio di sviluppo e progres- sione dell’OB all’esordio del MB. La L-selenometionina è efﬁcace nel prevenire l’OB lieve nei pazienti a rischio [1]. L’uso di lubriﬁcanti corneali è utile nei casi di sindrome del- l’occhio secco e congiuntivite limbica. Se presente riduzione della QV è indicato l’uso dei corticosteroidi. Forme inattive 2. Le Moli R, Muscia V, Tumminia A et al (2015) Type 2 diabetic pa- tients with Graves’ disease have more frequent and severe Graves’ orbitopathy. Nutr Metab Cardiovasc Dis 25:452–457 In questi casi la terapia medica è inefﬁcace ed è possibile ricorrere alla chirurgia estetico-riabilitativa. 3. Lanzolla G, Vannucchi G, Ionni I et al (2020) Cholesterol serum levels and use of statins in Graves’ orbitopathy: a new starting point for the therapy. Front Endocrinol 10:933 Funding Note Open access funding provided by Università degli Studi di Catania within the CRUI-CARE Agreement. 4. Le Moli R, Baldeschi L, Saeed P et al (2007) Determinants of li- ver damage associated with intravenous methylprednisolone pulse therapy in Graves’ ophthalmopathy. Thyroid 17:357–362 Conﬂitto di interesse L’autore Rosario Le Moli dichiara di non avere conﬂitti di interesse. 5. Bartalena L, Wiersinga WM (2020) Proposal for standardization of primary and secondary outcomes in patients with active, moderate- to-severe Graves’ orbitopathy. Eur Thyroid J 9(Suppl 1):3–16 Consenso informato Lo studio presentato in questo articolo non ha richiesto sperimentazione umana. Consenso informato Lo studio presentato in questo articolo non ha richiesto sperimentazione umana. Consenso informato Lo studio presentato in questo articolo non ha richiesto sperimentazione umana. 6. Kahaly GJ, Riedl M, König J et al for the European Group on Graves’ Orbitopathy (EUGOGO) (2018) Mycophenolate plus methylprednisolone versus methylprednisolone alone in active, moderate-to-severe Graves’ orbitopathy (MINGO): a randomised, observer-masked, multicentre trial. Lancet Diabetes Endocrinol 6(4):287–298 Studi sugli animali L’autore di questo articolo non ha eseguito studi sugli animali. Studi sugli animali L’autore di questo articolo non ha eseguito studi sugli animali. Nota della casa editrice Springer Nature rimane neutrale in riguar- do alle rivendicazioni giurisdizionali nelle mappe pubblicate e nelle afﬁliazioni istituzionali. 7. Wang Y, Patel A, Douglas RS (2019) Thyroid eye disease: how a novel therapy may change the treatment paradigm. Ther Clin Risk Manag 15:1305–1318
W2044282129.txt	https://wwwnc.cdc.gov/eid/article/9/12/pdfs/03-0270.pdf	en		Noninvasive Method for Monitoring<i>Pneumocystis carinii</i>Pneumonia	Emerging infectious diseases	2,003	cc-by	2,516	DISPATCHES Noninvasive Method for Monitoring Pneumocystis carinii Pneumonia Michael J. Linke,* Sandy Rebholz,† Margaret Collins,† Reiko Tanaka,† and Melanie T. Cushion† The progression of Pneumocystis carinii pneumonia was temporally monitored and quantified by real-time polymerase chain reaction of P. carinii–specific DNA in oral swabs and lung homogenates from infected rats. DNA levels correlated with the number of P. carinii organisms in the rats’ lungs, as enumerated by microscopic methods. This report is the first of a noninvasive, antemortem method that can be used to monitor infection in a host over time. P neumocystis pneumonia remains a leading opportunistic infection associated with AIDS patients, even in the era of highly active antiretroviral therapy (1). In developing countries, the incidence of infection has increased dramatically, with mortality rates ranging from 20% to 80% (2). An important limitation in its clinical management has been the inability to evaluate therapeutic response or to temporally measure the organism numbers because of the absence of an in vitro culture system. Our laboratory recently showed that the presence of Pneumocystis carinii–specific amplicons obtained from swabs of the oral cavities of nonimmunocompromised adult rats (Rattus norvegicus) was predictive of the development of P. carinii pneumonia after corticosteroid-induced immunosuppression (3). In the present study, we applied the oral swab technique in combination with quantification of organism-specific DNA using real-time polymerase chain reaction (PCR) to monitor the progression of infection in the rat model. The Study Thirty-two male Long Evans rats (140–160 g) known to harbor P. carinii were obtained from Room 004 at the Cincinnati Veterinary Medical Unit (4). All rats produced P. carinii amplicons from initial oral swab samples taken Veterans Affairs Medical Center, Cincinnati, Ohio, USA; and †University of Cincinnati College of Medicine, Cincinnati, Ohio, USA before immunosuppression. After sampling, 8 of the 32 rats were euthanized and their lungs were removed and processed as described below. The remaining 24 rats were removed from the room and individually caged under barrier conditions, as described previously (3), to prevent transmission of infection that might occur between cage mates or from the environment. Barrier conditions consisted of the following: microisolator tops for each shoebox cage, which was then housed within a BioBubble (The Colorado Clean Room Company, Fort Collins, CO); autoclaved water, into which a sterile solution of cephadrine (Velosef; E.R. Squibb and Sons, Inc., Princeton, NJ) was injected for a final concentration of 0.200 mg/mL; autoclaved cages, bedding, and tops; and irradiated Lab Chow (Tekmar Irradiated Lab Chow, Harlan Industries, Indianapolis, IN). To provoke P. carinii pneumonia, 4 mg/kg of methylprednisolone acetate (Depo Medrol; The Upjohn Co., Kalamazoo, MI) was administered to the rats weekly for 10 weeks. At 4 and 7 weeks, swab samples were obtained from groups of eight rats; the rats were then euthanized. Their lungs were removed for quantification by microscopic enumeration of organism nuclei expressed as log nuclei/mL (5) and real-time PCR analysis under aseptic conditions. Six rats survived the 10 weeks of immunosuppression and were processed in an identical manner. DNA was extracted from the oral swabs (OS) and lung homogenate (LH), as previously described (4). LH DNA was evaluated by spectrophotometric analysis at 260 and 280 nm. RC primers directed to a region of the mitochondrial large subunit rRNA (mtLSU) were used for amplification of P. carinii–specific DNA (6). Real-time PCR was performed and results were analyzed on the iCycler iQ Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA) under conditions of rapid melting at 95°C, annealing for 5 s at 55°C, and collection at 76°C for 10 s with 40 cycles of amplification. Five microliters of a 1/5 dilution of OS DNA or 2.5 ng of LH DNA were used in the reactions. Taq DNA (1.25 U) polymerase (Promega, Madison, WI) was used in the realtime PCR with a concentration of 2.5 mM MgCl2 in 25-µL reactions. To monitor the accumulation of the products, 0.4 µL of 1/1,000 dilution of concentrated SYBR Green (Molecular Probes, Eugene, OR) was included in the reactions. All reactions were performed in triplicate. The mtLSU product was cloned into the TOPO-TA PCR cloning vector (InVitrogen, Carlsbad CA) (mtLSU-T-TA), quantified by spectrophotometry, and used to generate a standard curve. The cloned PCR product, ranging from 0.0005 pg to 0.5 pg per reaction, was used as a template; the threshold cycles (CTs) of these reactions were then plotted against the log amount of plasmid per reaction in picograms. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 12, December 2003 1613 DISPATCHES P. carinii DNA in the LH and OS samples was quantified by linear regression analysis of the CTs relative to the standard curve (3). The concentration of P. carinii DNA in the LH and OS samples, determined from the standard curve in picograms, was converted to copies per milliliter by multiplying by the dilution factor based on the original concentration of DNA. The LH copies were log transformed and expressed as log copies per milliliter. The specificity of the reactions was verified by analysis of the product-melting curves and by gel electrophoresis. All products were of the expected size (137 bp) and produced a single peak with a Tm of approximately 78°C. Microscopic enumeration of nuclei of the lung homogenates was compared to real-time PCR lung homogenate results by using Tukey-Kramer Multiple Comparisons post-test to assess significance (InStat version 3; GraphPad Software, Inc., San Diego, CA). Pre- and postimmunosuppression OS samples were analyzed with the Mann-Whitney test (InStat v. 3). Spearman Rank Correlation was used to evaluate the correlation between microscopic enumeration and the real-time PCR output (Instat v.3). To ensure accurate and reproducible results, the efficiency of the real-time PCR with the RC primer set was evaluated for each type of sample used in this study: mtLSU/T-TA, LH DNA, and OS DNA (Table 1). The exponential amplification and efficiency of the reactions were determined by evaluating the slope of the curve generated by plotting the log of known concentrations of template DNA vs. their CTs (7). The RC primer set demonstrated acceptable levels of exponential amplification and efficiency with all three templates. The organism numbers in lung tissue, quantified by microscopic enumeration, increased from log 4.69 after 4 weeks of immunosuppression to log 9.35 after 10 weeks of immunosuppression (Figure, A.). No organisms were detected in the lungs of the eight rats euthanized before the study began (level of sensitivity = ~10,000 nuclei per lung). The amount of P. carinii–specific DNA quantified by real-time PCR in the LH samples increased substantially from 0 to 7 weeks, with similar levels after 7 and 10 weeks of immunosuppression (Figure, B). Only one of eight rats euthanized at the initiation of the experiment produced quantifiable copies of P. carinii–specific DNA, with a level similar to those after 4 weeks of immunosuppression (data not shown). In every case, the postimmunosuppression OS taken from the rats at 4, 7, and 10 weeks Table 1. Efficiencies of the real-time PCR reactionsa Template Range mtLSU 0.5 to 0.0005 pgs/rx LH 12.5 to 1.25 x 10-5 ngs/rx OS Undiluted to 1:8 dilution a r2 0.999 0.966 0.973 A B C Figure. Progression of Pneumocystis carinii pneumonia measured by enumeration of organisms and real-time PCR of DNA extracted from lung homogenates and oral swabs. Panel A: Log P. carinii nuclei per mL of homogenized rat lung assessed by microscopic enumeration of lung homogenates; 4 wk vs. 7 wk, p < 0.001; 4 wk vs. 10 wk, p < 0.001; 7 wk vs. 10 wk, p < 0.001. Panel B: Logtransformed copies of P. carinii-specific DNA (mtLSU) per mL of lung homogenate; 4 wk vs. 7 wk, p < 0.001; 4 wk vs. 10 wk, p < 0.001; 7 wk vs. 10 wk, p > 0.05. Panel C: Copies of P. cariniispecific DNA/mL in oral swabs taken between immunosuppression (4, 7, and 10 wk preimmunosuppression) and at the time of euthanasia (4, 7, and 10 wk postimmunosuppression); pre- and post-4 wk, p = 0.0006; pre- and post-7 wk, p = 0.0037; pre- and post-10 wk, p = 0.0221. The ranges in value for the preimmunosuppression oral swabs at 4, 7, and 10 wk were, respectively, 165–487 copies/mL; 98–816 copies/mL; and 380–1,667 copies/mL. Ranges for the postimmunosuppression copies/mL after 4, 7, and 10 wk were 342–1,254; 517–5,256; and 637–6,279. (Number of rats in the 4- and 7- wk postimmunosuppression group = 8; number of rats in the 10-wk group = 6.) Slope -3.220 -3.570 -3.328 Amplification 2.043 1.906 1.997 Efficiency 1.043 0.906 0.997 PCR, polymerase chain reaction; mtLSU, mitochondrial large ribosomal subunit RNA; LH, rat lung homogenate; OS, oral swab P. carinii–specific DNA; rx, reaction. 1614 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 12, December 2003 DISPATCHES had significantly more P. carinii–specific DNA than the preimmunosuppression OS taken at the initiation of the study (Figure, C). The amount of P. carinii–specific DNA in the OS samples also increased over time (Figure, C). No significant correlation was found between the amount of P. carinii DNA detected in the preimmunosuppression OS samples and the amount in the postimmunosuppression OS samples, the lung homogenates, or nuclei number, suggesting that the rats had equivalent but low levels of organisms at the initiation of the study. To determine the relationship between quantitation of P. carinii by real-time PCR and by microscopic enumeration, results were analyzed by Spearman rank correlation (Table 2). A significant correlation was found between both the amount of P. carinii DNA detected in the postimmunosuppression OS samples and in the LH versus the number of P. carinii nuclei. A significant correlation was also detected between the real-time PCR quantitation of P. carinii DNA in the OS and the LH. Conclusions The combination of antemortem oral swab sampling and real-time PCR amplification and quantification reported here should be useful for the study of the Pneumocystis infections in other experimental models and provides a rationale for similar studies to be conducted in the clinical setting. Real-time PCR previously has been shown to be useful for quantitation of the level of infection in the lungs of infected rats and mice, but the studies were performed on postmortem samples or purified organisms (8,9) P. jiroveci DNA levels from oral washes, induced sputa, and bronchoalveolar lavage fluids from humans have been quantified by using various real-time PCR techniques (10–13) as well, but the findings were used for diagnosis, detection, or quantification and did not obtain samples from individual hosts over time. In our study, the levels of P. carinii DNA in the oral cavities of the rats were measured temporally and shown to correlate with the numbers of organisms in the lungs, establishing the oral swab real-time PCR technique as a surrogate means of following the progress of the infection. Successful application of this method to the human infection would enhance epidemiologic studies, permit sensitive and rapid assessment of therapeutic response, and allow basic biologic questions of carriage length and potential reservoirs to be addressed. These studies were supported by a grant from the National Institutes of Health: RO1 AI29839-10 awarded to MTC. Dr. Linke is a research microbiologist at the Veterans Affairs Medical Center in Cincinnati, Ohio. His major research interest is the role of the innate immune response in the prevention and clearance of Pneumocystis infection. References 1. Jones JL, Hanson DL, Dworkin, MS, Alderton DL, Fleming PL, Kaplan, JE, et al. Surveillance for AIDS-defining opportunistic illnesses 1992-1997. MMWR CDC Surveill Summ 1999;48:1–22. 2. Fisk DT, Meshnick S, Kazanjian PH. Pneumocystis carinii pneumonia in patients in the developing world who have acquired immunodeficiency syndrome. Clin Infect Dis 2003;36:70–8. 3. Icenhour CR, Rebholz SL, Collins MS, Cushion MT. Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs. J Clin Microbiol 2001;39:3437–41. 4. Icenhour CR, Rebholz SL, Collins MS, Cushion MT. Early acquisition of Pneumocystis carinii in neonatal rats as evidenced by PCR and oral swabs. Eukaryot Cell 2002;1:414–9. 5. Cushion MT, Ruffolo JJ, Linke MJ, Walzer PD. Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines. Exp Parasitol 1985;60:43–54. 6. Palmer RJ, Cushion MT, Wakefield AE. Discrimination of rat-derived Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. ratti using the polymerase chain reaction. Mol Cell Probes 1999;13:147–55. 7. Stahlberg A, Aman P, Ridell B, Mostad P, Kubista M. Quantitative real-time PCR method for detection of β-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression. Clin Chem 2003;49:51–9. 8. Zheng M, Shellito JE, Marrero L, Zhong Q, Julian S, Ye P, et al. CD4+ T cell-independent vaccination against Pneumocystis carinii in mice. J Clin Invest 2001;108:1469–74. 9. Larsen HH, Kovacs JA, Stock F, Vestereng VH, Lundgren B, Fischer SH, et al. Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f. sp. carinii. J Clin Microbiol 2002;40:2989–93. Table 2. Comparisons of Pneumocystis carinii–specific DNA levels in pre- and postimmunosuppression samples, lung homogenates, and organism numbers in lung homogenates assessed by microscopic enumerationa Groupsb No. points Spearman r 95% Confidence interval p value Significant LH Pc DNA vs. post-OS Pc DNA 22 0.5576 0.1648 to 0.7978 0.0070 Yes LH Pc DNA vs. Pc nuclei 22 0.9035 0.7731 to 0.9606 <0.0001 Yes Post-OS Pc DNA vs. Pc Nuclei 22 0.4636 0.0388 to 0.7465 0.0298 Yes Pre-OS Pc DNA vs. post-OS Pc DNA 21 0.3707 -0.0863 to 0.6988 0.0980 No Pre-OS Pc DNA vs Pc Nuclei 21 0.4123 -0.0374 to 0.7232 0.0633 No Pre-OS Pc DNA vs. LH Pc DNA 21 0.2939 -0.1712 to 0.6519 0.1960 No a 21 data points were included in these analyses because 2 rats from the 10-wk group died and 1 preimmunosuppression oral swab sample in the 7-wk group was lost in processing. b Pre-OS Pc DNA, P. carinii –specific DNA from oral swabs taken prior to immunosuppression; post-OS Pc DNA, P. carinii–specific DNA from oral swabs taken at the time of euthanasia; LH Pc DNA, P. carinii–specific DNA from lung homogenates of rats at the 3 different time points; log Pc Nuclei, P. carinii organism number assessed by microscopic enumeration. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 12, December 2003 1615 DISPATCHES 10. Helweg-Larsen J, Jensen JS, Benfield T, Svendsen UG, Lundgren JD, Lundgren B. Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples. J Clin Microbiol 1998;36:2068–72. 11. Helweg-Larsen J, Jensen JS, Lundgren B. Non-invasive diagnosis of Pneumocystis carinii pneumonia by PCR on oral washes. Lancet 1997;350:1363. 12. Palladino S, Kay I, Fonte R, Flexman J. Use of real-time PCR and the LightCycler system for the rapid detection of Pneumocystis carinii in respiratory specimens. Diagn Microbiol Infect Dis 2001;39:233–6. 13. Helweg-Larsen J, Masur H, Kovacs JA, Gill VJ, Silcott VA, Kogulan P, et al. Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 2002;40:490–4. Address for correspondence: Melanie T. Cushion, Department of Internal Medicine, Division of Infectious Diseases, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0560, USA; fax: 513-475-6415; email: Melanie.Cushion@med.va.gov Search past issues of EID at www.cdc.gov/eid 1616 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 12, December 2003
https://openalex.org/W1932411203	https://revistacomsoc.pt/index.php/revistacomsoc/article/download/1135/1116	Portuguese	null	A liberdade académica e os seus inimigos	Comunicação e sociedade	2,015	cc-by	8,529	1 Já em 1989, Boaventura Sousa Santos (1994: 163-201) se referia à crise por que passava então a universidade e acentuava três aspetos: tratava-se de uma crise de hegemonia, de uma crise de legitimidade e de uma crise institucional. Porque a universidade não promovia já suficientemente a desejada mobilidade social, razão pela qual via decrescer o seu contributo para a democratização do país. Porque o discurso universitário não passava de um discurso entre muitos outros, nada o garantindo como tribunal da razão. E porque, diante das crescentes exigências sociais, que sobre ela caíam em tropel, a universidade se mostrava absolutamente incapaz de lhes dar resposta. Mas, transcorrido um quarto de século, é Zara Pinto-Coelho e Anabela Carvalho (2013: 4) quem chama a atenção para o facto de as transformações de sentido pelas quais têm passado “identidade, princípios e práticas” universitárias, de tão “profundas e contraditórias”, estarem agora a gerar tensões e disputas dentro da universidade e nas relações que esta estabelece com o estado e a sociedade. Ver também, no mesmo sentido, Martins (2013: 61-72) e Nóvoa (2014: 11-21). 2 Estas conferências foram reunidas na obra de Max Weber, Le savant et le politique [O homem de estudos e o político], com introdução de Raymond Aron. Teve a primeira edição em 1959, nas Recherches en Sciences Humaines da Librairie Plon. A edição que consultámos é da Union Générale d’Éditions, Plon, Col. 10/18. As conferências aparecem como capítulos deste livro, com as seguintes designações: “A profissão e a vocação do homem de estudos” e “A profissão e a vocação do homem político”. Universidade; modernidade; vocação e missão universitárias; avaliação de desempenho; governação da universidade; liberdade académica Universidade; modernidade; vocação e missão universitárias; avaliação de desempenho; governação da universidade; liberdade académica Comunicação e Sociedade, vol. 27, 2015, pp. 405 – 420 doi: http://dx.doi.org/10.17231/comsoc.27(2015).2109 Comunicação e Sociedade, vol. 27, 2015, pp. 405 – 420 doi: http://dx.doi.org/10.17231/comsoc.27(2015).2109 A liberdade académica e os seus inimigos Moisés de Lemos Martins Resumo Em nome da certificação da ‘qualidade’ e da ‘excelência’, a Universidade parece hoje con denada a celebrar apenas procedimentos, que no ensino e na investigação certificam rotinas e conformidades, eficiências e utilidades, confirmando a hegemonia da razão instrumental. É meu propósito, neste ensaio, todavia, refletir sobre a liberdade académica na universidade. Esta ques tão impõe que façamos recair a nossa interrogação sobre a natureza da própria universidade, sobre a profissão académica, e também sobre a sua vocação e missão. O que é, hoje, a univer sidade? Que forças a atravessam? Que abalos tem sofrido? A que ameaças está sujeita? Que contradições são as suas? Com que exigências se confronta? Que respostas têm que ser as suas? 4 A palavra é, por excelência, o grande mito da civilização ocidental. É este o ponto de vista que defendo em “Ce que peu vent les images. Trajet de l’un au multiple” (Martins, 2011 a). Com efeito, a nossa razão é discursiva, tanto na tradição greco-latina, como na tradição judaico-cristã. Para Aristóteles, por exemplo, o homem define-se pela linguagem. E como a linguagem é o caminho que nos conduz a outro, o homem é um “animal político”, expressão que encontramos tanto na Política, como na Ética a Nicómaco. Mas já os pré-socráticos olhavam para a palavra como aquilo que salva. Veja-se, por exemplo, o que diz Roland Barthes (1970) em “L’ancienne réthorique”, a propósito da criação do primeiro tratado de argu mentação, por Córax e Tísias. Quanto à tradição judaico-cristã, deparamos logo no início do Evangelho de São João (1, 1) com a proclamação de uma razão discursiva: “No começo era o Verbo e o Verbo era Deus”. Esta herança acompanhou-nos sempre e com ela atingimos a modernidade. Vemos isso em Nietzsche (1887, II, parág. 1), para quem somos animais de promessa, os únicos animais capazes de prometer. Vemo-lo também em Jorge Luís Borges, com a promessa a cumprir-se na dimensão ilocucionária da linguagem. No poema Unending Gift, Borges assinala que “na promessa alguma coisa existe de imortal”. E George Steiner (1993: 127) não diz coisa diferente em Presenças Reais: “A linguagem existe […] porque existe ‘o outro’”. Ou seja, a palavra é o caminho do encontro e o outro é o nosso destino. 3 O ponto de vista que temos sobre a verdade desfundamenta, todavia, o conceito de verdade, que ainda parece presente no texto de Raymond Aron. Subscrevemos, com efeito, um princípio de historicidade e um princípio hermenêutico, em que, pela invasão total do campo de conhecimento pelo discurso, a verdade é uma simples função discursiva (veja-se Martins, 1994: 5-18). Introdução Embora a reflexão sobre a vocação e missão universitárias estejam, hoje, na ordem do dia, como o demonstram, em obra recente, Zara Pinto Coelho e Anabela Carvalho (2013: 4- 14)1, o meu ponto de vista retoma um debate introduzido, há quase um século, por Max Weber, em duas conferências que proferiu, uma em 1917, outra em 1919: “Wis senchaft als Beruf”; e “Politik als Beruf”2. Retomo Max Weber, porque no debate sobre a universidade me interessa compreender, tanto aquilo que separa a ciência e a política, como, do mesmo modo, aquilo que as une. Como assinala Raymond Aron (1974: 8), na introdução à obra Le savant et le Politique, de Max Weber: “Não se pode ser ao mesmo tempo homem de ação e homem de estudos, sem comprometer a dignidade de uma e Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins de outra profissão, sem falhar a vocação de uma e de outra. Mas podemos assumir po sições políticas fora da universidade, e a posse do saber objetivo, não sendo porventura indispensável, é certamente favorável a uma ação razoável”. Uma teoria da ação é sempre “uma teoria do risco, e também uma teoria da causa lidade” (Ibidem), e é por essa razão, exatamente porque “o real não está escrito antecipa damente”, que a marcha da história depende de pessoas concretas e de circunstâncias específicas (Ibid.: 9). Mas a necessidade de fazer opções contextuais não obriga a que o pensamento viva de decisões “essencialmente irracionais” e que a existência se cumpra numa liberdade “que recuse submeter-se à Verdade” (Aron, 1959: 52)3. A nossa época é atravessada por uma força que a domina e lhe dá forma. Refiro-me à mobilização tecnológica para o mercado. A essa cinética do mun do chamámos-lhe, primeiro com Jünger (1930[1990]), depois com Sloterdijk (2000), uma mobilização ‘total’ e ‘infinita’ para o mercado. Por outro lado, desfez-se o mito que fundou o ocidente, o mito da palavra, um mito associa do a um espaço de promessa4. A promessa projetava uma ideia de futuro e dava-nos garantias sobre ele. Introdução Lançava um propósito par diante e dava-nos um fundamento seguro (essência, substância, Deus, transcendência, sujeito, homem, existência, consciência…) (Derrida, 1967: 410-411), um território conhecido (entre uma génese e um apocalipse, uma história da salvação, por exemplo, o Reino de Deus, a sociedade sem classes, uma sociedade esclarecida pe las Luzes do Progresso, com a razão a sobrepor-se às superstições)5 e uma identidade estável (sendo nós à imagem de Deus, ou então, aspi rando à fraternidade, o homem não sendo mais o lobo do homem…). A promessa projetava uma ideia de futuro e dava-nos garantias sobre ele. Lançava um propósito par diante e dava-nos um fundamento seguro (essência, substância, Deus, transcendência, sujeito, homem, existência, consciência…) (Derrida, 1967: 410-411), um território conhecido (entre uma génese e um apocalipse, uma história da salvação, por exemplo, o Reino de Deus, a sociedade sem classes, uma sociedade esclarecida pe las Luzes do Progresso, com a razão a sobrepor-se às superstições)5 e uma identidade estável (sendo nós à imagem de Deus, ou então, aspi rando à fraternidade, o homem não sendo mais o lobo do homem…). As tecnologias mobilizam-nos, em contrapartida, para as urgências do presente – é esta a cinética do mundo, uma mobilização para o presente (Martins, 2010). Numa 3 O ponto de vista que temos sobre a verdade desfundamenta, todavia, o conceito de verdade, que ainda parece presente no texto de Raymond Aron. Subscrevemos, com efeito, um princípio de historicidade e um princípio hermenêutico, em que, pela invasão total do campo de conhecimento pelo discurso, a verdade é uma simples função discursiva (veja-se Martins, 1994: 5-18). 5 A ideia de território conhecido, com uma narrativa que se desenvolve entre uma génese e um apocalipse, pode ser escla recida pela noção de “verdade”, como origem e fim de uma história de sentido, em que a origem é desocultada na forma de uma arqueologia e o fim antecipado na forma de uma escatologia. Sobre este assunto, ver Martins (1994). 406 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins civilização tecnológica, uma civilização centrada nos números, às palavras da promessa sucedem os números da promessa, que são sempre os números do crescimento eco nómico, os do Produto Interno Bruto (PIB) e os números das exportações, ou seja, os números do superavit da Balança Comercial. 6 Como já referimos, Raymond Aron, na introdução a Le Savant et le Politique, não deixa de enquadrar o pensamento de Max Weber por relação à grande categoria da Verdade, ao advogar que a existência não pode cumprir-se numa liberdade “que recuse submeter-se à Verdade” (Aron, 1959: 52). O nosso ponto de vista advoga, todavia, a desfundamantação do concei to de verdade, afastando-se de Aron. Como refere Derrida (1967: 412), que nos serve de referência, a desfundamentação do conceito de verdade é uma conquista do nosso tempo e entre os nomes mais emblemáticos desta conquista talvez devamos referir Nietzsche (e a sua critica da metafisica, o que quer dizer, a sua ideia de jogo, de interpretação e de signo sem verdade presente); assim como Freud (e a sua crítica da presença-a-si-próprio, quer dizer, a critica da consciência, do sujeito, da identidade em si próprio, da proximidade e da propriedade de si próprio); e ainda Heidegger (e a destruição da metafísica, a destruição da onto-teologia, a destruição da determinação do ser como presença). Introdução Para a promessa temos agora, fundamen talmente, os economistas, os engenheiros e os gestores; são eles os magos do presente, e não mais os políticos, os padres e os juristas. A atual cinética do mundo e a universidade É este o contexto em que vamos encontrar as universidades. Elas estão sujeitas à mesma cinética do mundo, a da mobilização tecnológica para o mercado, o que quer dizer, a da resposta às exigências de uma civilização dos números (Martins, 2013, 2003, 1993). Tradicionalmente, a universidade tinha como promessa: servir a Verdade6. Daí de corria o seu principal objetivo, a investigação, porque a verdade apenas a alcança quem a procura sistematicamente. Mas a verdade era mais do que ciência, pelo que a univer sidade retirava daí o seguinte objetivo: servir a cultura, mostrando-se capaz de educar o homem no seu todo. E depois, a verdade transmite-se, pelo que a universidade tinha que se consagrar ao ensino. Até o ensino das profissões era ordenado pelo princípio de uma formação integral. Mas o que vemos, agora, é a ideia de marketing ser aplicada ao sistema de ensino. Trata-se de a universidade colocar no mercado produtos com forte probabilidade de se rem comprados. E daí decorre que o ensino seja convertido em comércio, os professores se tornem profissionais de serviços e consultores, com os diretores comerciais, ou seja, os diretores das Escolas e Faculdades, a centralizar a direção deste comércio. A avaliação do produto, o seu ‘perfil’, é determinada a partir de cima, segundo critérios burocráticos, dependentes das leis do mercado, do comércio e do marketing, e também da sua visibili dade mediática. E os projetos de ensino considerados mais ‘frágeis’, aqueles que se des tinam a grupos demasiado restritos de ‘consumidores’, são eliminados impiedosamente. E podemos dizer a mesma coisa da investigação fundamental. Desde o começo dos anos 90 que não tem parado de crescer a tendência para fazer depender a validade científica dos projetos de investigação das suas virtualidades para responder a necessi dades sociais práticas. Mesmo nas ciências sociais e humanas, os projetos de investi gação não escapam às injunções do mercado, para a ‘qualidade’, a ‘excelência’, a ‘com petitividade’, a ‘eficiência’, a ‘relevância’, o ‘empreendedorismo’, a ‘empregabilidade’, 407 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins o ‘desenvolvimento económico e a criação de emprego’, o uso do inglês como língua única de ciência (Shore & Wright, 1999; Power, 2000; Martins, 2008, 2012 a, 2012 b, 2013; Martins & Oliveira, 2013; Nóvoa, 2014). A atual cinética do mundo e a universidade Com efeito, no nosso mundo não parece haver mais mundo que não sejam ne cessidades de mercado e injunções financeiras. É também a conclusão a que se chega ao depararmo-nos com o novo European Union Framework Programme for Research and Innovation, Horizon 2020. O ‘desafio-chave’ aí enunciado é o de “estabilizar o sistema económico e financeiro, enquanto se tomam medidas para criar oportunidades eco nómicas” (European Commission, 2013). Com efeito, do que se trata, agora, é da inteira submissão da política científica europeia a uma estratégia empresarial. Essa dependência sai reforçada no recente documento da Comissão, intitulado Research and innovation as sources of renewed growth (COM(2014)339 final). A secção “Increasing impact and value for money”, é precisa nos objetivos: “Raising the quality of public spending on research and innovation”. E entre as conclusões apontadas pelo documento merece especial des taque a seguinte: “O investimento [em I&D] precisa de ser acompanhado por reformas que desenvolvam a qualidade, a eficiência e os impactos da despesa pública em I&D, incluindo por alavancagem de investimento das empresas em I&D” (p. 12)7. Por sua vez, além da Comissão Europeia, outras Agências de financiamento (refiro as brasileiros, CAPES e CNPq, e a portuguesa FCT), e também as empresas não contem porizam mais com o que consideram não ter utilidade social. E por certo a sociedade civil também não, o mesmo se passando com as editoras, que não querem ouvir falar da publicação de investigação fundamental, argumentando que não vão ter leitores. É um facto, todos os setores da vida coletiva colocam, hoje, a universidade sob vigilância, em nome da ‘accountability’, sendo medida esta em termos de “valor económico” (Barr, 2012: 438-508). Em síntese, o que está a acontecer com a explosão da técnica é que o nosso tem po acelerou e foi mobilizado para o mercado. E também nas universidades, através das atuais políticas para o ensino e a investigação, ocorre idêntico processo, com o controle tecnológico da ciência da informação. Refiro, por exemplo, a convocação permanente a que se está sujeito, através das plataformas informáticas, na mobilização acelerada de professores e alunos para o mercado e para o ranking. Como bem salienta Hermínio Martins, fomos capturados pelo discurso da “Universidade da Excelência-como-negó cio” do máximo de “Throughput” (H. 7 A submissão da política europeia a uma estratégia empresarial é a linha geral deste documento da Comissão Europeia, de que respigo mais uma passagem: “Os desenvolvimentos na qualidade e eficiência da despesa podem contribuir para a criação de um ciclo virtuoso, através do alavancamento de níveis de maior investimento com origem em setores privados, gerando retornos económicos crescentes. As reformas para desenvolver a qualidade e a eficiência da despesa pública são importantes para todos os Estados Membros. Sobretudo, no caso daqueles que se encontram mais constrangidos financeira mente e são menos eficientes na despesa, é vital acrescentar impacto, com reformas de longo alcance, e aumentar sabiamente o investimento, à medida que as suas economias recuperem. Por outro lado, para aqueles que têm um espaço fiscal adequado e alta eficiência, vão surgir apoios que tornem mais esclarecidos os seus investimentos, no sentido de dar mais valor ao dinheiro” (COM(2014)339 final), p.5) [é nosso o destaque em itálico]. 10 De acordo com o “Diagnóstico do sistema de investigação e inovação: Desafios, forças e fraquezas rumo a 2020”, rea lizado pela Fundação para a Ciência e a Tecnologia (FCT), a percentagem de doutores nas empresas nacionais é de 2,6% [http://www.fct.pt/esp_inteligente/diagnostico]. O Diário de Notícias, de 13 de maio de 2013, deu conta da conferência públi ca onde foram apresentados os resultados deste Diagnóstico, assinalando que “Portugal tem a mais baixa percentagem de emprego de doutorados de um conjunto de 10 países europeus, com 2,6%, contra, por exemplo, 34% na Holanda ou Bél gica”. http://www.dn.pt/inicio/portugal/interior.aspx?content_id=3216596&page=-1 (consulta feita a 13 de maio de 2013). 8 A figura de ‘travessia’ tomo-a de João Guimarães Rosa (2001) [1967], em O Grande Sertão: Veredas. Podemos fazer, por exemplo, a passagem de um rio de uma para outra margem. Nessa experiência, não se esperam sobressaltos nem grandes obstáculos a transpor; espera-se uma viagem tranquila, a menos que a façamos a nado, como assinala João Guimarães Rosa (2001: 51). Nas passagens existe, com efeito, a habitualidade de um caminho conhecido. Coisa diferente é, todavia, a experiência de uma travessia, que nos coloca sempre em sobressalto pela sua perigosidade. É o perigo que a caracteriza fundamente: fazemos a travessia de um oceano; de um mar de tentações; de um deserto... (ver também, Martins, 2011 b: 60-61). A atual cinética do mundo e a universidade Martins, 2004), uma descrição de universidade que já nada tem a ver com a descrição que delas fazia Eliot Freidson (1986: 436): “in venções sociais notáveis para apoiar o trabalho que não tem valor comercial imediato”. 408 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Sendo o nosso tempo de mobilização tecnológica, exige-se, hoje, um novo tipo de professor e de aluno, e também um novo tipo de investigador. Cada vez com menos di reitos sociais, professores, investigadores e alunos têm, de ora em diante, por condição a mobilidade permanente, fazendo a travessia das necessidades do mercado8. E eles aí estão, os novos investigadores, em programas de mobilidade, de país em país e de universidade em universidade. É-lhes imposto que sejam competitivos e em preendedores, que promovam o auto-emprego, ou o emprego em geral, que criem spin off, por exemplo. E exige-se-lhes que sejam produtivos, realizadores de sucesso9. E é então que cresce a legião dos doutorandos e pós-doutorados, jovens à procura da redenção de uma bolsa de investigação, que o mais que lhes permite é a possibili dade de andarem de congresso em congresso, de projeto de investigação em projeto de investigação, batendo à porta das revistas científicas e correndo atrás de um ranking qualquer ou da miragem de um prémio científico. A justificar esta travessia, própria de uma condição nómada, sem direitos sociais, o discurso oficial ganha novos argumen tos: acrescenta-se que a economia, ou seja, as empresas, não os absorvem, que in vestigadores, professores e estudantes estão a mais no mercado de trabalho, que são dispensáveis, que são bons é para a emigração10. 9 As próprias universidades estão a tornar-se em incubadoras de empresas, apoiando os seus ex-alunos, com capitais próprios e a fundo perdido, no desenvolvimento de atividades empresariais. O quotidiano académico e a governação das universidades Sendo este o atual contexto, reflitamos sobre o quotidiano académico, e também sobre a governação das universidades. Aquilo que faz a natureza da universidade, hoje, é a ideologia comercial: as uni versidades são empresas; a educação são serviços; o ensino e a investigação são opor tunidades de negócios; os professores são profissionais de serviços ou consultores; os alunos são clientes. E com o mercado financeiro e o mercado de trabalho a ribombar fantasticamente por cima da sua cabeça, a universidade faz manchete da ‘excelência’ dos seus cursos e professores, ou seja, faz manchete da sua ‘qualidade’. Mas o que é a ‘excelência’ de que tanto se fala? A excelência é medida pelos índices de procura de uma instituição. São, também, as classificações de entrada numa dada 409 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins universidade. São, além disso, as taxas de sucesso escolar. E ainda, os índices de empre gabilidade dos antigos alunos, assim como uma rede constituída e alargada de alumni de sucesso. A ‘excelência’ de uma universidade não dispensa, hoje, um lugar no ranking das 500 melhores universidades, segundo o Times Higher Education, ou o ranking das 100 melhores universidades com menos de 50 anos. Como também não dispensa um lugar no Academic Ranking of World Universities (também conhecido como Ranking de Xangai), das mil melhores universidades do mundo, ou no mais recente CWTS Leiden Ranking, estabelecido na base de citações ISI. Mas a ‘qualidade’ académica não para aí. Ela mede-se, igualmente, pelos artigos ISI, da Thomson Reuters, ou pelos artigos Scopus, da Elsevier, ou ainda pelas citações no Google Scholar. E não dispensa os prémios dos docentes, pelas citações em revistas de fator de impacto, a capacidade de captação de fundos e de obtenção de projetos inter nacionais, e ainda, a visibilidade que a universidade possa ter no espaço público, sendo tal visibilidade estabelecida pelas notícias que sobre ela são publicadas nos média. p q p Para dar um exemplo, o site institucional da Universidade do Minho, no Norte de Portugal, consagra oito rubricas à presença da Universidade no espaço público. (1) Nós – jornal on-line. Os editores apresentam-no assim: “A UMinho em revista todos os meses. Aqui pode encontrar reportagens, entrevistas, percursos, opiniões e a agenda com os principais acontecimentos”. (2) Em agenda. 11 Na era dos média, sem fundamento seguro, território conhecido e identidade estável (Martins, 2002 a, 2002 b), a condi ção e o desempenho universitários apenas podem ser melancólicos (Martins, 2003). O quotidiano académico e a governação das universidades Ou seja “Dia-a-dia, o calendário académico e todos os restantes acontecimentos: congressos, seminários, campanhas, cerimónias, prémios e eventos diversos”. (3) Atualidade: “A cada instante, informação atualizada so bre o que de mais relevante acontece na Universidade do Minho”. (4) Clipping: “O olhar dos média sobre a UMinho. Tudo o que é publicado na TV, rádio, imprensa escrita e in ternet está aqui disponível”. (5) Perfil: “Vai conhecer aqui histórias de alunos, docentes, investigadores e funcionários da UMinho que se destacam nas mais diferentes áreas”. (6) Galeria de fotos: “As imagens que mostram a UMinho”. (7) Área Press: “Esta área é dedicada aos profissionais de comunicação social. Contacte-nos e coloque questões, dúvidas, sugestões”. E, finalmente, (8) O que os media dizem de nós. Universidade em notícia: semana a semana, um repositório de informações, que assinalam a passagem dos professores pelos plateaux da notícia: pelos estúdios de rádio e televisão, e também pelas redações dos jornais.11 Entretanto, na governação das universidades, os modelos “gerencialistas e econo micistas” levam hoje a melhor sobre os “modelos colegiais clássicos” (Ruão, 2008: 15). E é assim que a identidade universitária adquiriu um formato meramente instrumental e as estratégias de comunicação desenvolvidas na universidade passaram a ter uma preocu pação, cada vez mais acentuada, com a produção de efeitos estratégicos (Ruão, Ibid.: V). Nestas circunstâncias, controlar a comunicação para produzir efeitos estratégicos é hoje a tarefa dos Gabinetes de Imprensa das universidades, também denominados Gabinetes de Comunicação e Imagem. A maior parte das universidades tem hoje uma 410 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Pró-Reitoria de Comunicação e Imagem. E o seu objetivo é administrar no espaço públi co as políticas das universidades. Entretanto, as universidades mostram-se cada vez mais incapazes de dar resposta à pressão crescente das exigências sociais - porque tudo lhes é pedido. Pede-se às uni versidades que deem resposta: às necessidades de desenvolvimento económico; à cria ção de empregos; à modernização do país; à inovação tecnológica; à competitividade internacional; à necessidade de promoção da coesão social; ao combate às assimetrias étnicas e de género; à promoção da inclusão de minorias; enfim, à necessidade de com bate à iliteracia mediática e digital. E nós resignamo-nos a que as políticas académicas se confinem, hoje, a estratégias de gestão e as necessidades de crescimento se acomodem a respostas de caráter mera mente tecno-instrumental. 12 Veja-se, neste sentido: http://www.uminho.pt/uminho/qualidade/documentacao-do-sigaq-um 13 O processo de avaliação está, por seu lado, indexado a um regime remuneratório, que determinará as subidas de escalão na categoria académica. No entanto, este regime remuneratório não chegou a entrar em vigor, dado o facto de as carrei ras na função pública, em Portugal, se encontrarem congeladas, desde a primavera de 2011, momento em que este país passou a reger-se por um programa de austeridade, decretado pelas instituições internacionais a quem solicitou “ajuda financeira”. Foi a 3 de maio de 2011 que o primeiro-ministro de Portugal, José Sócrates, anunciou as medidas de austeri dade decididas pelo Comissão Europeia, Banco Central Europeu e Fundo Monetário Internacional (Troika), no quadro de um programa de “assistência financeira”. O quotidiano académico e a governação das universidades Com efeito, nada na universidade aponta, hoje, para aprender e ensinar a ver, nem para aprender e ensinar a pensar, como ensinou Nietzsche, no Crepús culo dos Ídolos (Nietzsche, 1988/1888: 67-68). Aprender e ensinar a ver, ou seja: habituar os olhos à calma, à paciência, deixar que as coisas se aproximem de nós; aprender a adiar o juízo, a rodear e a abarcar o caso particular a partir de todos os lados. E aprender e ensinar a pensar significa aprender e ensinar uma técnica, um plano de estudos, uma vontade de mestria - que o pensar deve ser aprendido como é aprendido o dançar, como uma espécie de dança... Readings inquieta-se, todavia: como é possível pensar numa instituição “cujos de senvolvimentos tendem a tornar o pensamento cada vez mais difícil e cada vez menos necessário?” (Readings, 1996: 175). E, todavia, o ideal académico não pode satisfazer-se com a atual mobilização operativa, financista e economicista, sem pensamento, sem comprometimento social e político, e sem o critério ético do desassossego crítico. Penso que a universidade deve ser encarada como um lugar de liberdade irrestrita. A universidade tem como missão a salvaguarda das possibilidades da (a) aventura do pensamento. Cabe-lhe fazer do ensino e da ciência uma ideia, que encarne um princípio de resistência crítica e uma força de dissidência, ambos comandados por aquilo a que Jacques Derrida (2001: 21) chamou, em tempos, “uma justiça do pensamento”. No entanto é neste contexto, em que as políticas académicas se confinam a estra tégias de gestão e as necessidades de crescimento se acomodam a respostas de caráter meramente tecno-instrumental, que foram criadas as Vice-Reitorias para a Qualidade e a Excelência nas universidades portuguesas. A Universidade do Minho foi, em Portugal, uma das primeiras universidades a ter um SIGAQ (Sistema Interno de Garantia da Qualidade) e uma Vice-Reitoria, que vela pelo funcionamento deste sistema, com a institucionalização de um Plano da Qualidade e de um Manual da Qualidade12. Este Sistema Interno de Garantia da Qualidade foi au ditado em Outubro de 2012 pela A3ES – Agência de Avaliação e Acreditação do Ensino Superior, em Portugal, e certificado por esta Agência, em Janeiro de 2013, por um perío do de seis anos. 411 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . O quotidiano académico e a governação das universidades Moisés de Lemos Martins Cingindo-nos a um ponto de vista meramente académico, diria todavia que os SIGAQ têm como efeito prático estender sobre os professores a dominação, ou seja, o controle tecnológico e a mobilização para o mercado (e para o ranking, que é uma con sequência do mercado). Aquilo que um SIGAQ produz no quotidiano académico é a entronização de proce dimentos corretivos e ortopédicos, que certificam no ensino e na investigação rotinas e conformidades, eficiências e utilidades. E no que diz respeito aos projetos e à extensão universitária, registam e arquivam informação, de modo a garantir os overheads institu cionais, hoje tão importantes na política de auto-financiamento de uma universidade, quando os financiamentos públicos parecem ter entrado em fase de irreversível restrição. Regulamentos de avaliação do desempenho dos docentes Não propriamente no quadro dos SIGAQ, mas articulados com ele, foram criados os RAD – Regulamentos de Avaliação do Desempenho dos docentes. Resultam de uma lei geral, uma Lei do Estado português (Lei n. 205/2009, de 31 de Agosto), que está em continuidade com uma profunda reforma do Ensino Superior, o Regime Jurídico das Instituições do Ensino Superior (RJIES) (Lei n.º 62/2007, de 10 de Setembro). Os RAD adequam esta Lei às condições concretas de cada uma das universidades do país, e mesmo a cada Faculdade ou Escola de uma universidade. O Regulamento de Avaliação do Desempenho dos Docentes da Universidade do Minho (RAD-UM) foi aprovado em Diário da República, a 18 de junho de 2010. O pro cesso compreende uma auto-avaliação dos docentes, de resposta expressa quantitativa mente, a um sem-número de quesitos, fixados por um conselho de avaliação interno à universidade. Cada uma das Escolas adequa ao seu contexto específico as determinações gerais. E os professores catedráticos intervêm no processo, homologando-o, podendo, todavia, alterar a pontuação, no caso de acharem que a auto-avaliação não é rigorosa. Nos termos da Lei, todos os Regulamentos de Avaliação do Desempenho se es praiam por quatro rubricas, que compreendem a investigação, o ensino, a extensão univer sitária e a gestão universitária. O desempenho académico consiste no cumprimento pelo docente do conjunto de quesitos, fixados por um conselho de avaliação, em cada uma das rubricas13. Vou tomar, como exemplo, dois modelos de Regulamento de Avaliação de Desem penho dos docentes. O primeiro modelo é o do Instituto de Ciências Sociais da Universi dade do Minho. Trata-se de um modelo que permite que todos os docentes, sem grande esforço, se auto-avaliem com um desempenho excelente (para cima de 80 pontos em 100, em cada uma das dimensões: investigação, ensino, extensão académica e gestão 412 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins académica). Diria que é um modelo burocrático, que serve propósitos administrativos, e que é, portanto, um modelo sem grande critério académico14. O segundo modelo é da Universidade da Beira Interior. Vou cingir-nos às partes comuns a todas as Faculdades, e também às especificidades que o modelo comporta, no que respeita às Faculdades de Ciências Sociais e Humanas, e Artes e Letras. 14 Junto, em anexo, as peças que compõem, no Instituto de Ciências Sociais da Universidade do Minho, a ficha de avaliação do docente, de acordo com o Regulamento de Avaliação e Desempenho dos Docentes da Universidade do Minho (RAD -UM), aprovado no Diário da República, 2.ª série, n. 117, de 18 de junho de 2010. Regulamentos de avaliação do desempenho dos docentes É um modelo que assenta no princípio da “diferenciação qualitativa da produção científica”, um princí pio que determina que “classificações de desempenho científico mais elevado correspon dam a patamares de produção científica mais exigentes, em detrimento da massificação da produção científica em patamares considerados cientificamente menos relevantes”. A proposta, manifestando embora uma grande preocupação académica, tem um caráter didático. Estipula o seguinte: “os degraus sucessivos de exigência devem saturar através de uma atividade científica digna e moderada”. Mas o que se pretende é que “os docentes, em especial os ainda mais frágeis em termos de produção científica, não sejam forçados a desperdiçar muito do seu tempo nesses patamares”, procurando-se antes que se sintam “motivados a alcançar o patamar seguinte até à categoria A, neces sariamente exigente, mas não inalcançável, caso contrário tenderia a ser ignorada”. Ainda no mesmo sentido, de uma manifesta preocupação, simultaneamente aca démica e didática, o Regulamento propõe que seja possível “saturar a soma das pontua ções das categorias D, C e B”, atribuindo “pontuações relativamente elevadas a tarefas fundamentais da atividade docente”, mas que, “se consideradas em pé de igualdade com atividades internacionalmente mais relevantes teriam que ser contabilizadas com muita parcimónia, como atividades científicas internas e nacionais”. São propostas quatro categorias de classificação, sendo a categoria A de grande exigência. Nestas circunstâncias, a proposta de Regulamento estipula o seguinte: “a ca tegoria A é o ‘rosto’ mais visível das opções estratégicas da Universidade, e do nível de exigência da UBI”. Por essa razão, “será decidida centralmente pelo conselho coordena dor da avaliação, que harmonizará o mesmo grau de exigência para todas as faculdades”. Fixo-me na categoria A de avaliação do desempenho académico, no que respeita à rubrica Investigação. Prémio científico internacional relevante * Livro autoral científico/coletânea de autor com mérito compatível, assinalado por peritagem solicitada pela comissão avaliadora 70 Edição e/ou tradução de fontes e de clássicos, com introdução e apara to crítico, aferida por peritagem solicitada pela comissão avaliadora 50 Capítulo de livro em obra de referência internacional (até dois autores) 25 Regulamentos de avaliação do desempenho dos docentes De acordo com um ideal universitário comum a todas as Faculda des da Universidade, este ideal é considerado na sua complexidade e abrangência, tendo presente designadamente, o critério da exigência da internacionalização da ciência, o critério da comparabilidade internacional, e ainda, o critério financeiro, que sinaliza a importância dos projetos científicos: Prémio científico internacional relevante * Livro autoral científico/coletânea de autor com mérito compatível, assinalado por peritagem solicitada pela comissão avaliadora 70 Edição e/ou tradução de fontes e de clássicos, com introdução e apara to crítico, aferida por peritagem solicitada pela comissão avaliadora 50 Capítulo de livro em obra de referência internacional (até dois autores) 25 14 Junto, em anexo, as peças que compõem, no Instituto de Ciências Sociais da Universidade do Minho, a ficha de avaliação do docente, de acordo com o Regulamento de Avaliação e Desempenho dos Docentes da Universidade do Minho (RAD -UM), aprovado no Diário da República, 2.ª série, n. 117, de 18 de junho de 2010. 413 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Artigo científico indexado à Qualis A1-B1 ou inde xado à ESF na categoria INT 1 ou INT 2 25 Artigo científico com revisão indexado à ISI ou Scopus numa re vista do 1º quartil do IF ou do RIP da sub área da revista. 25 Coordenador de um projeto Europeu H2020 ou de um projeto internacional que inclua um mínimo de universidades ou centros de investigação de três países diferentes e um montante de financiamento superior a 150 000 euros 40 Coordenador nacional de um projeto Europeu ou de um projeto internacional que inclua um mínimo de universidades ou centros de investigação de três países diferentes e um montante de financiamento superior a 150 000 euros 20 Bolsa individual internacional obtida em ambiente competitivo 15 Relatórios técnicos em grandes colaborações inter nacionais (mais de três países envolvidos) 15 Exposição ou exibição em eventos internacionais (congressos, mu seus, galerias de arte, festivais, mostras, etc.), individuais ou coleti vas, aferida por peritagem solicitada pela comissão avaliadora 50 Tabela 1 * - Classificação variável até pontuação máxima de 100 pontos proposta pela Comissão Avaliadora e homologada pelo Conselho Coordenador de Avaliação. Regulamentos de avaliação do desempenho dos docentes - A proposta de classificação de livro autoral na categoria A deve vir acompanhada de parecer solicitado pela Comissão avaliadora. - Obras de referência internacional são obras publicadas no estrangeiro em editora de referência, reconhecida como tal pela comissão de avaliação. - Exposição ou exibição internacional é uma exposição ou exibição realizada no estrangeiro ou, sendo em Portugal, com a participação de pelo menos 50% de artistas estrangeiros ou coorganizada por uma entidade estrangeira. 15 Aron, A. (1974) [1959]. “Introduction”. Weber, M., Le savant et le politique. Paris: Union Générale d’Éditions, Plon, col. 10/18, pp. 5-52. 15 Ver Regulamento de avaliação do desempenho dos docentes da Universidade da Beira Interior, Despacho n.º 10129/2014, publicado Diário da República, 2.ª série — N.º 150 — 6 de agosto de 2014. Uma nota final A nossa modernidade viu a razão instrumental tornar-se hegemónica. Foi, com efeito, a hegemonia do paradigma epistemológico que conduziu à racionalidade técnica e ao economicismo (Martins, 1993: 345). A Universidade converteu-se, então, numa rea lidade simultaneamente local e total. Ela tanto é uma realidade heterogénea e específica, como uma realidade homogénea e global. Sem dúvida, a universidade deveio uma reali dade fragmentada, como consequência da crise das teorias da verdade e do fundamento. Mas, simultaneamente, é uma realidade fecundada por uma condição translocal, porque é essa, de igual modo, a sua condição e a missão de sempre. Esperemos, todavia, que a desenfreada mobilização tecnológica para o mercado, para a estatística e para o ranking, assim como a entronização de procedimentos corretivos e ortopédicos, que certificam no ensino e na investigação rotinas e conformidades, eficiências e utilidades, não afun dem o pensamento, e desse modo não afundem a própria ideia de universidade. Aron, A. (1974) [1959]. “Introduction”. Weber, M., Le savant et le politique. Paris: Union Générale d’Éditions, Plon, col. 10/18, pp. 5-52. Referências 414 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Barr, E. (2012). “The Higher Education White Paper: The Good, the Bad, the Unspeakable – and the next White Paper”. Social Policy and Administration, (46, n. 5), pp. pp. 438-508. Barthes, R. (1970). “L’ancienne rhétorique”. Communications, n. 16, pp. 172-229. Borges, J. L. (1998) [1969]. “The Unending Gift”, Elogio da Sombra, in Obras Completas (1952-1972), II. Lisboa: Teorema. Conselho Científico das Ciências Sociais e das Humanidades da FCT (2011). Ciências Sociais e Humanidades, mais excelência, maior impacte. Relatório final (28.12.2011). http://www.fct.pt/conselhos_cientificos/ docs/rel_final_cccsh_2011_pdf. Derrida, J. (2001). L’université sans condition. Paris: Galilée. Derrida, J. (2001). L’université sans condition. Paris: Galilée. Derrida, J. (1967). “La structure, Ie signe et Ie jeu dans Ie discours des sciences humaines”, L ‘écriture de la différence. Paris: Seuil, pp. 409-428. European Commission (2013). Horizon 2020: The new EU framework programme for research and innovation, 2014-2020. [Presentation]. Retrieved October, 5, 2013 from http://ec.europa.eu/research/horizon2020/ pdf/press/horizon2020-presentation. pdf European Commission (2014). Reserch and innovation as sources of renewed grouth. Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of Regions. COM (2014)330 final Freidson, E. (1986). “Les peofessions artistiques comme défi à l’analyse sociologique”. Revue Française de Sociologie, (27, n.3). Paris: pp. 431-443. Guimarães Rosa, João (2001) [1967]. 0 Grande Sertão: Veredas. Rio de Janeiro: Nova Fronteira. Na base da 5.” Edição. Jünger, E. (1914) [1990]. La mobilisation totale, in L’État universel – suivi de La mobilisation totale. Paris: Galimmard. Martins, H. (2004). “The marketisation of universities and some cultural contradictions of academic capitalism”. Metacrítica. http://www.adelinotorres.com/sociologia.htm Martins, M. L. (2013). “Interview with Moisés de Lemos Martins”. “Pinto-Coelho, Z. & Carvalho, Anabela (Eds.) Academics Responding to Discourses of Crisis in Higher Education and Research. Braga: CECS, Universidade do Minho, pp. 61-72. http://repositorium.sdum.uminho.pt/handle/1822/29224 Martins, M. L.; Oliveira, M. (2013). Política científica de comunicação em Portugal: desafios e oportunidades para os doutoramentos. In: Kunsch., M. (Org.). La comunicación en Iberoamérica: políticas científicas y tecnológicas, postgrado y difusión de conocimiento. Quito: Ciespal/Confibercom, pp. 47-101. Martins, M. L. (2012 a). “Revistas científicas de ciências da comunicação em Portugal: da divulgação do conhecimento à afirmação do Português como língua de pensamento e conhecimento”. Intercom - Revista Brasileira de Ciências da Comunicação, (35, n.1). São Paulo, pp. 233- 251. http://repositorium. sdum.uminho.pt/handle/1822/23768 Martins, M. L. (2012 b). Referências “A política científica e tecnológica em Portugal e as ciências da comunicação: prioridades e indecisões”. In: Kunsch, M. & Melo, J. M. (Org.). Comunicação Ibero-americana: sistemas midiáticos, diversidade cultural, pesquisa e pós-graduação. 1ed.São Paulo: Confibercom & Escola de Comunicação e Artes da Universidade de São Paulo, v. 1, pp. 331-345. http://repositorium.sdum. uminho.pt/handle/1822/23931 415 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Martins, M. L. (2011 a). “Ce que peuvent les imagens. Trajet d e l’un au multiple”. Les Cahiers Internationaux de l’Imaginaire, (v. 1). Paris, CNRS, pp. 158-162. http://repositorium.sdum.uminho.pt/ handle/1822/24132 Martins, M. L. (2011 b). “Media e melancolia – o trágico, o grotesco e o barroco”. Acciaiuoli, M. & Babo, M. A. (Eds.) Arte e Melancolia. Lisboa: Instituto de História de Arte/Estudos de Arte Contemporânea e Centro de Estudos de Comunicação e Linguagem, pp. 53-65. http://repositorium.sdum.uminho.pt/ handle/1822/24106 Martins, M. L. (2010). “A mobilização infinita num movimento de meios sem fins”. Álvares, C. & Damásio, M. (Eds.) Teorias e práticas dos média. Situando o local no global. Lisboa: Edições Lusófonas. http:// repositorium.sdum.uminho.pt/handle/1822/24250 Martins, M. L. (2008) [2004, “Comunicação de Abertura”]. “As ciências sociais e a política científica”. Torres, A. & Baptista, L., Sociedades Contemporâneas. Reflexividade e Acção. Porto: Afrontamento, pp. 27-29. http://repositorium.sdum.uminho.pt/handle/1822/1059 Martins, M. L. (2003). Ensino Superior e melancolia. Viana do Castelo: Instituto Politécnico de Viana do Castelo. http://repositorium.sdum.uminho.pt/handle/1822/1288 Martins, M. L. (2002 a). “De animais da promessa a animais em sofrimento de finalidade”, in O Escritor, n. 18/19/20, Revista da Associação Portuguesa de Escritores, Lisboa, pp. 351-354. http://repositorium. sdum.uminho.pt/handle/1822/1676 Martins, M. L. (2002 b). “O trágico como imaginário da era mediática”. Comunicação e Sociedade, 4: 73-79. http://repositorium.sdum.uminho.pt/handle/1822/25340 Martins, M. L. (1994). “A verdade e a função de verdade nas ciências sociais”. Cadernos do Noroeste, 7, 2. Braga, pp. 5-18. http://repositorium.sdum.uminho.pt/handle/1822/25385 Martins, M. L. (1993). “As incertezas da nossa modernidade e o impasse universitário”. Cadernos do Noroeste, 6, 1-2: 341-348. http://repositorium.sdum.uminho.pt/handle/1822/25330 Nietzsche, F. (1998) [1887]. Genealogia da Moral, São Paulo: Companhia das Letras. Nietzsche, F. (1988) [1888]. O crepúsculo dos ídolos. Lisboa: Ed. 70. Nóvoa, A. (2014). “Em busca da liberdade nas universidades: Para que serve a investigação em educação?”. Revista Lusófona de Educação, 28, pp. 11-21. Pinto-Coelho, Z. & Carvalho, A. (Eds.) (2013). Diplomas legais Regime Jurídico das Instituições do Ensino Superior (RJIES), Lei n.º 62/2007, de 10 de Setembro. Regime Jurídico das Instituições do Ensino Superior (RJIES), Lei n.º 62/2007, de 10 de Setembro. Regulamento de Avaliação e Desempenho dos Docentes, Lei n. 205/2009, de 31 de agosto. egulamento de Avaliação e Desempenho dos Docentes, Lei n. 205/2009, de 31 de agosto. Regulamento de Avaliação e Desempenho dos Docentes da Universidade do Minho (RAD-UM), aprovado no Diário da República, 2.ª série – N.º 117 - 18 de junho de 2010. Regulamento de Avaliação e Desempenho dos Docentes da Universidade do Minho (RAD-UM), aprovado no Diário da República, 2.ª série – N.º 117 - 18 de junho de 2010. Regulamento de avaliação do desempenho dos docentes da Universidade da Beira Interior. Despacho n.º 10129/2014, publicado Diário da República, 2.ª série — N.º 150 — 6 de agosto de 2014. Regulamento de avaliação do desempenho dos docentes da Universidade da Beira Interior. Despacho n.º 10129/2014, publicado Diário da República, 2.ª série — N.º 150 — 6 de agosto de 2014. Endereços eletrónicos http://www.fct.pt/esp_inteligente/diagnostico http://www.dn.pt/inicio/portugal/interior.aspx?content_id=3216596&page=-1 http://www.uminho.pt/uminho/qualidade http://www.uminho.pt/uminho/qualidade/documentacao-do-sigaq-um http://www.fct.pt/esp_inteligente/diagnostico http://www.dn.pt/inicio/portugal/interior.aspx?content_id=3216596&page=-1 http://www.uminho.pt/uminho/qualidade http://www.uminho.pt/uminho/qualidade/documentacao-do-sigaq-um Referências Academics Responding to Discourses of Crisis in Higher Education and Research. Braga: CECS, Universidade do Minho. Power, M. (1999). The audit society: rituals of verification. Oxford: Oxford University Press. Readings, B. (1996). The university in ruins. Cambridge, Mass: Harvard University Press. Ruão, T. (2008). A comunicação organizacional e os fenómenos de identidade: a aventura comunicativa da formação da Universidade do Minho, 1974-2006. Tese de doutoramento, Universidade do Minho. Santos, B. S. (1994). “Da ideia de universidade à universidade das ideias”. Pela mão da Alice. O social e o político na pós-modernidade. Porto: Afrontamento, pp. 163: 201. Sloterdijk, P. (2000). La mobilisation infinie. Christian Bourgeois. Sloterdijk, P. (2000). La mobilisation infinie. Christian Bourgeois. Steiner, G. (1993 [1989]). Presenças Reais, Lisboa: Presença. 416 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins TOB (Traduction Oecuménique de la Bible). Nouveau Testament (1978). Paris: Les Editions du Cerf. TOB (Traduction Oecuménique de la Bible). Nouveau Testament (1978). Paris: Les Editions du Cerf. Weber, M. (1974) [1959]. Le savant et le politique. Paris: Union Générale d’Éditions, Plon, col. 10/18. Weber, M. (1974) [1959]. Le savant et le politique. Paris: Union Générale d’Éditions, Plon, col. 10/18. * Submetido: 30-11-2014 * Aceite: 15-3-2015 * Aceite: 15-3-2015 Nota biográfica Moisés de Lemos Martins é professor de sociologia da cultura e da comunicação na Universidade do Minho (Braga, Portugal), sendo nesta universidade Diretor do Cen tro de Estudos de Comunicação e Sociedade (CECS), que fundou em 2001. É autor de vários livros, entre os quais: O Olho de Deus no discurso salazarista (1990) e Para uma inversa navegação – O discurso da identidade (1996), ambos nas Edições Afrontamento; A linguagem, a verdade e o poder – Ensaio de semiótica social (2002, Fundação Calouste Gulbenkian e Fundação para a Ciência e a Tecnologia); Crise no Castelo da Cultura – Das estrelas para os ecrãs (2011, Grácio Editor). Fundou, em 1999, a revista Comunicação e Sociedade e, em 2013, Anuário Lusófono de Estudos Culturais. Preside, desde 2012, à Con federação Iberoamericana das Associações Científicas e Académicas de Comunicação, depois de ter presidido, de 2005 a 2015, à Associação Portuguesa de Ciências da Comu nicação (Sopcom). E-mail: moisesm@ics.uminho.pt; moiseslmartins@gmail.com Centro de Estudos de Comunicação e Sociedade, Campus de Gualtar, Universidade do Minho, Braga 4710-057, Portugal Centro de Estudos de Comunicação e Sociedade, Campus de Gualtar, Universidade do Minho, Braga 4710-057, Portugal * Submetido: 30-11-2014 * Aceite: 15-3-2015 417 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Comunicação e Sociedade, vol. Nota biográfica 27, 2015 Anexo Anexo Investigação Vertente I - Investigação Pontua ção Coefi ciente Total 1 – Produção científico-cultural 0,00 0,6 0,00 2 – Participação, reconhecimento e coordenação/direção científica 0,00 0,4 0,00 0,00 3- Fez o doutoramento ou obteve mudança de grau 0,00 Instrumentos Pontua ção base Quan tidade Pontua ção Pontua ção base Quan tidade Pon tuação Tabela 6 – produção cientifico-cultural Nacional Internacional Livros publicados 25 30 Artigos em Revistas com Referee 18 20 Artigos em Revistas 15 18 Capítulos de livros 18 20 Edição de livros 15 20 Artigos em atas de conferências 10 15 0,00 0,00 Relatórios científicos 10 Outras publicações 5 0,00 Tabela 7 – Participação, reconhecimento e coordenação/direção científica Participação Nacional Internacional Membro de equipa de investigação em proje tos científicos com financiamento 15 20 0,00 0,00 Membro da equipa de investigação em pro jetos sem financiamento externo 10 Membro da comissão para a criação/al teração de ciclos de estudos 10 Membro da comissão para a criação/al teração de outros ciclos 8 0,00 Reconhecimento Anexo Investigação Vertente I - Investigação Pontua ção Coefi ciente Total 1 – Produção científico-cultural 0,00 0,6 0,00 2 – Participação, reconhecimento e coordenação/direção científica 0,00 0,4 0,00 0,00 3- Fez o doutoramento ou obteve mudança de grau 0,00 Instrumentos Pontua ção base Quan tidade Pontua ção Pontua ção base Quan tidade Pon tuação Tabela 6 – produção cientifico-cultural Nacional Internacional Livros publicados 25 30 Artigos em Revistas com Referee 18 20 Artigos em Revistas 15 18 Capítulos de livros 18 20 Edição de livros 15 20 Artigos em atas de conferências 10 15 0,00 0,00 Relatórios científicos 10 Outras publicações 5 0,00 Tabela 7 – Participação, reconhecimento e coordenação/direção científica Investigação Nacional Internacional 15 20 0,00 0,00 10 10 8 0,00 418 Comunicação e Sociedade, vol. 27, 2015 A liberdade académica e os seus inimigos . Nota biográfica Moisés de Lemos Martins Interno Externo 1,5 1,5 11,25 15 0,00 0,00 Interno Externo 1,2 1,2 21 21 9 15 0,00 0,00 Interno Externo 10 12,5 5 6,25 10 12,5 0,00 0,00 Interno Externo 1 1 17,5 17,5 7,5 10 0,00 0,00 Interno Externo 0,5 0,5 8,75 8,75 3,75 5 0,00 0,00 Interno Externo 0,3 0,3 5,25 5,25 2,25 3 0,00 0,00 Nacional Internacional 10 15 30 37,5 10 12,5 5 6,25 0,00 0,00 tão coloque o valor 1 Interno Externo Membro de júri para concurso de recruta mento nas carreiras - presidente 1,5 1,5 Membro de júri para concurso de recru tamento nas carreiras - vogal 11,25 15 0,00 0,00 Interno Externo Membro de júri de provas de agregação - presidente 1,2 1,2 Membro de júri de provas de agregação - arguente 21 21 Membro de júri de provas de agregação - vogal 9 15 0,00 0,00 Interno Externo Participação em peritagens e referees 10 12,5 Membro de corpo editorial (revista própria ou externa) 5 6,25 Membro de comissão de organização de evento científico 10 12,5 0,00 0,00 Interno Externo Membro de júri de provas de doutoramento - presidente 1 1 Membro de júri de provas de doutoramento - arguente 17,5 17,5 Membro de júri de provas de doutoramento - vogal 7,5 10 0,00 0,00 Interno Externo Membro de júri de provas de mestrado - presidente 0,5 0,5 Membro de júri de provas de mestrado - arguente 8,75 8,75 Membro de júri de provas de mestrado - vogal 3,75 5 0,00 0,00 Interno Externo Membro de júri de provas de licenciatura - presidente 0,3 0,3 Membro de júri de provas de licenciatura - arguente 5,25 5,25 Membro de júri de provas de licenciatura - vogal 2,25 3 0,00 0,00 Nacional Internacional Comunicação em eventos científicos 10 15 Agraciamento com prémios 30 37,5 Cargos em organizações científicas 10 12,5 Outros reconhecimentos de natureza científica 5 6,25 0,00 0,00 Coordenação/direção Caso tenha executado uma destas atividades no ano em questão coloque o valor 1 Responsável geral de projetos científi cos com financiamento internacional 30 Responsável local de projetos científi cos com financiamento internacional 25 Membro de júri para concurso de recruta mento nas carreiras - presidente Membro de júri para concurso de recru tamento nas carreiras - vogal Membro de júri de provas de agregação - presidente Membro de júri de provas de agregação - arguente Membro de júri de provas de agregação - vogal Membro de júri de provas de agregação - presidente Membro de júri de provas de agregação - arguente Membro de júri de provas de agregação - vogal Participação em peritagens e referees Membro de corpo editorial (revista própria ou externa) Membro de comissão de organização de evento científico Membro de júri de provas de doutoramento - presidente Membro de júri de provas de doutoramento - arguente Membro de júri de provas de doutoramento - vogal Membro de júri de provas de mestrado - presidente Membro de júri de provas de mestrado - arguente Membro de júri de provas de mestrado - vogal Membro de júri de provas de licenciatura - presidente Membro de júri de provas de licenciatura - arguente Membro de júri de provas de licenciatura - vogal Caso tenha executado uma destas atividades no ano em questão coloque o valor 1 419 Comunicação e Sociedade, vol. Nota biográfica 27, 2015 A liberdade académica e os seus inimigos . Moisés de Lemos Martins Responsável de projetos científicos na cionais com financiamento 20 Responsável de projetos científicos na cionais sem financiamento 15 Coordenação de evento científico-cultural 15 Coordenação de comissão para a criação/ alteração de ciclos de estudos 15 Coordenação de comissão para a criação/ alteração de ciclos de estudos 10 Supervisão de bolseiros de investigação 3 0,00 20 15 15 15 10 3 0,00 Responsável de projetos científicos na cionais com financiamento Responsável de projetos científicos na cionais sem financiamento Coordenação de evento científico-cultural Coordenação de comissão para a criação/ alteração de ciclos de estudos Coordenação de comissão para a criação/ alteração de ciclos de estudos Supervisão de bolseiros de investigação 420
https://openalex.org/W4388543076	https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/79/e3sconf_isffs2023_02038.pdf	English	null	New emerging viral disease on Giant Gourami (<i>Osphronemus goramy</i> Lac.) in Java, Indonesia	E3S web of conferences	2,023	cc-by	4,412	* Corresponding author: tauk001@brin.go.id © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). New emerging viral disease on Giant Gourami (Osphronemus goramy Lac.) in Java, Indonesia Taukhid Taukhid1,*, Hessy Novita2, Tuti Sumiati2, Domenico Caruso3, and Edy Barkat Kholidin1 1Research Center for Fisheries, National Research and Innovation Agency, Indonesia 2Research Center for Veterinary Science, National Research and Innovation Agency, Indonesia 3Institute of Research for Development (IRD) Univ. Montpellier, France Abstract. This study investigates the role of pathogens in the disease outbreaks and mass mortality affecting giant gourami (Osphronemus goramy) populations from 2018 to 2020, leading to significant economic losses. Presumptive diagnoses, based on clinical symptoms and references, implicated two viruses: Tilapia Lake Virus (TiLV) and Infectious Spleen and Kidney Necrosis Virus (ISKNV). Samples from four Indonesian provinces (West Java, Central Java, East Java and Yogyakarta) were tested molecularly for these viruses. The results revealed widespread viral pathogen involvement, with ISKNV prevalence ranging from 20% to 100% and TiLV prevalence consistently at 100%. These findings strongly suggest that the outbreaks were primarily caused by TiLV and ISKNV infections, potentially with other co-infecting pathogens. Bacterial pathogens, notably Aeromonas spp., Mycobacterium spp., and Pseudomonas spp., may have exacerbated the disease. This research highlights the emergence of viral diseases as a significant threat to giant gourami populations in Java, Indonesia. E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 https://doi.org/10.1051/e3sconf/202344202038 1 Introduction The giant gourami, Osphronemus goramy, is a freshwater fish native to Southeast Asia [1]. Global production of giant gourami was recorded at 145,000 tons in 2017, of which Indonesia contributed 98% of the total production, equivalent to 142,100 tons [2]. It was also noted that only five other countries reported production of this species (Thailand, Myanmar, Malaysia, Philippines, and Singapore). However, Vietnam and Laos are known to produce this fish but do not report it. Using conventional systems, the fish have been farmed in Indonesia for centuries, mainly in Central Java and West Java [3]. Giant gourami farming has grown rapidly in Java and Sumatra and has even spread to other areas such as Kalimantan, Sulawesi, Bali, and West Nusa Tenggara [4]. Giant gourami's community preference and economic value continuously increase and generally remain above the average of other freshwater-farmed fish species. These facts motivate farmers to promote more efficient production by changing the farming system from traditional to semi-intensive with higher stocking densities, using commercial feeds E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 E3S Web of Conferences 442, 02038 (2023) https://doi.org/10.1051/e3sconf/202344202038 and shorter growing periods. As a result, as giant gourami farming becomes more intensive, disease issues have arisen as one of the challenges producers face. A parallel health management system has yet to accompany changes in the increasingly intensive farming system. In addition, giant gourami farming is generally implemented in an aquatic environment that is challenging to manage, where the process of infectious disease transmission is more difficult to control. and shorter growing periods. As a result, as giant gourami farming becomes more intensive, disease issues have arisen as one of the challenges producers face. A parallel health management system has yet to accompany changes in the increasingly intensive farming system. In addition, giant gourami farming is generally implemented in an aquatic environment that is challenging to manage, where the process of infectious disease transmission is more difficult to control. In 2018-2020, giant gourami farming in Indonesia experienced a disease outbreak that resulted in mass mortality, particularly in the production centers covering Java, with economic losses estimated at 40 billion rupiahs [5, 6, 7]. The impact of the disease outbreaks was reflected in the national production of this species. Production of giant gourami increased from 2011 to 2017; however, there was a significant decline of more than 30% from 2018 to 2020 [8]. 1 Introduction Epidemiological studies related to disease outbreaks have not been conducted comprehensively, so the primary causes still need to be discovered and fully described, including the determinant factors of the disease [7]. This study aimed to investigate pathogen involvement in disease outbreaks and mass mortality of giant gourami (O. goramy) in Java, Indonesia, during 2018-2020. Based on presumptive diagnosis (clinical symptoms, mortality patterns, and available references), this study highlighted the presence of two viral pathogens, Tilapia Lake Virus (TiLV) and Infectious Spleen and Kidney Necrosis Virus (ISKNV). 2.3 Detection of virus Virus analysis was conducted on two viral agents strongly implicated as potential primary etiological agents in emerging disease outbreaks. Specifically, these viruses were identified as TiLV and ISKNV. The fish specimens for this analysis were selected based on their manifestation of distinctive clinical symptoms indicative of the suspected viral infections. 2.1 Sample and data collection The diseased giant gouramis were collected from West Java, Central Java, Yogyakarta, and East Java. The number and size of samples collected from each sampling point varied depending on the availability of the population at risk of disease and survivors in the recovery process. The number of sampling sites and the total number of samples collected in each province are shown in Table 1. p In addition to laboratory analysis, secondary information was collected from relevant resource persons (farmers and fishery health officers) through interviews guided by a simple questionnaire. The information collected in the questionnaire included the size of the affected fish, clinical signs, and mortality. Estimated losses due to the outbreak were obtained from the report prepared by the Fisheries Department in each area where samples were collected. Table 1. The number and body weight of giant gourami (Osphronemus gouramy) samples collected from each affected location. Province Spot sampling Number of samples Size (gram) Culture unit West Java 4 40 50-100 400-600 Grow-out Grow- out/broodstock Central Java 2 9 2,500-4,000 Broodstock Yogyakarta 2 5 300-550 2,500-4,000 Grow-out Broodstock East Java 3 9 300-500 Grow-out able 1. The number and body weight of giant gourami (Osphronemus gouramy) samples collected from each affected location. 2 2 https://doi.org/10.1051/e3sconf/202344202038 E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 2.3.1 Tilapia Lake Virus (TiLV) The organs and tissues chosen to detect TiLV included the eye, brain, liver, spleen, and kidney [13]. Subsequently, these organ samples were meticulously preserved by immersion in RNA later solution or a solution comprised of absolute ethanol and glycerol in an 80:20 (v/v) ratio. The preserved samples were then securely stored at -70 oC until they were prepared for further analytical procedures. The methodology employed for TiLV detection followed the procedure established by a prior study [14]. This involved using a semi-nested Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique. In Step 1 of the nested RT-PCR, the specific primers were ext-1 (TATGCAGTACTTTCCCTGCC) and ME1 a prior study [14]. This involved using a semi-nested Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique. In Step 1 of the nested RT-PCR, the specific primers were ext-1 (TATGCAGTACTTTCCCTGCC) and ME1 (GTTGGGCACAAGGCATCCTA). The target PCR amplicon size for this step was 415 base pairs. In Step 2, a semi-nested RT-PCR approach was utilized, and it featured the following primers: ME2 (TATCACGTGCGTACTCGTTCAGT) and ME1 (GTTGGGCACAAGGCATCCTA). This step aimed to generate a PCR product with a size of 250 base pairs. (GTTGGGCACAAGGCATCCTA). The target PCR amplicon size for this step was 415 base pairs. In Step 2, a semi-nested RT-PCR approach was utilized, and it featured the following primers: ME2 (TATCACGTGCGTACTCGTTCAGT) and ME1 (GTTGGGCACAAGGCATCCTA). This step aimed to generate a PCR product with a size of 250 base pairs. 2.2 Identification of bacteria The manufacturer's instructions prepared sterile media for isolating pathogenic bacteria. Bacterial isolation was performed primarily on samples with obvious clinical signs, such as ulcers and kidney abnormalities, liver, spleen, and muscle abnormalities. Bacteria from each organ were then isolated aseptically on tryptic soy agar (TSA), Brain Heart Infusion Agar (BHIA), and Lowenstein-Jensen medium. Incubations were 24-72 h at 28 oC, while Mycobacterium spp. were incubated for at least seven days. Bacterial growth and morphology were observed. The bacterial analysis included Gram stain, motility, oxidase, Rimmler-Shott, catalase, O/F test, Triple Sugar Iron Agar (TSIA) test, and API 20E test (Biomereux). Bacterial identification was performed according to [9, 10, 11, 12]. 2.3.2 Infectious Spleen and Kidney Necrosis Virus (ISKNV) The investigation involved the aseptic procurement of organs and tissues, specifically the liver, brain, spleen, and kidney, for the precise detection of ISKNV, as meticulously detailed in scholarly references [15, 16]. These biological specimens were individually harvested or arranged into various combinations and submerged in an appropriate preservative solution. The analytical assessment of ISKNV was carried out in strict adherence to the rigorous methodological framework established by the authors in reference [17]. This method incorporated the judicious design of primers, derived from the nucleotide sequence of a 959-base pair Pst I restriction fragment, which was uniquely identified by its DDBJ accession number AB006954, originating from the genomic DNA of the Red Seabream Iridovirus (RSIV). These primers denoted as 1F (Forward Primer) with the sequence 5'CTCAAACACTCTggCTCATC'3 and 1R (Reverse Primer) with the sequence 3 E3S Web of Conferences 442, 02038 (2023) E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 https://doi.org/10.1051/e3sconf/202344202038 5'GCACCAACACATCTCCTATC'3, were thoughtfully tailored to specifically target a molecular weight of 570 base pairs within the RSIV genome DNA. 2.4 Analysis The study's data and information underwent a comprehensive analysis encompassing qualitative and quantitative methodologies. Subsequently, the results were thoughtfully organized and presented in a meticulously structured tabular format, adhering to established academic standards. 3 Results and discussion The giant gourami disease outbreak, from early 2018 to the mid-2020s, resulted in thousands of productive and candidate broodstock mortality. The extraction of data/ information collected from the local fisheries office and the farmers is presented in Table 2. It was indicated that the size of giant gourami affected mainly by the disease and causing large economic losses at that time, was from the grow-out to broodstock sizes, with mortality rates of 20% - 60% with an estimated total loss of ≈ 48 billion rupiahs (US$ 3,139,307). Table 2. Summary of data/information collected from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). Location District Stage Size (gram) Mortality rate (%) Estimated loss (million rupiahs) West Java Bogor, Tasikmalaya, Banjar, Ciamis Grow-out Brood stocks 100-600 1,500-4,000 40-50 20-25 1,500 9,000 Central Java Banyumas, Banjarnegara Grow-out Brood stocks 300-700 2,000-5,000 25-30 50-60 5,351 24,873 Yogya- karta Bantul, Sleman, Kulon Progo, Gunung Kidul Grow-out Brood stocks 100-600 2,000-5,000 20-25 30-35 1,000 4,500 East Java Tulung Agung, Kediri Grow-out 100-450 20-25 2,000 Total estimated financial loss 48,224 ble 2. Summary of data/information collected from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). Behavioral observations made by farmers and direct observations in the field of diseased and moribund giant gourami generally show the following behavior a few days earlier: slow swimming movements, less responsiveness, and drastically decreased appetite. Concurrently, the clinical manifestations encompassed a spectrum of discernible symptoms, including but not limited to altered pigmentation characterized by a deepening of the body coloration, ocular anomalies such as endophthalmitis and cataracts, dermal irregularities such as scales appearing loose or skin exhibiting signs of peeling, as well as erosions and ulcerations. Most of these clinical symptoms are consistent with those described in tilapia exposed to TiLV as described by [13, 18, 19, 20, 21, 22]. Other clinical symptoms were also observed during the outbreak, including loss of equilibrium, excess mucus, bleeding petechiae on the skin, swimming upside down, abdominal distension, respiratory distress, darkening of body color, loss of scales, 4 E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 https://doi.org/10.1051/e3sconf/202344202038 enlargement and discoloration of internal organs, especially the spleen, and kidneys. These clinical signs were also reported in giant gourami infected by ISKNV, as described by [15, 16, 23]. enlargement and discoloration of internal organs, especially the spleen, and kidneys. 3 Results and discussion These clinical signs were also reported in giant gourami infected by ISKNV, as described by [15, 16, 23]. During the examination of ailing specimens, we observed that when lifted, there was a noticeable excess of mucus and blood oozing from between the scales. To help clarify these observations, we've included visual representations of moribund giant gourami and those in the early stages of expiration in Figure 1. These images serve as visual aids to support our findings. Fig. 1. Diseased giant gourami (Osphronemus gouramy) collected from the affected area in Yogyakarta (2019): pale/dark body color, scale protrusion, skin redness, and fin rot. Fig. 1. Diseased giant gourami (Osphronemus gouramy) collected from the affected area in Yogyakarta (2019): pale/dark body color, scale protrusion, skin redness, and fin rot. Analysis of samples obtained from outbreak-afflicted regions spanning 2018 to 2020 unveiled a notable prevalence of bacterial infections. Mycobacteriosis and motile aeromonad septicemia (MAS) emerged as the principal afflictions affecting giant gourami. Additionally, diverse strains of bacteria were identified within the collected samples. In tandem with bacterial infections, the analysis also disclosed the presence of two viruses, TiLV and ISKNV, within the same set of test samples. The findings about identifying and detecting these pathogens have been systematically cataloged and delineated in Table 3. able 3. Bacteria identified and virus detected on giant gourami (Osphronemus gouramy) samples from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). Table 3. Bacteria identified and virus detected on giant gourami (Osphronemus gouramy) samples from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). Table 3. Bacteria identified and virus detected on giant gourami (Osphronemus gouramy) samples from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). No. Location ∑ samples Bacteria Virus Genus/species Prevalence (%) Species Prevalence (%) 1. West Java 40 Aeromonas spp. Mycobacterium spp. Pseudomonas spp. 8.0 12.0 8.0 TiLV ISKNV 100.00 38.46 2. Central Java 9 Aeromonas spp. Mycobacterium spp. Pseudomonas spp. nd TiLV ISKNV 55.56 20.00 5 5 https://doi.org/10.1051/e3sconf/202344202038 E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 3. Yogyakarta 5 Aeromonas spp. Mycobacterium spp. Pseudomonas spp. nd TiLV ISKNV 55.56 20.00 4. East Java 9 Mycobacterium spp. Pseudomonas spp. 3 Results and discussion 10.0 10.0 TiLV ISKNV 100.00 100.00 Notes: TiLV = Tilapia Lake Virus, ISKNV = Infectious Spleen and Kidney Necrosis Virus, and nd = not done The tabulated data reveals the substantial impact of viral infections on the afflicted giant gourami population in the surveyed regions. Specifically, TiLV infection exhibited a remarkable prevalence of 100% in both West Java and East Java. In the neighboring regions of Central Java and Yogyakarta, the prevalence was 55.56%. In parallel, ISKNV infection was evident, with a prevalence of 38.46% in West Java and 20% in Central Java and Yogyakarta, while the prevalence reached 100% in East Java. As recognized worldwide, TiLV disease was initially known to affect only tilapia [13, 18, 19, 20, 22]. The spreading of the disease was very fast, and in 2020, [22] it was suspected that at least 16 countries across four continents had been exposed to TiLV. The virus has been detected in Indonesia [22, 24, 25]. Thus, it is likely that the virus was indeed present and widespread in tilapia farm areas in Indonesia [24, 25, 26]. However, the disease has generally not led to serious fatalities and losses compared to similar diseases in several other affected countries [21, 27, 28]. This is also based on the no reports of significant disease and mortality among tilapia farms, and this is also reflected in the national production of tilapia, which has been increasing steadily from 2010 to the present day [4]. The evidence of TiLV being able to infect giant gourami with significantly higher mortality rates than in tilapia is a serious concern to be aware of [29], until finally, it is relevant to the emergence of a disease outbreak in giant gourami culture, which closely coincided with the first detection of TiLV on tilapia in Indonesia [24, 25]. The outbreak of TiLV infection in giant gouramis is a new occurrence, and all samples collected from the affected areas revealed a high prevalence. The impact of disease outbreaks was reflected in the national production achievements for this species. The national production of giant gourami increased during 2011-2017; however, there was a significant decline of more than 30% from 2018-2020 [8]. To date, diseased fish showing the same clinical symptoms are still encountered with varying degrees of loss [30]. In all samples collected from the outbreak areas, ISKNV infection was also involved with a relatively lower prevalence than TiLV. 3 Results and discussion ISKNV infection in giant gourami in Indonesia has been reported since a decade ago [15], as well as in other species of labyrinth fish [15, 31, 32]. However, the impact of the losses associated with this virus is less massive than after co-infection with TiLV as the outbreak occurred in 2018-2020. Outside Indonesia, the ISKNV infection has also been reported in giant gourami in India [16]. The heightened prevalence of both these viruses within the sampled population was further substantiated through molecular analysis. Notably, all samples from the affected regions returned positive results, as the data in Figures 2 and 3 corroborated. These findings underscore the substantial impact of these viral pathogens on the health of the giant gourami population under study. 6 6 https://doi.org/10.1051/e3sconf/202344202038 E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 M P N WJ-1 WJ-2 CJ-1 CJ-2 EJ-1 EJ-2 Y-1 Y-2 Fig 2. Gel electrophoresis profile of Tilapia Lake Virus (TiLV) detection of giant gourami (Osphronemus gouramy) samples collected from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). M = DNA ladder, P = positive control, N = negative control, WJ-1 & 2 = West Java, CJ-1 & 2 = Central Java, EJ-1 & 2 = East Java, and Y-1 & 2 = Yogyakarta. Fig 2. Gel electrophoresis profile of Tilapia Lake Virus (TiLV) detection of giant gourami (Osphronemus gouramy) samples collected from affected areas in Java (West Java, Central Java, Yogyakarta, and East Java). M = DNA ladder, P = positive control, N = negative control, WJ-1 & 2 = West Java, CJ-1 & 2 = Central Java, EJ-1 & 2 = East Java, and Y-1 & 2 = Yogyakarta. M P N WJ-1 WJ-2 CJ-1 CJ-2 EJ-1 EJ-2 Y-1 Y-2 Fig 3. Gel electrophoresis profile of Infectious Spleen and Kidney Necrosis Virus (ISKNV) detection of giant gourami (Osphronemus gouramy) samples collected from affected areas in Java (West Java, Central Java, Yogyakarta and East Java). M = DNA ladder, P = positive control, N = negative control, WJ-1 & WJ-2 = West Java, CJ-1 & CJ-2 = Central Java, EJ-1 & EJ-2 = East Java, Y-1 & Y-2 = Yogyakarta. 570 bp M P N WJ 1 WJ 2 CJ 1 CJ 2 EJ 1 EJ 2 Y 1 Y 2 Fig 3. Gel electrophoresis profile of Infectious Spleen and Kidney Necrosis Virus (ISKNV) detection of giant gourami (Osphronemus gouramy) samples collected from affected areas in Java (West Java, Central Java, Yogyakarta and East Java). M = DNA ladder, P = positive control, N = negative control, WJ-1 & WJ-2 = West Java, CJ-1 & CJ-2 = Central Java, EJ-1 & EJ-2 = East Java, Y-1 & Y-2 = Yogyakarta. Based on the analysis of the samples collected immediately after the onset of the disease, the clinical signs observed, and the mortality rate, there is a strong suspicion that the main aetiological agent causing disease and mortality is a single infection by either TiLV or ISKNV and co-infection with both species of viruses. This suspicion is not only based on clinical symptoms and mortality patterns but also confirmed by the results of molecular diagnosis and available references [16, 28, 30]. The involvement of other 7 7 E3S Web of Conferences 442, 02038 (2023) ISFFS 2023 https://doi.org/10.1051/e3sconf/202344202038 pathogens belonging to the bacterial group also exacerbates the disease, particularly Mycobacterium spp. and Aeromonas spp. Consequently, a strong and well-founded suspicion exists that Tilapia Lake Virus (TiLV) and Infectious Spleen and Kidney Necrosis Virus (ISKNV) are the primary etiological agents responsible for these infections. This suspicion is grounded in a comprehensive assessment encompassing anamnesis, clinical observation, and a thorough diagnostic analysis. Furthermore, it is imperative to underscore that TiLV and ISKNV consistently manifest as the dominant pathogens, bearing substantial prevalence within the affected giant gourami population. This prevalence pertains to singular viral infections and co-infections with other microbial agents, such as Mycobacterium spp., Aeromonas spp., and Pseudomonas spp. Determining the primary pathogenic agents of a newly emerging disease requires the completion of pathogenicity (Koch's postulates), virological (cytopathic effect/CPE), and histopathological assays. In this study, these diagnostics were not performed. M P N WJ-1 WJ-2 CJ-1 CJ-2 EJ-1 EJ-2 Y-1 Y-2 Therefore, the gold standard diagnosis is still needed for confirmation. Nevertheless, base on this study, we posit with substantial confidence that both viral species, TiLV and ISKNV, act as primary pathogenic agents, harboring considerable potential as emerging diseases in giant gourami within Java and, conceivably, in gourami culture regions beyond the confines of Java Island. Authors’ contributions All authors have made substantial contributions to the refinement of the final manuscript. 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https://openalex.org/W4383557805	https://www.frontiersin.org/articles/10.3389/fphar.2023.1157229/pdf	English	null	Editorial: Herbal medicines for gastrointestinal and hepatic diseases - novel pharmacological and toxicological approaches, Volume I	Frontiers in pharmacology	2,023	cc-by	2,148	TYPE Editorial PUBLISHED 07 July 2023 DOI 10.3389/fphar.2023.1157229 TYPE Editorial PUBLISHED 07 July 2023 DOI 10.3389/fphar.2023.1157229 TYPE Editorial PUBLISHED 07 July 2023 DOI 10.3389/fphar.2023.1157229 Front. Pharmacol. 14:1157229. doi: 10.3389/fphar.2023.1157229 Front. Pharmacol. 14:1157229. doi: 10.3389/fphar.2023.1157229 herbal medicines, traditional medicines, active ingredients from herbal medicines, GIT, liver, toxicology COPYRIGHT COPYRIGHT © 2023 Hasnat, Baig, Saleem, Ullah, Nadeem, Durazzo and Lucarini. 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. © 2023 Hasnat, Baig, Saleem, Ullah, Nadeem, Durazzo and Lucarini. 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. Editorial on the Research Topic Herbal medicines for gastrointestinal and hepatic diseases - novel pharmacological and toxicological approaches, Volume I OPEN ACCESS OPEN ACCESS EDITED BY Javier Echeverria, University of Santiago, Chile REVIEWED BY Hari Prasad Devkota, Kumamoto University, Japan CORRESPONDENCE Muhammad Hasnat, muhammad.hasnat@uvas.edu.pk RECEIVED 02 February 2023 ACCEPTED 22 June 2023 PUBLISHED 07 July 2023 CITATION Hasnat M, Baig MMFA, Saleem M, Ullah A, Nadeem MF, Durazzo A and Lucarini M (2023), Editorial: Herbal medicines for gastrointestinal and hepatic diseases - novel pharmacological and toxicological Muhammad Hasnat1, Mirza Muhammad Faran Ashraf Baig2, Mohammad Saleem3, Aftab Ullah4, Muhammad Faisal Nadeem5, Alessandra Durazzo6 and Massimo Lucarini 6 Muhammad Hasnat1*, Mirza Muhammad Faran Ashraf Baig2, Mohammad Saleem3, Aftab Ullah4, Muhammad Faisal Nadeem5, Alessandra Durazzo6 and Massimo Lucarini 6 RECEIVED 02 February 2023 ACCEPTED 22 June 2023 PUBLISHED 07 July 2023 CITATION Hasnat M, Baig MMFA, Saleem M, Ullah A, Nadeem MF, Durazzo A and Lucarini M (2023), Editorial: Herbal medicines for gastrointestinal and hepatic diseases - novel pharmacological and toxicological approaches, Volume I. 1Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan, 2The Hong Kong University of Science and Technology, Hong Kong, Hong Kong SAR, China, 3University of the Punjab Lahore, Lahore, Pakistan, 4Jiangsu University, Zhenjiang, China, 5University of Veterinary and Animal Sciences, Lahore, Pakistan, 6CREA-Research Centre for Food and Nutrition, Rome, Italy KEYWORDS Front. Pharmacol. 14:1157229. doi: 10.3389/fphar.2023.1157229 Editorial on the Research Topic H b l di i f i This study reported that MLG increased the levels of antioxidant enzymes and reduced the levels of TNF-α and IL-1β. MLG increased the expressions of Nrf-2 and HO-1 and decreased the expressions of COX-2 and NF-κB. This Research Topic includes the following dimensions: Pre- clinical and clinical studies of herbal products and their biologically active metabolites in the management of GIT and liver diseases. Novel in vitro assays for the identiﬁcation of potentially biologically active compounds for the treatment of gastric and hepatic cancer. Novel cellular and molecular mechanisms that describe the therapeutic effects as well as toxic effects of herbal medicines and their active metabolites on the GIT and liver. The role of the GI- microbiome in preventing and treating gastrointestinal and hepatic diseases. The papers within this Research Topic carry out interesting themes in the area of research. As instance, Lee et al. found that treatment with Chunggan syrup (CGX) reduced tumor nodules in normal and HFD fed mice. Molecular biology studies explained that CGX antitumor effect was associated with the activation of E- Cadherin and reduction in the expression of VE- Cadherin in liver under MC38 free condition. CGX also reduced liver steatosis via modulating AMPk and PPARα. Yuan et al. showed that triptolide (TP), one of the fat-soluble components extracted from the Chinese medicinal herb Tripterygium wilfordii Hook F. (TWHF), induced cell death in TNFα-pretreated MKN45 cells and AGS cells. Both TP and TNFα, in combination promoted the gastric cancer cell death through inﬂuencing H19/miR-204-5p/NF-κB/ FLIP axis. Other studies on herbal medicines treating gastrointestinal and liver diseases have also signiﬁcant ﬁndings. Chen et al. published a clinical evidence base study and expressed that SLBZP had capacity to treat ulcerative colitis. Kang et al. reported the randomized, double-blind, placebo-controlled, multi-center clinical trial study for the assessment of the efﬁcacy and safety of traditional Chinese medicine external washing for treating the postoperative wounds in diabetic patients with anal ﬁstula. Yao et al. proposed the general guidelines about the use of herbal preparations in the treatment of gastrointestinal and liver complications. Sheng et al. showed that lithocholic acid had signiﬁcant protective activities on the intestinal environment, including the maintenance of tight junctions, anti-bacterial and anti- inﬂammatory responses. Gao et al. showed that epimedin B caused the Epimedii Folium (EF)-induced Idiosyncratic drug- induced liver injury (IDILI) and the glycogen concentration in EF is not dependent on NLRP3 inﬂammasome activity. Editorial on the Research Topic H b l di i f i Editorial on the Research Topic Herbal medicines for gastrointestinal and hepatic diseases - novel pharmacological and toxicological approaches, Volume I After respiratory tract diseases, acute gastrointestinal infections are the second most common infections among infants and children and are responsible for morbidity and mortality (Ferguson et al., 2020). These infections are caused by a variety of microorganisms with the most common species are Helicobacter pylori, Salmonella species, Clostridium difﬁcile, Shigella species, Giardia lamblia and Escherichia coli (Shariati et al., 2019). Gastrointestinal system is also associated with the hepatic complications including NAFLD and gastrointestinal malignancies, i.e., HCC (Younossi et al., 2018). Worldwide, liver cancer causes second most cancer related deaths (Jemal et al., 2011). For the management of hepatic and gastrointestinal diseases, long-term strategies are required from government and international bodies because hepatitis B virus and hepatitis C virus infected subjects are 370 million and 130 million, respectively (Alter, 2006). Traditional and complementary medicines (TCMs) are clinical practices that are used in the diagnosis, treatment and prevention of diseases. They are not completely merged into the healthcare system, however they are affordable, accessible and culturally accepted by the people. Herbal products are one of the major part of TCM. It is reported that market share of natural preparations is up to several billions of dollars in developing and developed nations, showing the trust of people on these products (Hitl et al., 2019). Herbal medicines treat gastrointestinal diseases by affecting intestinal barrier, microbial composition and metabolites, and inﬂammation (Wang et al., 2023). 01 Frontiers in Pharmacology Frontiers in Pharmacology frontiersin.org Hasnat et al. 10.3389/fphar.2023.1157229 Hasnat et al. blocking NF-κB pathway and autophagy markers. HLD showed anti-inﬂammatory effects by increasing the level of IL-10 and decreasing the level of pro-inﬂammatory cytokines. HLD also reduced the levels of LC3II/I and Beclin 1. In the Research Topic of papers under the Research Topic “Herbal Medicines for Gastrointestinal and Hepatic Diseases - Novel Pharmacological and Toxicological approaches - Volume I″, 15 papers were published., mainly focusing the herbal medicines for gastrointestinal and hepatic diseases. Couple of studies reported ethanol-induced gastric complications, i.e., Ke et al. ﬁrst time reported that LDOP-1 had protective effect against ethanol-induced gastric mucosal damage by controlling AMPK/mTOR pathway. Xie et al. performed a study on ethanol-induced gastric lesions and showed that MLG could enhance defensive mechanism through NF-κB/Nrf2/HO-1 pathway and had protective effect against Ethanol-induced gastric lesions. Acknowledgments We would like to acknowledge the authors for their valuable publications in this Research Topic. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Several studies have focused the autophagy pathway like, Zhang et al. expressed that Qingluo Tongbi Formula (QTF), a Chinese herbal formula, reduced TWHF-induced hepatotoxicity. TWHF caused an increase in endoplasmic reticulum stress (ERS) that regulated the mitochondrial autophagy through PERK-ATF4 pathway. QTF reduced the TW-induced ERS and mitophagy. Wang et al. concluded that chrysophanol-8-O-glucoside (CPOG) protected mice from LPS/D-galactosamine-induced acute liver damage by decreasing inﬂammation, oxidative stress and autophagy. CPOG inhibited the levels of p-IκB, p-p65, TNF-α and IL-1β upregulated by LPS. CPOG also reduced the levels of LC3B, P62, ATG5 and Beclin 1 by reducing reactive oxygen species and MAPK pathway. Cheng et al. reported that Huangkui lianchang decoction (HLD), a Chinese herbal preparation, treated colitis by Editorial on the Research Topic H b l di i f i They studied that epimedin B increased the secretion of IL-1β, played a role in the maturation of caspase-1 and activated the NLRP3 inﬂammasome by increasing the level of ROS. Ivyna de Araújo Rêgo et al. reported the role of ﬂavonoids in the prevention of gastric cancer by treating H. pylori infection and explored that ﬂavonoid rich extracts had anti-H. pylori action by inhibiting urease, distortion of genetic material, decreasing protein synthesis and adhesion of microorganism to host cells. Tan et al. published a systematic review and meta-analysis on Chinese herbal medicine with oxaliplatin and showed that these agents improve the tumor response in advance gastric cancer. Brockmueller et al. showed for the ﬁrst time that β1 integrin partly suppressed the inhibitory effects of resveratrol on the metastasis of colorectal cancer (CRC) cells. Resveratrol inhibited TME induced phosphorylation and nuclear shift of NF- κB which is associated with CXCR-4, MMP-9, FAK and caspase-3. However, β1 integrin inhibited the anti-invasive and anti-metastatic effects of resveratrol. In conclusion, this Research Topic provided recent advances in the scientiﬁc knowledge on the gastrointestinal system. Frontiers in Pharmacology frontiersin.org Younossi, Z., Anstee, Q. M., Marietti, M., Hardy, T., Henry, L., Eslam, M., et al. (2018). Global burden of NAFLD and NASH: Trends, predictions, risk factors and prevention. Nat. Rev. Gastroenterology hepatology 15, 11–20. doi:10.1038/nrgastro.2017.109 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. 02 frontiersin.org 10.3389/fphar.2023.1157229 10.3389/fphar.2023.1157229 Hasnat et al. Publisher’s note 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. 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. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated Hitl, M., Gavarić, N., Kladar, N., Brkić, S., Samojlik, I., Dragović, G., et al. (2019). Herbal preparations use in prevention and treatment of gastrointestinal and hepatic disorders—data from vojvodina, Serbia. Complementary Ther. Med. 43, 265–270. doi:10.1016/j.ctim.2019.02.018 Wang, L., Gou, X., Ding, Y., Liu, J., Wang, Y., Wang, Y., et al. (2023). The interplay between herbal medicines and gut microbiota in metabolic diseases. Front. Pharmacol. 14, 1105405. doi:10.3389/fphar.2023.1105405 Shariati, A., Fallah, F., Pormohammad, A., Taghipour, A., Safari, H., Chirani, A. S., et al. (2019). The possible role of bacteria, viruses, and parasites in initiation and exacerbation of irritable bowel syndrome. J. Cell. physiology 234, 8550–8569. doi:10.1002/jcp.27828 Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., and Forman, D. (2011). Global cancer statistics. CA a cancer J. Clin. 61, 69–90. doi:10.3322/caac.20107 Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., and Forman, D. (2011). Global cancer statistics. CA a cancer J. Clin. 61, 69–90. doi:10.3322/caac.20107 Shariati, A., Fallah, F., Pormohammad, A., Taghipour, A., Safari, H., Chirani, A. S., et al. (2019). The possible role of bacteria, viruses, and parasites in initiation and exacerbation of irritable bowel syndrome. J. Cell. physiology 234, 8550–8569. doi:10.1002/jcp.27828 Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., and Forman, D. (2011). Global cancer statistics. CA a cancer J. Clin. 61, 69–90. doi:10.3322/caac.20107 Shariati, A., Fallah, F., Pormohammad, A., Taghipour, A., Safari, H., Chirani, A. S., et al. (2019). The possible role of bacteria, viruses, and parasites in initiation and exacerbation of irritable bowel syndrome. J. Cell. physiology 234, 8550–8569. doi:10.1002/jcp.27828 Wang, L., Gou, X., Ding, Y., Liu, J., Wang, Y., Wang, Y., et al. (2023). The interplay between herbal medicines and gut microbiota in metabolic diseases. Front. Pharmacol. 14 1105405 doi:10 3389/fphar 2023 1105405 References Alter, M. J. (2006). Epidemiology of viral hepatitis and HIV co-infection. J. Hepatology 44, S6–S9. doi:10.1016/j.jhep.2005.11.004 Alter, M. J. (2006). Epidemiology of viral hepatitis and HIV co-infection. J. Hepatology 44, S6–S9. doi:10.1016/j.jhep.2005.11.004 Ferguson, N., Laydon, D., Nedjati-Gilani, G., Imai, N., Ainslie, K., and Baguelin, M. (2020). 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https://openalex.org/W2941022421	https://www.journals.vu.lt/nonlinear-analysis/article/download/12885/11729	English	null	A new eigenvalue problem for the difference operator with nonlocal conditions	Nonlinear analysis	2,019	cc-by	10,259	eISSN: 2335-8963 ISSN: 1392-5113 eISSN: 2335-8963 ISSN: 1392-5113 https://doi.org/10.15388/NA.2019.3.9 Nonlinear Analysis: Modelling and Control, 2019, Vol. 24, No. 3, 462–484 Received: November 11, 2018 / Revised: March 7, 2019 / Published online: April 19, 2019 Abstract. In the paper, the spectrum structure of one-dimensional differential operator with nonlocal conditions and of the difference operator, corresponding to it, has been exhaustively investigated. It has been proved that the eigenvalue problem of difference operator is not equivalent to that of matrix eigenvalue problem Au = λu, but it is equivalent to the generalized eigenvalue problem Au = λBu with a degenerate matrix B. Also, it has been proved that there are such critical values of nonlocal condition parameters under which the spectrum of both the differential and difference operator are continuous. It has been established that the number of eigenvalues of difference problem depends on the values of these parameters. The condition has been found under which the spectrum of a difference problem is an empty set. An elementary example, illustrating theoretical expression, is presented. Keywords: eigenvalue problem, nonlocal condition, difference operator. Mifodijus Sapagovasa, Regimantas ˇCiupailab, Kristina Jakub˙elien˙ec, Stasys Rutkauskasd aFakulty of Mathematics and Informatics, Vilnius University, Akademijos str. 4, LT-08412 Vilnius, Lithuania mifodijus.sapagovas@mii.vu.lt aFakulty of Mathematics and Informatics, Vilnius University, Akademijos str. 4, LT-08412 Vilnius, Lithuania mifodijus.sapagovas@mii.vu.lt aFakulty of Mathematics and Informatics, Vilnius University, Akademijos str. 4, LT-08412 Vilnius, Lithuania mifodijus.sapagovas@mii.vu.lt bVilnius Gediminas Technical University, Saul˙etekio ave. 11, LT-10223 Vilnius Lithuania regimantas ciupaila@vgtu lt bVilnius Gediminas Technical University, Saul˙etekio ave. 11, LT-10223 Vilnius Lithuania regimantas.ciupaila@vgtu.lt bVilnius Gediminas Technical University, Saul˙etekio ave. 11, LT-10223 Vilnius Lithuania regimantas.ciupaila@vgtu.lt cDepartment of Applied Mathematics, Kaunas University of Technology, Studentu˛ str. 50, LT-51368 Kaunas, Lithuania kristina.jakubeliene@ktu.lt cDepartment of Applied Mathematics, Kaunas University of Technology, Studentu˛ str. 50, LT-51368 Kaunas, Lithuania kristina.jakubeliene@ktu.lt dInstitute of Data Science and Digital Technologies, Vilnius University, Akademijos str. 4, LT-08412 Vilnius, Lithuania 1 Introduction and problem statement During last several decades of the development of differential equations theory and nu- merical analysis, there is an increased interest in problems with various types of nonlocal conditions. A separate class of these problems is eigenvalue problems of differential and difference operators. Eigenvalue problems of differential operator with nonlocal condi- tions can be interpreted as a separate case of the non-self-adjoint operators theory [12]. c⃝Vilnius University, 2019 463 A new eigenvalue problem for the difference operator with nonlocal conditions In papers [4,16,18,20,21], eigenvalue problems of one- and two-dimensional differential operators with various nonlocal boundary conditions were analyzed. Eigenvalue problems of difference operators with nonlocal conditions usually arise when solving boundary problems by the ﬁnite difference method. The spectrum proper- ties of difference operators with various nonlocal boundary conditions were explored for investigation of the stability of difference schemes [1,2,4,7,8,10,11]. Another sphere of such a spectrum analysis application is convergence of iterative methods for the systems of difference equations [17, 19, 22], in particular, for nonlinear elliptic equations with integral boundary conditions [22,23]. Many articles on the investigation of the partial differential equations with various types of nonlocal conditions were published presenting new mathematical models in heat conduction, thermoelasticity, underground water ﬂow, biochemistry and so on. References to the original papers can be found in [2, 19, 21]. Solving these problems by the ﬁnite difference method, we meet unavoidably the problem of the structure of the spectrum of difference operator. Therefore, the eigenvalue problems could be interpreted as one of the methods of modeling. In [5, 6], the eigenvalue problem was investigated in connection with the existence, uniqueness and multiplicity of the solution of differential problems with nonlocal condi- tions. The spectrum of differential and difference operators with nonlocal conditions is much more diverse and complicated as compared to the spectrum in the case of the classical boundary conditions (Dirichlet or Neumann). Let us take such an eigenvalue problem with the Bitsadze–Samarskii nonlocal condition [20]: d2u dx2 + λu = 0, x ∈(0, 1), u(0) = 0, u(1) = γu(ξ), ξ ∈(0, 1), where γ, ξ are given real numbers. It has been proved that, depending on the values of these parameters, in the spectrum of both differential and difference operators, there can be zero, positive, negative or complex values. 1 Introduction and problem statement Besides, though the matrix of a difference problem is non-symmetrical (except the case γ = 0), we can determine intervals of γ and ξ in which all the eigenvalues are real and positive. Next, let us take the corresponding difference problem ui−1 −2ui + ui+1 h2 + λui = 0, i = 1, . . . , N −1, u0 = 0, uN = γus, ui−1 −2ui + ui+1 h2 + λui = 0, i = 1, . . . , N −1, u0 = 0, uN = γus, where h = 1/N, ξ = Sh, which is equivalent to (N −1)-order matrix A eigenvalue problem Au = λu, u = {ui}, i = i, . . . , N −1. It has been proved that, under certain values of γ and ξ, matrix A may have a parasitic eigenvalue without any correspondence as h →0. For example, if as h →0. For example, if γ = (−1)N−S ξ , Nonlinear Anal. Model. Control, 24(3):462–484 464 M. Sapagovas et al. 464 M. Sapagovas et al. M. Sapagovas et al. there exists a matrix eigenvalue λ = 4/h2 with the corresponding eigenvector u = {ui} = (−1)icih , i = 1, . . . , N −1, where c ̸= 0 is any real number. As h →0, the limit of the eigenvector does not exist. where c ̸= 0 is any real number. As h →0, the limit of the eigenvector does not exis In paper [18], one more singularity of the spectrum of a difference operator with non- local conditions is indicated. Let us take a differential eigenvalue problem with integral conditions d2u dx2 + λu = 0, x ∈(0, 1), u(0) = γ1 1 Z 0 u(x) dx, u(1) = γ2 1 Z 0 u(x) dx. The following difference problem corresponds to it with the approximation error O(h2): ui−1 −2ui + ui+1 h2 + λui = 0, i = 1, . . . , N −1, u0 = γ1h u0 + uN 2 + N−1 X i=1 ui ! , uN = γ2h u0 + uN 2 + N−1 X i=1 ui ! . This difference eigenvalue problem for all the values γ1, γ2 and h, except one case as h = 2/(γ1 + γ2), is equivalent to the eigenvalue problem Au = λu, where A is the (N −1)-order matrix. 1 Introduction and problem statement If γ1 + γ2 > 2 and h < 2/(γ1 + γ2), then all eigenvalues of difference operator are positive except one that is negative. This negative eigenvalue tends to inﬁnity (−∞) as h →2/(γ1+γ2). This fact is well illustrated by numerical experiment when γ1 + γ2 is quite large positive number. If h > 2/(γ1 + γ2), then all eigenvalues are positive, and one of them tends to inﬁnity (+∞) as h →2/(γ1 + γ2). In the case h = 2/(γ1 + γ2), difference eigenvalue problem cannot be written in the matrix form Au = λu. In this paper, we consider a differential eigenvalue problem d2u dx2 + λu = 0, x ∈(0, 1), (1) u(0) = γ1u(1), (2) u(ξ) = γ2u(1 −ξ), 0 < ξ < 1, (3) (2) (3) ( ) (3) (3) and a difference eigenvalue problem, corresponding to it. Such a difference eigenvalue problem was investigated in paper [3] in which some necessary conditions for the pa- rameters γ1, γ2 and ξ were obtained in order that zero, positive, negative or complex eigenvalues might exist. In this paper, we have investigated in detail the spectrum of differential and difference operators and drew a new qualitative conclusions. Particularly, we have proved that, de- pending on the parameters γ1, γ2, ξ and h, the spectrum structure of a difference operator can be essentially different from the spectrum structure both of differential operator and that of matrix. https://www.mii.vu.lt/NA https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions 465 2 Eigenvalue problem of a differential operator As λ > 0, the general solution of (1) is u(x) = c1 cos αx + c2 sin αx, α = √ λ > 0. u(x) = c1 cos αx + c2 sin αx, α = √ λ > 0. After substituting this solution into nonlocal conditions (2) and (3), we have a system (1 −γ1 cos α)c1 −(γ1 sin α)c2 = 0, cos αξ −γ2 cos α(1 −ξ) c1 + sin αξ −γ2 sin α(1 −ξ) c2 = 0. (8) (8) For nontrivial solution (c1, c2) of this system, the necessary and sufﬁcient condition is is D = 1 −γ1 cos α −γ1 sin α cos αξ −γ2 cos α(1 −ξ) sin ξ −γ2 sin α(1 −ξ) = 0. After elementary rearrangements, hence it follows (7). Theorem 2. For all values of γ1 and γ2, except two cases γ1 =γ2 =1 and γ1 =γ2 =−1, and all values of ξ, there exist a countable set of positive eigenvalues of form (6). Theorem 2. For all values of γ1 and γ2, except two cases γ1 =γ2 =1 and γ1 =γ2 =−1, and all values of ξ, there exist a countable set of positive eigenvalues of form (6). Proof. Let us consider three qualitatively different cases of the parameters γ1 and γ2. (i) γ1 = γ2 ̸= ±1, i.e., γ1 −γ2 = 0, γ1γ2 −1 ̸= 0. In this case, equation (7) becomes as follows: sin αξ = 0. sin αξ = 0. Hence, we derive αk = kπ ξ , k = 1, 2, . . . . (ii) γ1 = 1/γ2, γ1 ̸= ±1, i.e., γ1γ2 −1 = 0, γ1 −γ2 ̸= 0. From (7) we obtain sin α(1 −ξ) = 0, αk = kπ 1 −ξ , k = 1, 2, . . . . αk = kπ 1 −ξ , k = 1, 2, . . . . (iii) γ1 −γ2 ̸= 0, γ1γ2 −1 ̸= 0. Denote ϕ1(α) = (γ1γ2 −1) sin αξ, ϕ2(α) = (γ1 −γ2) sin α(1 −ξ). The functions ϕ1(α) and ϕ2(α) are continuous periodical functions with the periods 2π/ξ and 2π/(1 −ξ), respectively. Since ξ < 1 −ξ, in a longer interval α ∈(0, 2π/ξ), the graphs of both functions intersect at least one time (or several times) in case ξ, γ1 and γ2 are ﬁxed. 2 Eigenvalue problem of a differential operator We investigate the spectrum structure of a differential operator, deﬁned by formu- las (1)–(3). First, we analyze when there exist real eigenvalues by separate three cases: λ = 0, λ > 0 and λ < 0. Theorem 1. The number λ = 0 is an eigenvalue of differential problem (1)–(3) if and only if the following condition is true: (γ1γ2 −1)ξ = (γ1 −γ2)(1 −ξ). (4) (4) Proof. As λ = 0, the general solution of equation (1) is Proof. As λ = 0, the general solution of equation (1) is u(x) = c1x + c2, where c1 and c2 are arbitrary constants. By substituting this expression into nonlocal conditions (2) and (3), we obtain a system of equations with two unknowns c1 and c2 −γ1c1 + (1 −γ1)c2 = 0, ξ −γ2(1 −ξ) c1 + (1 −γ2)c2 = 0. (5) (5) ξ −γ2(1 −ξ) c1 + (1 −γ2)c2 = 0. This system has a nontrivial solution if and only if This system has a nontrivial solution if and only if D = −γ1 1 −γ1 ξ −γ2(1 −ξ) 1 −γ2 = 0. After elementary rearrangement, it follows (4) from this equality. After elementary rearrangement, it follows (4) from this equality. Remark 1. In the coordinate plane (γ1, γ2), as ξ is a ﬁxed number, equation (4) deter- mines a hyperbola. Points (−1, −1) and (1, 1) always belong to the hyperbola indepen- dent of the ξ value (see Fig. 1). Figure 1. The graph of hyperbola (4) in the case ξ = 0.4. The grey areas correspond to the values of γ1, γ2 for which there exists one negative eigenvalue. Figure 1. The graph of hyperbola (4) in the case ξ = 0.4. The grey areas correspond to the values of γ1, γ2 for which there exists one negative eigenvalue. Nonlinear Anal. Model. Control, 24(3):462–484 466 M. Sapagovas et al. Lemma 1. Any positive eigenvalue of differential operator (1)–(3) is deﬁned by the formula 2 Lemma 1. Any positive eigenvalue of differential operator (1)–(3) is deﬁned by the formula 2 λk = α2 k, (6) (6) where αk are roots of the equation where αk are roots of the equation (γ1γ2 −1) sin αξ = (γ1 −γ2) sin α(1 −ξ). (7) (7) Proof. As λ > 0, the general solution of (1) is Proof. where β0 > 0 is the root of the equation where β0 > 0 is the root of the equation (γ1γ2 −1) sinh βξ = (γ1 −γ2) sinh β(1 −ξ). (10) (10) Proof. As λ < 0, the general solution of equation (1) is Proof. As λ < 0, the general solution of equation (1) is u(x) = c1 cosh βx + c2 sinh βx, β = √ −λ > 0. After substituting this expression into nonlocal condition (2) and (3), we get After substituting this expression into nonlocal condition (2) and (3), we get c1 = γ1(c1 cosh β + c2 sinh β), c1 cosh βξ + c2 sinh βξ = γ2 c1 cosh β(1 −ξ) + c2 sinh β(1 −ξ) . (11) (11) The necessary and sufﬁcient condition for the existence of a nontrivial solution (c1, c2) of this system is as follows: D = 1 −γ1 cosh β −γ1 sinh β cosh βξ −γ2 cosh β(1 −ξ) sinh βξ −γ2 sinh β(1 −ξ) = 0. As earlier, it follows (10) from this equality. As earlier, it follows (10) from this equality. As earlier, it follows (10) from this equality. As earlier, it follows (10) from this equality. Next, we explore when equation (10) has at least one root. Next, we explore when equation (10) has at least one root. Theorem 3. Except two cases γ1 = γ2 = 1 and γ1 = γ2 = −1, equation (10) has a unique root β0 ∈(0, ∞) if and only if Theorem 3. Except two cases γ1 = γ2 = 1 and γ1 = γ2 = −1, equation (10) has a unique root β0 ∈(0, ∞) if and only if γ1γ2 −1 γ1 −γ2 > 1 −ξ ξ , γ1 ̸= γ2. (12) (12) If condition (12) is satisﬁed, there exists a unique negative eigenvalue of differential problem (1)–(3), and it is deﬁned by formula (9). If condition (12) is satisﬁed, there exists a unique negative eigenvalue of differential problem (1)–(3), and it is deﬁned by formula (9). Proof. According to the assumption of the theorem, let us consider all real values of γ1 and γ2 except γ1 = γ2 = ±1. Thus, if γ1 = γ2, then equation (10) has only a root β = 0. Therefore, the condition γ1 −γ2 ̸= 0 is the necessary condition for the existence of the root β > 0. 2 Eigenvalue problem of a differential operator Thus, in all three cases, equation (7) has a countable set of roots αk, i.e., there exists a countable set of positive eigenvalues λk = α2 k. https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions A new eigenvalue problem for the difference operator with nonlocal conditions 467 467 Corollary 1. If γ1 = γ2 = 1 or γ1 = γ2 = −1, equations (7) become an identity for all values of λ. In other words, any positive number λ is an eigenvalue. It means that the spectrum of differential problem (1)–(3) is continuous. The phenomenon of continuous spectrum also takes a place in the theory of boundary value problems for degenerate elliptic equations [14,15]. Lemma 2. The negative eigenvalue of differential operator (1)–(3), if it exists, is deﬁned by the formula λ = −β2 0, (9) (9) Nonlinear Anal. Model. Control, 24(3):462–484 where β0 > 0 is the root of the equation In this case, we can rewrite equation (10) as follows: sinh β(1 −ξ) sinh βξ = γ1γ2 −1 γ1 −γ2 . (13) (13) Nonlinear Anal. Model. Control, 24(3):462–484 Nonlinear Anal. Model. Control, 24(3):462–484 468 M. Sapagovas et al. Figure 2. The graph of the function ϕ(β) = sinh β(1−ξ)/ sinh βξ in the case ξ = 0.4, γ1 = 2.4, γ2 = 1.4. Figure 2. The graph of the function ϕ(β) = sinh β(1−ξ)/ sinh βξ in the case ξ = 0.4, γ1 = 2.4, γ2 = 1.4. The function ϕ(β) = sinh β(1 −ξ)/ sinh βξ, as ξ ∈(0, 1/2), has in the interval β ∈(0, ∞) the following properties: (i) it is continuous, and ϕ(β) > 1; (i) it is continuous, and ϕ(β) > 1; (i) it is continuous, and ϕ(β) > 1; (ii) it is monotonously increasing function since (ii) it is monotonously increasing function since ϕ′(β) = (1 −ξ) cosh β(1 −ξ) sinh βξ −ξ sinh β(1 −ξ) cosh βξ sinh2 (βξ) = cosh β(1 −ξ) −ξ sinh βξ > 0; (iii) ϕ(0) = lim β→0 sinh β(1 −ξ) sinh βξ = lim β→0 (1 −ξ) cosh β(1 −ξ) ξ cosh βξ = 1 −ξ ξ > 1; (iv) lim β→∞ sinh β(1 −ξ) sinh βξ = +∞. = cosh β(1 −ξ) −ξ sinh βξ > 0; (iii) ϕ(0) = lim β→0 sinh β(1 −ξ) sinh βξ = lim β→0 (1 −ξ) cosh β(1 −ξ) ξ cosh βξ = 1 −ξ ξ > 1; (iv) lim β→∞ sinh β(1 −ξ) sinh βξ = +∞. (iv) It follows from these properties that equation (13) has a unique root in interval (0, ∞) if and only if condition (12) is fulﬁlled (see Fig. 2). Corollary 2. It follows from Lemma 2 that, in the case γ1 = γ2 = ±1, i.e., if γ1γ2−1 = 0 and γ1 −γ2 = 0, equation (10) turns into identity for all values of β > 0. Thus, if γ1 = γ2 = ±1, then any negative number λ = −β2 is the eigenvalue of differential problem (1)–(3). 3 Eigenvalue problem of a difference operator Positive eigenvalues of difference problem (14)–(15) satisfying the inequality 0 < λ < 4 h2 (17) 0 < λ < 4 h2 (17) (17) are deﬁned by the formula are deﬁned by the formula λk = 4 h2 sin2 αkh 2 , (18) (18) 3 Eigenvalue problem of a difference operator Let us write a difference problem of eigenvalues that approximates differential prob- lem (1)–(3) with the approximation error O(h2): ui−1 −2ui + ui+1 h2 + λui = 0, i = 1, . . . , N −1, (14) u0 = γ1uN, uS = γ2uN−S. (15) (14) (15) u0 = γ1uN, uS = γ2uN−S. https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal condition A new eigenvalue problem for the difference operator with nonlocal conditions 469 469 We denote here h = 1/N, ξ = Sh, 1 −ξ = (N −S)h; N and S are integer numbers. In other words, the grid is uniform, and ξ is a mesh point (1 −ξ is also a mesh point). Note that 1 ⩽S ⩽N/2 −1 and h ⩽ξ ⩽1/2 −h. We denote here h = 1/N, ξ = Sh, 1 −ξ = (N −S)h; N and S are integer numbers. In other words, the grid is uniform, and ξ is a mesh point (1 −ξ is also a mesh point). Note that 1 ⩽S ⩽N/2 −1 and h ⩽ξ ⩽1/2 −h. We call the number λ an eigenvalue of the difference problem, if with this number there exists a nontrivial solution (eigenvector) of problem (14)–(15). Let us analyze the spectrum (the set of all eigenvalues) of difference problem (14)– (15). The proofs of lemmas and theorems presented below, according to methodology are analogous to that used in Section 2. Therefore, in our proofs, we emphasize only that what is different. Theorem 4. The number λ = 0 is the eigenvalue of difference problem (14)–(15) if and l if (16) (γ1γ2 −1)ξ = (γ1 −γ2)(1 −ξ). (16) Proof. First, let us pay attention that condition (16) is coincident with condition (4) of Theorem 1. As λ = 0, the general solution of difference equation (14) is ui = c1ih + c2, i = 1, . . . , N −1, where c1 and c2 are arbitrary constants. By substituting this expression of ui into nonlocal conditions (15), we obtain a system just like system (5) in the proof of Theorem 1. Remark 1 is right for difference operator (14)–(15) as well as for differential opera- tor (1)–(3). Lemma 3. where αk are roots of the equation (γ1γ2 −1) sin αξ = (γ1 −γ2) sin α(1 −ξ) (19) (19) in the interval (0, π/h). Proof. First of all, note that equation (19) is coincident with equation (7) in Lemma 1. However, expressions and numbers of eigenvalues are different than in Lemma 1. Si h i li Proof. First of all, note that equation (19) is coincident with equation (7) in Lemma 1. However, expressions and numbers of eigenvalues are different than in Lemma 1. Si th i lit Since the inequality 1 −λh2 2 < 1 follows from condition (17), we can introduce into equation (14) a new unknown α instead of λ: follows from condition (17), we can introduce into equation (14) a new unknown α instead of λ: cos αh = 1 −λh2 2 , 0 < α < π h. (20) (20) Nonlinear Anal. Model. Control, 24(3):462–484 Nonlinear Anal. Model. Control, 24(3):462–484 M. Sapagovas et al. 470 M. Sapagovas et al. M. Sapagovas et al. Hence, formula (18) follows. Now, equation (14) becomes as follows: Hence, formula (18) follows. Now, equation (14) becomes as follows: ui−1 −2(cos αh)ui + ui+1 = 0, ui−1 −2(cos αh)ui + ui+1 = 0, and its general solution is ui = c1 cos αih + c2 sin αih. (21) (21) By substituting this expression of ui into nonlocal conditions (15), we obtain a system coincident with system (8) in the demonstration of Lemma 1. So, the rest of this proof now is coincident with that one of Lemma 1. Theorem 5. The number of positive eigenvalues of form (18) is ﬁnite with all the values of γ1, γ2 except two cases: γ1 = γ2 = 1 and γ1 = γ2 = −1. Theorem 5. The number of positive eigenvalues of form (18) is ﬁnite with all the values of γ1, γ2 except two cases: γ1 = γ2 = 1 and γ1 = γ2 = −1. Proof. According to assumption of the theorem, all possible values of γ1 and γ2 can be separated into three qualitatively different cases. (i) γ1 = γ2 ̸= ±1; then γ1γ2 −1 ̸= 0 and γ1 −γ2 = 0. Equation (19), in this case, is sin αξ = 0, sin αξ = 0, and its roots are αk = kπ ξ , 0 < αk < π h, i.e., 0 < k < S. Thus, i.e., 0 < k < S. https://www.mii.vu.lt/NA where αk are roots of the equation Thereby we admit that from the equality ξ = h it not follows (1)–(3) is ill- possessed problem. The matter is that h →0, but ξ = const. Hence, the spectrum of the difference problem (14)–(15) is empty set with only one concrete value h, i.e., h = ξ (see Section 6, case 2)). With increasing or decreasing value h this phenomenon disappears. Corollary 3. If γ1 = γ2 = 1 or γ1 = γ2 = −1, then the spectrum of difference problem (14)–(15) is continuous for all the values of ξ and h, i.e., any number λ ∈ (0, 4/h2) is an eigenvalue. The conclusion follows directly from equation (19). The conclusion follows directly from equation (19). Let us ﬁnd an eigenvector as γ1 = γ2 = 1, i.e., in the case of continuous spectrum. Consider any ﬁxed number λ0 ∈(0, 4/h2) as an eigenvalue. In accordance with (20), we calculate α0 ∈(0, π/h) from the equality cos α0h = 1 −λ0h2 2 . (24) (24) The eigenvector is of form (21). Choosing c1 = 1, from system (8) we calculate c2 = 1 −cos α0 sin α0 . c2 = 1 −cos α0 sin α0 . Thus, the eigenvector corresponding to the eigenvalue λ0 is as follows: u = {ui} = cos α0ih + 1 −cos α0 sin α0 sin α0ih , i = 0, . . . , N, where α0 satisﬁes equality (24). Note that, differently than in the case of the differential problem (Theorem 2), we have found not all the positive eigenvalues of the difference problem, but only the eigenvalues from the interval (0, 4/h2). Under certain additional conditions, there may exist one more eigenvalue λ ⩾4/h2 of the difference problem (see below Theorems 7 and 8). As far as the authors are acquainted, for the ﬁrst time, the existence of such an eigenvalue in the case of nonlocal conditions was noticed most likely in paper [20]. Lemma 4. The negative eigenvalue of difference problem (14)–(15), if it exists, is deﬁned by the formula λ = −4 h2 sinh2 βh 2 , β > 0, (25) (25) where β > 0 is the root of the equation where β > 0 is the root of the equation (γ1γ2 −1) sinh βih = (γ1 −γ2) sinh β(N −i)h. (26) (26) Proof. where αk are roots of the equation Thus, λk = 4 h2 sin2 kπh 2ξ = 4 h2 sin2 kπ 2S , k = 1, . . . , S −1, (22) (22) where S ⩾2. (ii) γ1 = 1/γ2 ̸= ±1, i.e., γ1γ2 −1 = 0, γ1 −γ2 ̸= 0. Then equation (19) is sin α(1 −ξ) = 0. Analogously as in the ﬁrst case, we derive Analogously as in the ﬁrst case, we derive αk = kπ 1 −ξ , k < N −S, λk = 4 h2 sin2 πk 2(N −S), k = 1, . . . , N −S −1. (23) (23) (iii) γ1 −γ2 ̸= 0 and γ1γ2 −1 ̸= 0. As mentioned in the proof of Theorem 2, in the interval (0, 2π/ξ), equation (19) has one or several roots. Since α ∈(0, π/h), the number of roots in this interval is ﬁnite (in the general case it depends on four parameters: γ1, γ2, ξ and h). (iii) γ1 −γ2 ̸= 0 and γ1γ2 −1 ̸= 0. As mentioned in the proof of Theorem 2, in the interval (0, 2π/ξ), equation (19) has one or several roots. Since α ∈(0, π/h), the number of roots in this interval is ﬁnite (in the general case it depends on four parameters: γ1, γ2, ξ and h). We shall indicate one interesting fact. We shall indicate one interesting fact. https://www.mii.vu.lt/NA https://www.mii.vu.lt/NA https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions 471 Remark 2. If ξ = h, i.e., S = 1 and γ1 = γ2 ̸= ±1, then it follows from formula (22) that difference problem (14)–(15) has no positive eigenvalue λ that satisﬁes the inequality 0 < λ < 4/h2. Thereby we admit that from the equality ξ = h it not follows (1)–(3) is ill- possessed problem. The matter is that h →0, but ξ = const. Hence, the spectrum of the difference problem (14)–(15) is empty set with only one concrete value h, i.e., h = ξ (see Section 6, case 2)). With increasing or decreasing value h this phenomenon disappears. Remark 2. If ξ = h, i.e., S = 1 and γ1 = γ2 ̸= ±1, then it follows from formula (22) that difference problem (14)–(15) has no positive eigenvalue λ that satisﬁes the inequality 0 < λ < 4/h2. Nonlinear Anal. Model. Control, 24(3):462–484 where αk are roots of the equation If λ < 0, then 1 −λh2/2 > 1, therefore we can introduce a new unknown β by the relation 2 Proof. If λ < 0, then 1 −λh2/2 > 1, therefore we can introduce a new unknown β by the relation 2 cosh βh = 1 −λh2 2 , β > 0. cosh βh = 1 −λh2 2 , β > 0. Nonlinear Anal. Model. Control, 24(3):462–484 Nonlinear Anal. Model. Control, 24(3):462–484 472 M. Sapagovas et al. M. Sapagovas et al. M. Sapagovas et al. Hence, formula (25) follows. Equation (14) becomes as follows: Hence, formula (25) follows. Equation (14) becomes as follows: ui−1 −2(cosh βh)ui + ui+1 = 0. The general solution of this equation is ui = c1 cosh βih + c2 sinh βih. By substituting this expression into nonlocal conditions (15), we get system (11). A fur- ther proof of the lemma is coincident with that of Lemma 2. Like as Theorem 3, the next theorem is proved. Theorem 6. Except two cases γ1 = γ2 = 1 and γ1 = γ2 = −1, there exists a unique neg- ative eigenvalue of form (25) of difference problem (14)–(15) if and only if condition (12) is true. Corollary 4. If γ1 = γ2 = ±1, then any number λ < 0 is the eigenvalue of difference operator (14)–(15). Indeed, in this case, equation (26) is an identity 0 = 0 for all values of β. In Fig. 1, in the presence of the ﬁxed value ξ = 0.4, in the grey areas of the coordinate plane (γ1, γ2), there exists a negative eigenvalue of difference problem (14)–(15). According to Theorems 3 and 6, there exists a negative eigenvalue of both differential and difference operators under the same conditions (12). Now we can return to conditions under which there exists the positive eigenvalue λ ⩾4/h2. Theorem 7. The number λ = 4/h2 is the eigenvalue of difference problem (14)–(15) if and only if the condition (−1)S(γ1γ2 −1)ξ = (−1)N−S(γ1 −γ2)(1 −ξ) (27) (27) is satisﬁed. Proof. When λ = 4/h2, equation (14) becomes as follows: Proof. When λ = 4/h2, equation (14) becomes as follows: ui−1 + 2ui + ui+1 = 0. ui−1 + 2ui + ui+1 = 0. The general solution of this equation is The general solution of this equation is ui = (−1)i(c1ih + c2). Nonlinear Anal. Model. Control, 24(3):462–484 where αk are roots of the equation (28) (28) After substituting the expression of this solution into conditions (15), we obtain c2 = γ1(−1)N(c1 + c2), (−1)S(c1ξ + c2) = γ2(−1)N−Sc1(1 −ξ) + c2 . https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions 473 473 Hence, the necessary and sufﬁcient condition for the existence of nontrivial solution (c1, c2) is Hence, the necessary and sufﬁcient condition for the existence of nontrivial solution (c1, c2) is D = −(−1)Nγ1 1 −(−1)Nγ1 (−1)Sξ −(−1)N−Sγ2(1 −ξ) (−1)S −(−1)N−Sγ2 = 0. ementary rearrangement, hence we derive (27). After elementary rearrangement, hence we derive (27). After elementary rearrangement, hence we derive (27). Assume that N is an even number. Then condition (27) is coincident with condi- tion (16). Thus, we obtained the following conclusion. Corollary 5. As N is an even number, the existence condition of the eigenvalues λ = 0 and λ = 4/h2 is the same. Theorem 8. Except two cases γ1 = γ2 = 1 and γ1 = γ2 = −1, difference prob- lem (14)–(15) has one eigenvalue λ > 4/h2 if and only if (−1)N γ1γ2 −1 γ1 −γ2 > 1 −ξ ξ . (29) (29) If this condition is satisﬁed, then If this condition is satisﬁed, then λ = 4 h2 cosh2 βh 2 , (30) (30) where β is a unique root of the equation Remark 3. The result of Theorems 7 and 8 on the existence conditions for the eigenvalues λ = 4/h2 and λ > 4/h2 is proper on for a difference but not a differential problem. The matter is that eigenvectors (28) and (32), corresponding to these eigenvalues, have no limit as h →0. where β is a unique root of the equation where β is a unique root of the equation where β is a unique root of the equation (−1)N(γ1γ2 −1) sinh βξ = (γ1 −γ2) sinh β(1 −ξ) (31) (31) in the interval (0, ∞). Proof. If λ > 4/h2, then 1 −λh2/2 < −1. Therefore, we can introduce in equation (14) a new unknown β > 0 by the equality Proof. If λ > 4/h2, then 1 −λh2/2 < −1. Therefore, we can introduce in equation (14) a new unknown β > 0 by the equality cosh βh = λh2 2 −1, β > 0. Hence, expression (30) follows, and equation (14) becomes such as follows: Hence, expression (30) follows, and equation (14) becomes such as follows: ui−1 + 2(cosh βh)ui + ui+1 = 0. ui−1 + 2(cosh βh)ui + ui+1 = 0. The general solution of this equation is ui = (−1)i(c1 cosh βih + c2 sinh βih). (32) (32) After substituting this expression into (15), we obtain a system, analogous to system (11): After substituting this expression into (15), we obtain a system, analogous to system (11): c1 = γ1(−1)N(c1 cosh β + c2 sinh β), c1 cosh βξ + c2 sinh βξ = γ2(−1)Nc1 cosh β(1 −ξ) + c2 sinh β(1 −ξ) . 1 γ1( ) ( 1 β + 2 β), c1 cosh βξ + c2 sinh βξ = γ2(−1)Nc1 cosh β(1 −ξ) + c2 sinh β(1 −ξ) . By equating a determinant of this system to zero, after elementary rearrangement, we obtain (31). The further proof of the theorem is analogous to that of Theorem 3. By equating a determinant of this system to zero, after elementary rearrangement, we obtain (31). The further proof of the theorem is analogous to that of Theorem 3. Nonlinear Anal. Model. Control, 24(3):462–484 Nonlinear Anal. Model. Control, 24(3):462–484 474 M. Sapagovas et al. M. Sapagovas et al. The inferences analogous to Corollaries 3 and 5 are true. The inferences analogous to Corollaries 3 and 5 are true. Corollary 6. If γ1 = γ2 = ±1, then any number λ > 4/h2 is the eigenvalue of difference problem (14)–(15). Corollary 7. As N is an even number, the existence condition of the eigenvalues λ < 0 and λ > 4/h2 is the same. Corollary 7. As N is an even number, the existence condition of the eigenvalues λ < 0 and λ > 4/h2 is the same. 4 Complex eigenvalues By substituting this expression in nonlocal conditions (15) we obtain where c1 and c2 are arbitrary complex constants. where c1 and c2 are arbitrary complex constants. where c1 and c2 are arbitrary complex constants. where c1 and c2 are arbitrary complex constants. By substituting this expression in nonlocal conditions (15) we obtain By substituting this expression in nonlocal conditions (15) we obtain c1 = γ1(c1 cos q + c2 sin q), c1 cos qξ + c2 sin qξ = γ2 c1 cos q(1 −ξ) + c2 sin q(1 −ξ) . c1 = γ1(c1 cos q + c2 sin q), Hence just like in the proof of Lemma 3, we get that the nontrivial solution (c1, c2) exists if and only if condition (34) is fulﬁlled. In case this equation has complex roots qk = αk ± iβk, αk ̸= 0, βk ̸= 0, then the corresponding eigenvalue λk is deﬁned by (33). We can specify three elementary cases where difference problem (14)–(15) has no complex eigenvalues. We formulate these cases as the corollaries of Lemma 5. Corollary 8. If γ1 = γ2 ̸= ±1, there are no complex eigenvalues. Corollary 8. If γ1 = γ2 ̸= ±1, there are no complex eigenvalues. Indeed, in this case, γ1 −γ2 = 0, γ1γ2 −1 ̸= 0. Thus, equation (34) becomes as follows: Indeed, in this case, γ1 −γ2 = 0, γ1γ2 −1 ̸= 0. Thus, equation (34) becomes as follows: sin qξ = 0. sin qξ = 0. If ξ is a real numbers, then all the roots qk are real. Corollary 9. In case γ1 = 1/γ1 ̸= 1, there are no complex eigenvalues. Corollary 9. In case γ1 = 1/γ1 ̸= 1, there are no complex eigenvalues. In this case, γ1γ2 −1 = 0, γ1 −γ2 ̸= 0 and equation (34) is as follows: In this case, γ1γ2 −1 = 0, γ1 −γ2 ̸= 0 and equation (34) is as follows: sin q(1 −ξ) = 0, sin q(1 −ξ) = 0, Nonlinear Anal. Model. Control, 24(3):462–484 4 Complex eigenvalues Differential operator of (1)–(3) is not self-adjoint. Therefore, there may exist complex eigenvalues. Such a statement is also right for difference operator of (14)–(15). We investi- gate when there exist complex eigenvalues of a difference operator, since for a differential operator, both the investigation methods and their results are analogous. p g g In this section, we denote an imaginary unit by the letter i, i.e., i = √−1. Therefore, in equation (14), we shall use the index j instead of index i. Lemma 5. If there exist complex eigenvalues λk of difference problem (14)–(15), they are deﬁned by the following formula: λk = 4 h2 sin2 qkh 2 , (33) (33) where qk = αk ± iβ are complex roots of the equation (γ1γ2 −1) sin qξ = (γ1 −γ2) sin q(1 −ξ). (34) (34) Proof. If λ is a complex number in equation (14), we can introduce a new complex quantity q by the formula Proof. If λ is a complex number in equation (14), we can introduce a new complex quantity q by the formula cos qh = 1 −λh2 2 , (35) (35) where q = α ± iβ. Hence, it follows that λ = 4 h2 sin2 qh 2 . Note that, in the case where λ is a complex number, there must be α ̸= 0 and β ̸= 0. The condition β = 0 is coincident with the condition that q is a real number, so λ is positive. If α = 0, then λ = 4 h2 sin2 iβh 2 = 4 h2 i sinh βh 2 2 = −4 h2 sinh2 βh 2 , i.e., the case α = 0, β ̸= 0 corresponds to λ < 0. In case α = β = 0, it follows λ = 0. i.e., the case α = 0, β ̸= 0 corresponds to λ < 0. In case α = β = 0, it follows λ = 0. https://www.mii.vu.lt/NA 475 A new eigenvalue problem for the difference operator with nonlocal conditions After replacement (35), equation (14) becomes as follows: After replacement (35), equation (14) becomes as follows: uj−1 −2(cos qh)uj + uj+1 = 0. uj−1 −2(cos qh)uj + uj+1 = 0. Its general solution is uj = c1 cos qjh + c2 sin qjh, q = α ± iβ, where c1 and c2 are arbitrary complex constants. which implies that the roots qk are real numbers. which implies that the roots qk are real numbers. Corollary 10. In case γ1γ2 −1 = γ2 −γ1 = a ̸= 0, there are no complex eigenvalues. Corollary 10. In case γ1γ2 −1 = γ2 −γ1 = a ̸= 0, there are no complex eigenvalues. In this case, we can rewrite equation (34) as follows: In this case, we can rewrite equation (34) as follows: 2a sin q ξ + 1 2 cos q 2 = 0, the roots qk of which are only real. the roots qk of which are only real. An analogous proposition is right in the case q y An analogous proposition is right in the case γ1γ1 −1 = −(γ1 −γ2) = a ̸= 0. We analyze one more complicated case where all eigenvalues of the difference opera- tor are real. Nonlinear Anal. Model. Control, 24(3):462–484 M. Sapagovas et al. 476 M. Sapagovas et al. Let us introduce a generalized parameter ˜γ = γ1γ2 −1 γ1 −γ2 . (36) (36) This value was used in many lemmas and theorems. This value was used in many lemmas and theorems. This value was used in many lemmas and theorems. https://www.mii.vu.lt/NA Theorem 9. If \|˜γ\| ⩽1, then all the eigenvalues of difference operator (14)–(15) are real. Theorem 9. If \|˜γ\| ⩽1, then all the eigenvalues of difference operator (14)–(15) are real. Proof. Suppose that under condition \|˜γ\| ⩽1, equation (34) has a complex root q = α±iβ, α ̸= 0, β ̸= 0. Since \|˜γ\| ⩽1, it means that γ1 −γ2 ̸= 0. We rewrite equation (34) in the following form: sin (α ± iβ)(1 −ξ) −˜γ sin (α ± iβ)ξ = 0. sin α(1 −ξ) cosh β(1 −ξ) ± i sinh β(1 −ξ) cos α(1 −ξ) −˜γ(sin αξ cosh βξ ± i sinh βξ cos αξ) = 0. We separate the real and imaginary parts: We separate the real and imaginary parts: We separate the real and imaginary parts: sin α(1 −ξ) cosh β(1 −ξ) −˜γ sin αξ cosh βξ = 0, (37) sinh β(1 −ξ) cos α(1 −ξ) −˜γ sinh βξ cos αξ = 0. (38) (37) (38) (38) We express sin α(1 −ξ) from equation (37) and cos α(1 −ξ) from equation (38) and substitute the obtained expressions into the identity sin2 α(1 −ξ) + cos2 α(1 −ξ) = 1. By substituting, we get ˜γ cosh βξ cosh β(1 −ξ) 2 sin2 αξ + ˜γ sinh βξ sinh β(1 −ξ) 2 cos2 αξ = 1. (39) (39) Since ξ < 1 −ξ, β ̸= 0, \|˜γ\| ⩽1, we derive Since ξ < 1 −ξ, β ̸= 0, \|˜γ\| ⩽1, we derive Since ξ < 1 −ξ, β ̸= 0, \|˜γ\| ⩽1, we derive Since ξ < 1 −ξ, β ̸= 0, \|˜γ\| ⩽1, we derive ˜γ cosh βξ cosh β(1 −ξ) 2 < 1, ˜γ sinh βξ sinh β(1 −ξ) 2 < 1. Consequently, equality (39) is impossible. We have got a contradiction from which it follows that q cannot be complex. Corollary 11. In case γ1 = γ2 = ±1, any complex number q is the root of equation (34), i.e., any complex number λ is the eigenvalue of difference problem (14)–(15). Corollary 11. In case γ1 = γ2 = ±1, any complex number q is the root of equation (34), i.e., any complex number λ is the eigenvalue of difference problem (14)–(15). https://www.mii.vu.lt/NA 477 A new eigenvalue problem for the difference operator with nonlocal conditions Nonlinear Anal. Model. Control, 24(3):462–484 5 The difference eigenvalue problem as a generalized matrix eigen- value problem In paper [3], it is stated difference eigenvalue problem (14)–(15) can be written as a matrix eigenvalue problem (40) Au = λu, (40) where A is (N−1)-order matrix, u = (u1, u2, . . . , uN−1)T. The expression of matrix A is written as well. Though the expression of matrix A is written correctly, problem (14)–(15) is not equivalent to problem (40). We shall repeat these reasoning, presented in paper [3], and will correct one inaccu- racy in it. At the same time, we note that the authors of paper, after writing problem (14)– (15) in incorrect form (40), nowhere use such a form. We will write a slightly different matrix from of problem (14)–(15), and we will comment it in Section 6. p Let us write equation (14) more in detail: h−2(−u0 + 2u1 −u2) = λu1, h−2(−u1 + 2u2 −u3) = λu2, h−2(−uS−2 + 2uS−1 −uS) = λuS−1, h−2(−uS−1 + 2uS −uS+1) = λuS, (41) h−2(−uS + 2uS+1 −uS+2) = λuS+1, (41) h−2(−uN−3 + 2uN−2 −uN−1) = λuN−2, h−2(−uN−2 + 2uN−1 −uN) = λuN−1. We substitute the expression u0 = γ1uN from (15) into the ﬁrst equation of sys- tem (41). Analogously, we substitute the expression uS = γ2uN−S from (15) into three equations of system (41) as i = S −1, S, S + 1. After substitution, we obtain a new form of problem (14)–(15) h−2(2u1 −u2 −γ1uN) = λu1, h−2(−u1 + 2u2 −u3) = λu2, h−2(2u1 −u2 −γ1uN) = λu1, h−2(−u1 + 2u2 −u3) = λu2, . . . h−2(−uS−2 + 2uS−1 −γ2uN−S) = λuS−1, h−2(−uS−1 −uS+1 + 2γ2uN−S) = λuS, (42) h−2(2uS+1 −uS+2 −γ2uN−S) = λuS+1, (42) h−2(−uN−3 + 2uN−2 −uN−1) = λuN−2, h−2(−uN−2 + 2uN−1 −uN) = λuN−1. 478 M. Sapagovas et al. System (42), together with (15), is equivalent to system (14)–(15). Now system (42) can be written in the matrix form System (42), together with (15), is equivalent to system (14)–(15). Now system (42) can be written in the matrix form Au(1) = λu(2), Au(1) = λu(2), where where u(1) = (u1, u2, . . . , uS−1, uS+1, . . . , uN−1, uN)T, u(2) = (u1, u2, . . . , uS−1, uS, . . . , uN−2, uN−1)T. The expression of the matrix A is written correct in [3]. Since u(1) ̸= u(2), the problem Au(1) = λu(2) is not an eigenvalue problem. 5 The difference eigenvalue problem as a generalized matrix eigen- value problem Note that, neither by the method proposed in [3], not by any other way, difference eigenvalue problem (14)–(15) cannot be written in form (40). However, this problem (14)–(15) can be written as generalized matrix eigenvalue problem [9,13] (43) Au = λBu, (43) where A and B are the N-order matrices. A speciﬁc feature of such a problem is that B is a singular matrix (B−1 does not exists). Let us write system (14)–(15) in the following way: u0 = γ1uN, (44) ui−1 −2ui + ui+1 + λh2ui = 0, i = 1, . . . , N −1, (45) uS = γ2uN−S. (46) (44) (44) (45) (46) (46) In this system, we take two steps of equivalent rearrangement. In this system, we take two steps of equivalent rearrangement. In this system, we take two steps of equivalent rearrangement. The ﬁrst step: write the expression u0 from (44) into equation (45) in which i = 1. So we obtain −2u1 + u2 + γ1uN + λh2u1 = 0. The second step: subtract equation (46) from equation (45) in which i = S + 1. Instead of equation (46), we obtain the new equation −2uS+1 + uS+2 + γ2uN−S + λh2uS+1 = 0. https://www.mii.vu.lt/NA In this way, we get a new system In this way, we get a new system In this way, we get a new system −2u1 + u2 + γ1uN + λh2u1 = 0, ui−1 −2ui + ui+1 + λh2ui = 0, i = 2, . . . , N −1, −2uS+1 + uS+2 + γ2uN−S + λh2uS+1 = 0. (47) (47) By adding equation (44) this system is equivalent to initial system (44)–(46). Now system (47) can be written as generalised matrix eigenvalue problem (43), where B is https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions 479 A new eigenvalue problem for the difference operator with nonlocal conditions 479 singular matrix [16]. Next, we write expressions of N-order matrices A and B: A =          2 −1 −γ1 −1 2 −1 0 −1 2 −1 0 ... −1 2 −1 0 . . . 2 −1 . . . −γ2 . . . 0 0 0          , B = h2          1 0 1 0 1 0 ... 1 0 0 . . . . . . 0 1 0 . . . . . . 0          . Nonzero elements on the last row of matrix A are in columns with the numbers S +1, S + 2, N −S. Nonzero element on the last row of matrix B is in the column with the number S + 1. Matrix B is singular matrix, its last column consists only of zeros. The eigenvalues of eigenvalue problem (43) are the roots of a generalized characteristic equation det(A −λB) = 0. (48) (48) Since the order of matrix A and B is N, det(A −λB) is not higher than the N-order polynomial. It would be not right assert that the characteristic polynomial is of N-order because that is possible only in the case when exists B−1. Equation (48) yields the following assertion. Corollary 12. If the spectrum of difference problem (14)–(15) is not continuous (case γ1 = γ2 = ±1), then there exist no more than N eigenvalues. 6 Illustrative example Let us take a concrete example: ξ = 0.25, N = 4 and γ1, γ2 are varying parameters. By means of this rather elementary example we illustrate the substance and variety of the spectrum of difference operator with nonlocal conditions. So we get the following eigenvalue problem: u0 = γ1u4, (49) u0 −2u1 + u2 + λh2u1 = 0, (50) u1 −2u2 + u3 + λh2u2 = 0, (51) u2 −2u3 + u4 + λh2u3 = 0, (52) u1 = γ2u3. (53) Nonlinear Anal. Model. Control, 24(3):462–484 480 M. Sapagovas et al. We transform this eigenvalue problem into a matrix form Au = λBu in accordance with the methodology described in Section 5. To this end, we substitute u0 from (49) into (50), and we change equation (53) by a difference of (51) and (53). Thus, we derive −2u1 + u2 + γ1u4 + λh2u1 = 0, u1 −2u2 + u3 + λh2u2 = 0, u2 −2u3 + u4 + λh2u3 = 0, −2u2 + (1 + γ2)u3 + λh2u2 = 0 or, in matrix form,     −2 1 0 γ1 1 −2 1 0 0 1 −2 1 0 −2 1 + γ2 0         u1 u2 u3 u4    = λh2     −1 0 0 0 0 −1 0 0 0 0 −1 0 0 −1 0 0         u1 u2 u3 u4    . Next, we calculate the fourth-order determinant: Next, we calculate the fourth-order determinant: det(A −λB) = −2 + λh2 −2 + λh2 1 0 1 −2 + λh2 1 −2 + λh2 1 + γ2 0 − 1 0 γ1 1 −2 + λh2 1 −2 + λh2 1 + γ2 0 = −2 + λh2−2 + λh2 −(1 + γ2) −2 + λh2 − γ1(1 + γ2) −γ1 −2 + λh22 −(1 + γ2) = −2 + λh22(γ1 −γ2) −(γ1 −1)(γ2 + 1) = 0. (54 (54) In the general case, we obtain the second-order characteristic polynomials. To be pre- cise, depending on the values of γ1 and γ2, we obtain not higher than the second-order polynomial (we remind, N = 4). 6 Illustrative example It follows from (55) that both eigenvalues Case 5. We take γ1 and γ2 such that \|˜γ\| ⩽1. It follows from (55) that both eigenvalues λ1,2 = 2 h2 ± √˜γ + 1 h2 λ1,2 = 2 h2 ± √˜γ + 1 h2 are real (see Theorem 9). Note that the condition \|˜γ\| ⩽1 is not necessary for eigen- values to be real. In our particular case, the necessary and sufﬁcient condition for both eigenvalues to be real is ˜γ ⩾−1. are real (see Theorem 9). Note that the condition \|˜γ\| ⩽1 is not necessary for eigen- values to be real. In our particular case, the necessary and sufﬁcient condition for both eigenvalues to be real is ˜γ ⩾−1. Case 6. ˜γ < −1. It follows from (55) that both eigenvalues are complex conjugate numbers: λ1,2 = 2 h2 ± √˜γ + 1 h2 = 2 ± i p \|˜γ\| −1 h2 . 6 Illustrative example Besides, note that, in the case γ1 ̸= γ2, we can write equation (54) as follows: −2 + λh22 = γ1γ2 −1 γ1 −γ2 + 1 or −2 + λh2 = ± p ˜γ + 1. (55) (55) Let us take several concrete values of γ1 and γ2. Let us take several concrete values of γ1 and γ2. Let us take several concrete values of γ1 and γ2. Case 1. γ1 = γ2 = ±1. Characteristic equation (54) becomes an identity 0 = 0 for all the values λ. Thus, the spectrum of difference operator (49)–(53) is continuous (see Corollaries 3, 4, 6). Case 2. γ1 = γ2 ̸= ±1. Characteristic equation (54) becomes as follows: (γ1 −1)(γ2 + 1) = 0, (γ1 −1)(γ2 + 1) = 0, what it is not true. So the spectrum of difference equation is an empty set (see Remark 2). what it is not true. So the spectrum of difference equation is an empty set (see Remark 2). https://www.mii.vu.lt/NA https://www.mii.vu.lt/NA https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions A new eigenvalue problem for the difference operator with nonlocal conditions 481 481 Case 3. Let us choose γ1 and γ2 so that ˜γ = γ1γ2 −1 γ1 −γ2 = 1 −ξ ξ . Since ξ = 0.25, we have (1 −ξ)/ξ = 3. It follows from (55) that Since ξ = 0.25, we have (1 −ξ)/ξ = 3. It follows from (55) that −2 + λh2 = ±2 −2 + λh2 = ±2 or λ1 = 0, λ2 = 4/h2 (see Corollary 5). Note that there is an inﬁnite set of points of parameters γ1 and γ2 with the property ˜γ = 3. or λ1 = 0, λ2 = 4/h2 (see Corollary 5). Note that there is an inﬁnite set of points of parameters γ1 and γ2 with the property ˜γ = 3. Case 4. Let us choose γ1 and γ2 so that it were ˜γ = γ1γ2 −1 γ1 −γ2 > 1 −ξ ξ = 3. In this case, √˜γ + 1 > 2, and from (55) we derive In this case, √˜γ + 1 > 2, and from (55) we derive λ1 = 2 h2 + √˜γ + 1 h2 > 4 h2 , λ2 = 2 h2 − √˜γ + 1 h2 < 0 (see Corollary 7). Case 5. We take γ1 and γ2 such that \|˜γ\| ⩽1. Nonlinear Anal. Model. Control, 24(3):462–484 7 Remarks and generalization In this paper, the spectrum structure of differential and difference operators with nonlocal condition has been explored. The main aim was research of the eigenvalue problem of difference operator. It has been proved that the spectrum structure of both differential and difference opera- tors depends not only on the type on nonlocal conditions, but particularly on the parameter Nonlinear Anal. Model. Control, 24(3):462–484 482 M. Sapagovas et al. M. Sapagovas et al. M. Sapagovas et al. value under nonlocal conditions. Besides, the dependence is more clearly deﬁned not by the values of separate parameters γ1, γ2 and ξ, but using generalized parameter values ˜γ = γ1γ2 −1 γ1 −γ2 , ˜ξ = 1 −ξ ξ as well as interdependence of these generalized values. The conditions (equalities and inequalities) that include these generalized parameters in the coordinates plane (γ1, γ2) are related with a hyperbola. By comparing the published results of the considered subject, we have to note that, perhaps for the ﬁrst time, it has been proved that the spectrum of difference operators with nonlocal conditions can be continuous or coincident with an empty set. Namely, if the parameters γ1, γ2 satisfy the condition γ1 = γ2 = 1 or γ1 = γ2 = −1, any real or complex number is an eigenvalue of both the difference and differential operator. Meanwhile, the spectrum of the difference operator as an empty set can be in case ξ = h. In addition, the number of eigenvalues of difference operator (14)–(15) depends on the parameters γ1, γ2 and ξ rather in a complicated way. The eigenvalue problem of a difference operator with nonlocal conditions investigated in our paper differs by many properties from the eigenvalue problems of a differential operator and that of matrix. The fact that the spectrum of a difference operator with concrete values of parameters γ1, γ2 has some unusual properties is conditioned by form of nonlocal conditions. Without an exhaustive examination of this issue, we refer to one typical property of nonlocal conditions analyzed in this paper. Boundary interval points x = 0 and x = 1 are included only in one nonlocal condi- tion. Another nonlocal condition is deﬁned only at interior points of the interval under consideration. In essence, that is the main reason why the eigenvalue problem of differ- ence operator cannot be expressed in the matrix form Au = λu with the (N −1)-order matrix. https://www.mii.vu.lt/NA 7 Remarks and generalization Note that this property is typical not only of nonlocal conditions (15). Nonlocal conditions of different type can have such property, for example, integral conditions 1 Z 0 u(x) dx = 0, ξ2 Z ξ1 u(x) dx = 0, 0 < ξ1 < ξ2 < 1. If we exchange nonlocal condition (3) by a more general one If we exchange nonlocal condition (3) by a more general one u(ξ) = γ2u(η), 1 2 < η < 1, many propositions, proved in this paper, will be correct. Taking apart, conclusions about a continuous spectrum will be true also. Without doubt, in each concrete case of new nonlocal condition, more exhaustive researches are necessary. The spectrum structure of difference operators is rather important in the investigation of stability and convergence of difference schemes as well as iterative methods for systems of difference equations. These issues are not the research object of this article as well as https://www.mii.vu.lt/NA A new eigenvalue problem for the difference operator with nonlocal conditions 483 the spectrum structure of the corresponding two-dimensional differential operators. We left these problems for future research. The main aim of our paper was to pay attention to some unusual properties of the spectrum structure of difference operators with nonlocal conditions. The research we performed allows us to make the following conclusion. The spectrum of the difference operator with nonlocal conditions may essentially differ from the spectrum of the dif- ferential operators or the one of the matrices. Therefore, the eigenvalue problem of the difference operator with nonlocal conditions is worth to be self-contained object of the investigation. 13. J. Rommes, Arnoldi and Jacobi–Davidson methods for generalized eigenvalue problem Ax = λBx with singular B, Math. Comput., 77(262):995–1015, 2007. References 1. B. Cahlon, D.M. Kulkarni, P. Shi, Stepwise stability for the heat equation with a nonlocal constrain, SIAM J. Numer. Anal., 32(2):571–593, 1995. 2. R. ˇCiegis, N. Tumanova, Stability analysis of ﬁnite difference schemes for pseudoparabolic problems with nonlocal boundary conditions, Math. Model. Anal., 19(2):285–293, 2014. 3. A. Elsaid, S.M. Hedal, A.M.A. El-Sayed, The eigenvalue problem for elliptic partial differ- ential equation with two-point nonlocal conditions, J. Appl. Anal. Comput., 5(1):146–158, 2015. 4. A.V. Gulin, On the spectral stability in subspaces for difference schemes with nonlocal bound- ary conditions, Differ. Equations, 49(7):815–823, 2013. 5. J. 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Sapagovas, On the spectral properties of three-layer difference schemes for parabolic equations with nonlocal conditions, Differ. Equations, 48(7):1018–1027, 2012. 17. M. Sapagovas, V. Griškonien˙e, O. Štikonien˙e, Application of M-matrices theory to inves- tigation of a nonlinear elliptic equation with an integral condition, Nonlinear Anal. Model. Control, 22(4):489–504, 2017. 18. M. References Sapagovas, T. Meškauskas, F. Ivanauskas, Numerical spectral analysis of a difference op- erator with non-local boundary conditions, Appl. Math. Comput., 218(14):7513–7527, 2012. 19. M. Sapagovas, O. Štikonien˙e, R. ˇCiupaila, Ž. Jokšien˙e, Covergence of iterative methods for elliptic equations with integral boundary conditions, Electron. J. Differ. Equ., 2016(118):1–14, 2016. 20. M.P. Sapagovas, A.D. Štikonas, On the structure of the spectrum of a differential operator with a nonlocal condition, Differ. Equations, 41(7):961–969, 2005. 21. A. 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https://openalex.org/W4376629786	https://www.nature.com/articles/s41598-023-34746-z.pdf	English	null	Transcutaneous auricular vagus nerve stimulation as a potential novel treatment for polycystic ovary syndrome	Scientific reports	2,023	cc-by	10,987	Transcutaneous auricular vagus nerve stimulation as a potential novel treatment for polycystic ovary syndrome Shike Zhang 1,2, Hui He 3, Yu Wang 4, Xiao Wang 3 & Xiaofang Liu 5 OPEN Shike Zhang 1,2, Hui He 3, Yu Wang 4, Xiao Wang 3 & Xiaofang Liu 5 Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of childbearing age. The etiology of PCOS is multifactorial, and current treatments for PCOS are far from satisfactory. Recently, an imbalanced autonomic nervous system (ANS) with sympathetic hyperactivity and reduced parasympathetic nerve activity (vagal tone) has aroused increasing attention in the pathogenesis of PCOS. In this paper, we review an innovative therapy for the treatment of PCOS and related co-morbidities by targeting parasympathetic modulation based on non-invasive transcutaneous auricular vagal nerve stimulation (ta-VNS). In this work, we present the role of the ANS in the development of PCOS and describe a large number of experimental and clinical reports that support the favorable effects of VNS/ta-VNS in treating a variety of symptoms, including obesity, insulin resistance, type 2 diabetes mellitus, inflammation, microbiome dysregulation, cardiovascular disease, and depression, all of which are also commonly present in PCOS patients. We propose a model focusing on ta-VNS that may treat PCOS by (1) regulating energy metabolism via bidirectional vagal signaling; (2) reversing insulin resistance via its antidiabetic effect; (3) activating anti-inflammatory pathways; (4) restoring homeostasis of the microbiota-gut-brain axis; (5) restoring the sympatho-vagal balance to improve CVD outcomes; (6) and modulating mental disorders. ta-VNS is a safe clinical procedure and it might be a promising new treatment approach for PCOS, or at least a supplementary treatment for current therapeutics. The pathological mechanism of polycystic ovary syndrome. Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disorder with a prevalence of 4–10% in women of childbearing-age depending on the definitions that are used1. It’s typical clinical manifestations include menstrual irregulari- ties, clinical and/or biochemical hyperandrogenism (HA), and polycystic ovary morphology on ultrasound2,3. Besides reproductive dysfunction, PCOS may result in various metabolic co-morbidities affecting multiple aspects of a woman’s overall health. For example, insulin resistance (IR) is a key characteristic of PCOS, and this can be exacerbated by obesity, particularly visceral adiposity4. Women with PCOS also have a markedly increased risk for impaired glucose tolerance5,6, type 2 diabetes mellitus (T2DM)7,8, and cardiovascular disease (CVD)9 owing to the persistent effects of IR on metabolism. In addition, these women are more likely to suffer from psychological illnesses such as anxiety and depression than healthy controls10–15. www.nature.com/scientificreports www.nature.com/scientificreports Transcutaneous auricular vagus nerve stimulation as a potential novel treatment for polycystic ovary syndrome Shike Zhang 1,2, Hui He 3, Yu Wang 4, Xiao Wang 3 & Xiaofang Liu 5 OPEN The ovary is innervated by the sympathetic superior ovarian nerve and the ovarian plexus nerve from the upper lumbar segment via visceral nerve fibers and by the parasympathetic nerve through the VN, which is regulated by the central nervous system (CNS)33,34. Abnormalities in the ANS play an important role in the progression of ovarian pathological conditions, such as PCOS. Numerous previous experiments in rats with steroid-induced polycystic ovaries have shown that hyperactivity of the SNS innervating the ovary— which can be demonstrated through enhanced synthesis of SNS activity markers, such as nerve growth fac- tor, norepinephrine, and tyrosine hydroxylase—may contribute to the etiology of PCOS35–39. Furthermore, the peripheral SNS may be involved in the pathophysiology of PCOS by modulating immune-endocrine function40. In turn, hormonal and metabolic disturbances are also related to autonomic dysfunction in PCOS. Hashim et al. reported that obese women with PCOS exhibited more pronounced autonomic dysfunction and sympathoexci- tation than non-obese patients41. Shorakae et al. demonstrated that lower high-molecular-weight adiponectin, a biologically active form closely associated with insulin sensitivity and metabolic disorders, is inversely associ- ated with increased sympathetic activity in women with PCOS42. Sverrisdottir et al. showed that testosterone is positively associated with muscle sympathetic nerve activity in lean women with PCOS43, and Shorakae et al. found that chronic low-grade inflammation might play a potential role in mediating the effect of sympathetic dysfunction on HA and IR in PCOS24. y Based on the abundant evidence of increased sympathetic neural activity in PCOS documented in both animal and human studies, and the complicated and bidirectional associations of sympathetic activation with endocrinal and metabolic disorders, it is reasonable to speculate that sympathoexcitation may play a role in the progression of the syndrome. Therefore, treatments that seek to reduce sympathetic activity or increase parasympathetic activity in order to restore the sympatho-vagal balance may have the potential to improve the outcomes of PCOS. Indeed, conventional interventions, such as weight loss, pharmacotherapy with the insulin sensitizer metformin, electroacupuncture, etc., have been reported to play a role in suppressing sympathetic over-activation44, while parasympathetic (vagal) modulation has been largely neglected. Transcutaneous auricular vagus nerve stimulation (ta‑VNS). Vagus nerve stimulation (VNS), a kind of bioelectronic medicine, was first introduced by James Leonard Corning in the late eighteenth century and provided a new way of regulating the autonomic tone45. Transcutaneous auricular vagus nerve stimulation as a potential novel treatment for polycystic ovary syndrome Shike Zhang 1,2, Hui He 3, Yu Wang 4, Xiao Wang 3 & Xiaofang Liu 5 OPEN p y g y p y PCOS is a biologically heterogeneous condition involving multiple pathophysiological processes that lead to ovarian dysfunction. It is considered to be a neuroendocrine disease resulting from an aberrant hypothalamic- hypophyseal system16,17. Hypothalamus-pituitary-ovary axis dysregulation—including rapid gonadotrophin- releasing hormone pulse frequency associated with luteinizing hormone (LH) hypersecretion and increased ovarian androgen production—leads to impaired folliculogenesis and oocyte development18–20. Adrenally derived androgens due to disruption of the hypothalamic–pituitary–adrenal (HPA) axis may also contribute to the occurrence of PCOS21. IR and HA are the most significant hormonal disturbances and are usually regarded as the chief causes of PCOS22. In addition, chronic low-grade inflammation may be an important component in 1Southern University of Science and Technology Yantian Hospital, Shenzhen 518081, China. 2Shenzhen Yantian District People’s Hospital, Shenzhen 518081, China. 3First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China. 4First Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin 150040, China. 5Chinese People’s Liberation Army General Hospital, Beijing 100853, China. email: 1923725887@qq.com \| https://doi.org/10.1038/s41598-023-34746-z Scientific Reports \| (2023) 13:7721 www.nature.com/scientificreports/ the pathophysiology of PCOS and might have additional effects on the long-term metabolic disorders related to PCOS23,24. Recently, increasing studies have reported that autonomic dysfunction is also involved in the development of PCOS25–28. the pathophysiology of PCOS and might have additional effects on the long-term metabolic disorders related to PCOS23,24. Recently, increasing studies have reported that autonomic dysfunction is also involved in the development of PCOS25–28. The sympatho‑vagal balance. The sympathetic nervous system (SNS) and parasympathetic nervous sys- tem (PNS) are two primary branches of the autonomic nervous system (ANS). The vagus nerve (VN), which is the 10th and longest of the cranial nerves with 80%–90% afferent fibers, is a critical constituent of the PNS and acts as an important bidirectional conduit between the body and brain and mainly serves to maintain homeostasis29,30. The functions of the SNS and PNS are antagonistic and are in dynamic balance under physi- ological conditions, and imbalances in the interactions between the SNS and PNS can lead to various autonomic modulation-related disorders, including neurological, metabolic, inflammatory, cardiovascular, and psychiatric diseases31. In general, this imbalance involves relatively higher sympathetic activity associated with a paucity of parasympathetic activity32. Link between ANS dysfunction and PCOS. Ovary function is not only regulated by hormones, but also by neural signals. Potential pathways and action mechanisms by which ta‑VNS has Therapeutic effects PCOS Energy metabolism regulation via bidirectional vagal signaling. Although obesity is not necessary for the PCOS phenotype, most women with PCOS are overweight or obese. Obesity worsens IR and compensa- tory hyperinsulinemia (HI), which in turn promotes adipogenesis and reduces lipolysis. In addition, obesity increases the sensitivity of thecal cells to luteinizing hormone stimulation, leading to amplified ovarian androgen production4. Furthermore, obesity is often accompanied by systemic or tissue-specific chronic inflammation and oxidative stress, which is also true in the ovary, thus impairing oocyte development and maturation and reducing female fertility56. Generally, weight loss is the first step in the management of PCOS57, and various weight loss strategies like modified diet, regular exercise, behavioral changes, and even bariatric surgery in cases of severe obesity have been proposed. However, PCOS patients often fail to maintain these interventions and have high drop-out rates, thus decreasing the long-term effects of the interventions. The optimal methods for improving sustainability to achieve the recommended weight loss goal need further research57. y g g In particular, the VN establishes a bidirectional communication between the gastrointestinal tract and the CNS and thus plays an important role in regulating energy metabolism. There is evidence that disrupted vagal signaling is associated with the development of diet-induced weight gain, and targeting the VN with neuro- modulation provides a novel way to treat obesity58. Previously, several small clinical studies performed in patients with depression or epilepsy observed that VNS might have a significant effect on weight loss59–61. Subsequent experiments also showed that chronic VNS can effectively reduce food intake and decrease body weight by increasing brain satiety signals or by delaying gastric emptying mediated via the vagal afferents62–66. In addition, restoring or augmenting VN activity can attenuate obesity-associated conditions via inflammatory reflexes67. Recently, implanted VNS for obesity has been approved by the FDA68. As a non-invasive alternative approach to VNS, ta-VNS is now being investigated to evaluate its effect on weight loss. Li et al. observed that auricular VNS showed remarkable effects on reducing body weight and reducing visceral fat in obese rats69. Wang and Yu et al. reported that ta-VNS significantly ameliorates weight gain without changing food intake in Zucker diabetic fatty rats70–72. The mechanism might be that ta-VNS can accelerate energy expenditure by inhibiting hypothalamic P2Y1R expression, which is responsible for intracellular triglyceride accumulation in adipocytes mediated by extracellular ATP73. Transcutaneous auricular vagus nerve stimulation as a potential novel treatment for polycystic ovary syndrome Shike Zhang 1,2, Hui He 3, Yu Wang 4, Xiao Wang 3 & Xiaofang Liu 5 OPEN Emerging evidence has verified the improved outcomes of VNS in treating various diseases, and it has been approved by the Food and Drug Administration (FDA) as an alternative therapy for refractory epilepsy, refractory depression, cluster headaches, and migraines46. Fur- thermore, studies have expanded the use of VNS for a wider range of illnesses, including obesity, diabetes, CVD, and chronic inflammatory disorders such as sepsis, lung injury, rheumatoid arthritis, etc.47. Initially, VNS was performed by implanting stimulating or surface electrodes into the easily accessible vagus nerve in the neck for acute or chronic stimulation. However, this invasive VNS technique is associated with various surgical complica- tions, for example, infection, cardiac arrhythmia, device malfunction, cough, hoarseness, dyspnea, dysphagia, and so on48, thus limiting the application of VNS among larger patient populations. With advancements in medical technology, VNS can now be applied indirectly and non-invasively, including transcutaneous cervical VNS and ta-VNS49.i Specifically, the auricular concha area is the only place on the body surface where the VN sends its peripheral branch, called the auricular branch of the vagus nerve (ABVN)49. The ABVN forms a cutaneous receptive field in the pinna of the ear that is highly sensitive to external stimuli. The locations for the application of ta-VNS on the auricular surface are shown in Fig. 1. Evidence for the effects of ta-VNS on brainstem neuronal responses in healthy subjects has been confirmed by fMRI data demonstrating that ta-VNS of the cymba conchae projects to the nucleus tractus solitaries, which is a primary relay station for vagal afferent signals, and its main dopamin- ergic-downstream targets, the dorsal raphe nucleus, the substantia nigra, the subthalamic nucleus, and a region adjacent to the red nucleus50. It is precisely because of this direct anatomical pathway between the ABVN and the brainstem that makes it possible for ta-VNS to regulate bodily functions. Electricity has been an important tool in clinicians’ treatments over the past 2000 years, and using electrical stimulation has become one of the optimal way to perform non-invasive or minimally invasive stimulation of the outer ear51,52. Although still lacking FDA approval, current evidence suggests several advantages of ta-VNS, including beneficial effects comparable to implantable VNS, simple operation, greater accessibility, and milder side-effects53–55. Scientific Reports \| (2023) 13:7721 \| https://doi.org/10.1038/s41598-023-34746-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The locations for ta-VNS on the auricular surface. ta-VNS: transcutaneous auricular vagal nerve stimulation. Figure 1. Hypothesis yp Because ta-VNS is as effective as conventional VNS, we hypothesize that parasympathetic activity is increased by ta-VNS so as to suppress sympathetic overactivity and restore sympatho-vagal balance. Our hypothesis assumes that the severity of PCOS and its associated complications can be attenuated by "endogenous systemic braking", which will be naturally provided by the parasympathetic part of the ANS. Transcutaneous auricular vagus nerve stimulation as a potential novel treatment for polycystic ovary syndrome Shike Zhang 1,2, Hui He 3*, Yu Wang 4, Xiao Wang 3 & Xiaofang Liu 5 OPEN The locations for ta-VNS on the auricular surface. ta-VNS: transcutaneous auricular vagal nerve stimulation. www.nature.com/scientificreports/ glucose homeostasis that further develops into T2DM, and these alterations strongly correlate with the degree of adiposity. On the other hand, the increased androgen secretion associated with PCOS also plays a role in the development of prediabetes and T2DM by exacerbating IR78 or by stimulating low-grade inflammation79. Clini- cal evidence has suggested that administration of insulin-sensitizing agents, such as metformin and inositols, along with lifestyle modification can improve the endocrine and metabolic conditions in women with PCOS80. However, these agents are still controversial and are associated with some adverse effects such as diarrhea and stomachache81. Studies on how to prevent T2DM in women with PCOS are lacking.hhf p g The VN plays a necessary role in maintaining glucose homeostasis. The vagal afferent transmits nutrient- dependent signals generated from the upper small intestine to the liver and activates the vagal efferent system at the level of the dorsal vagal complex, thereby regulating hepatic glucose production82. Furthermore, the VN is involved in mediating early-phase insulin release as well as optimizing postprandial insulin release from the pancreas83. Numerous studies have suggested that ANS imbalance characterized by sympathetic activation or NV inactivity is associated with abnormal glucose metabolism84. For example, Poon et al. observed that high homeostasis model assessment index for insulin resistance is associated with lower high frequency spectral component, which is an indicator of vagal activity measured during daily activities85. The research by Saito et al. yielded similar results86. Chen et al. reported that muscle sympathetic nerve activity burst frequency is inversely correlated with liver insulin sensitivity in non-diabetic obese men87. Licht et al. found that high respiratory sinus arrhythmia (which reflects high PNS activity) is negatively associated with glucose levels88. Carnethon et al. indi- cated that lower heart rate variability, which has been used to non-invasively assess efferent vagal pathway activity, is independently associated with the risk of developing T2DM in healthy adults89. Given the above association between the VN and glucose metabolism, some studies have provided evidence that targeting vagal signaling to affect glucose metabolism may have therapeutic potential for reversing metabolic disorders. Chunchai et al. and Samniang et al. reported that VNS for 12 weeks significantly improved both peripheral and brain insulin sensitivity in obese IR rats and pigs90,91. Huang et al. www.nature.com/scientificreports/ reported that ta-VNS significantly reduced fasting plasma glucose, 2-h fasting plasma glucose, and glycosylated hemoglobin compared with the no-treatment control group and concluded that ta-VNS is a promising, simple, and cost-effective treatment for impaired glucose tolerance/ pre-diabetes with only mild side-effects92. Payne et al. and Meyers et al. further suggested that selective efferent VNS might be an effective therapy for treating T2DM93,94. As for the therapeutic mechanism, Deng et al. proposed that VNS may prevent IR by activating the α7nACh receptor to attenuate endoplasmic reticulum stress-induced inflammation, thus ameliorating hepatic IR in Kupffer cell95. Li et al. demonstrated that long-term ta-VNS treat- ment may prevent the progression of hyperglycemia possibly through up-regulating insulin receptor expression in various tissues96. Wang et al. also suggested that ta-VNS can regulate glucose metabolism by triggering the rhythmic secretion of melatonin, which plays a protective role in T2DM70. In addition, Yin et al. reported that VNS reduces blood glucose in diabetic rats by enhancing the release of glucagon-like peptide-1, which can be relayed by vagal afferent neurons to the brain to participate in satiation and glucoregulatory responses97. There- fore, we propose the use of ta-VNS to modulate IR in the management of PCOS. Activation of anti‑inflammatory pathways. Kelly et al. proposed for the first time that chronic low- grade inflammation is a novel mechanism contributing to coronary heart disease and T2DM in women with PCOS98. Since that work, inflammation has increasingly been recognized as a key contributor to the pathogen- esis of PCOS. The chronic low-level inflammation associated with PCOS generally does not show any obvious symptoms such as local redness or fever, but can result in the secretion of inflammatory factors, mainly char- acterized by elevated concentrations of C-reactive protein (CRP), tumor necrosis factor, interleukin 18 (IL-18), IL-6, white blood cell count, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, etc.99. In addition, all key features of PCOS, such as obesity, IR/HI, and HA aggravate the inflammatory state by pro- moting the oversecretion of proinflammatory factors100. There is also evidence that the uterine hyperinflamma- tory state in PCOS may induce major pregnancy complications ranging from recurrent miscarriage to placental dysfunction101. Potential pathways and action mechanisms by which ta‑VNS has Therapeutic effects PCOS Furthermore, the beneficial effects of ta-VNS on body weight and metabolic parameters were shown to produce lasting changes in brain networks74. Therefore, it is suggested that ta-VNS might be an alternative therapy for obesity in women with PCOS. Reversing insulin resistance via antidiabetic effect. IR is seen in 50% to 70% of women with PCOS75 and causes increased secretion of insulin in pancreatic β-islet cells and leads to compensatory HI. IR/HI stimu- lates increased production of androgens from ovarian theca cells76 and inhibits the production of sex hormone binding globulin in the liver, resulting in elevated free testosterone levels in the circulation77. IR and HA are considered to be the chief cause of PCOS22. It is likely that the presence of IR in PCOS contributes to abnormal https://doi.org/10.1038/s41598-023-34746-z Scientific Reports \| (2023) 13:7721 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Therefore, activation of the anti-inflammatory pathways might provide new therapeutic targets for treating PCOS.l g Since the late 1990s, the VN has been thought to be a core part of an anti-inflammatory regulatory pathway, which is a part of the inflammatory reflex consisting of both an afferent sensory and efferent effector arm102. The anti-inflammatory function of the VN might be mediated by several pathways103. The first is the anti-inflamma- tory hypothalamic–pituitary–adrenal axis (HPAA) pathway. In the HPAA, inflammatory signals are conveyed to the nucleus tractus solitaries via IL-1β receptors in vagal afferents, which activates the A2 noradrenergic group neurons and then sends information to the parvo-cellular region of the paraventricular nucleus of the hypothalamus surrounding corticotrophin releasing factor–containing neurons. These neurons then activate the release of adrenocorticotrophic hormones, which ultimately stimulate the release of glucocorticoids to exert anti- inflammatory effects. The second is the cholinergic anti-inflammatory pathway (ChAP). This pathway is initially activated through afferent VN stimulation, and the signal is then sent to the brain and processed in a muscarinic acetylcholine receptor-dependent manner, and the integrated anti-inflammatory signal is subsequently trans- mitted via VN efferent fibers to enteric neurons resulting in the release of acetylcholine (Ach), which activates the α7 nicotinic receptors in macrophages causing down-regulation of pro-inflammatory cytokines. The third is the splenic sympathetic anti-inflammatory pathway (SSAP). The inflammatory information is conveyed to the spleen through VN stimulation, and this results in the release of norepinephrine from splenic nerve terminals. Norepinephrine activates β2 adrenergic receptors in specialized T-lymphocytes and promotes the synthesis and expression of Ach. Ach binds to the α7 nicotinic receptors in macrophages, which inhibits the release of pro- inflammatory cytokines. Currently, several preliminary studies have shown a promising effect of the application of VNS as an anti-inflammatory treatment in a range of inflammatory diseases such as sepsis, obesity, CVD, Scientific Reports \| (2023) 13:7721 \| https://doi.org/10.1038/s41598-023-34746-z www.nature.com/scientificreports/ lung injury, diabetes, rheumatoid arthritis, and inflammation-related pain47. The development of ta-VNS, which does not need an implanted electrode and neurostimulator, is thus of therapeutic interest. Salama et al. showed that ta-VNS attenuates the acute postsurgical inflammatory response by reducing serum CRP, IL6, and IL10 in patients undergoing lobectomy104. Animals experiments have also suggested that ta-VNS can suppress inflamma- tory responses via the α7nAChR-mediated ChAP105,106. www.nature.com/scientificreports/ Therefore, it stands to reason that a sustained increase in vagal nerve activity through ta-VNS treatment may result in a strong anti-inflammatory response by activating the HPAA, ChAP, or SSAP and thus relieving the inflammatory state associated with PCOS. Restoring homeostasis of the gut‑microbiota‑brain axis. In the past two decades, the relationship between the gut microbiota (GM), also called the "second genome" in the human body, and metabolic disorders has become a research hotspot and has provided new insights into the pathogenesis of PCOS107. Increasing studies have investigated the potential role of GM dysregulation in the occurrence of PCOS. These studies have reported that compared with healthy controls, women with PCOS have reduced GM diversity and altered bar- rier function108,109, and dysbiotic GM in PCOS is not only associated with metabolic disorders such as IR and obesity110–112, but also with reproductive defects such as increased androgen concentrations113 and decreased estradiol levels108. Zhao et al. concluded in a recent literature review that the GM may promote the development of PCOS through several possible mechanisms, including energy absorption, short-chain fatty acids, lipopoly- saccharides, choline and bile acids, intestinal permeability, and the brain-gut axis107. Therefore, research target- ing the modulation of the GM as a novel therapy for the treatment of PCOS has aroused much interest.h The brain-gut axis is a bidirectional communication system between the brain and the GM through multiple pathways, including the VN, the immune system, neuroendocrine pathways, and bacteria-derived metabolites114, of which the afferent vagal pathway is suggested to be the most important115. The VN can sense the bacterial compounds or metabolites secreted from the GM through its afferent projections and can transmit this gut information to the CNS where it is integrated and adaptive or inappropriate responses are generated, the latter of which can perpetuate pathological conditions in the digestive tract or favor neurodegenerative diseases116,117. Therefore, focusing on the VN might help restore the homeostasis of the microbiota-gut-brain axis to treat these diseases. Compelling evidence have shown that VNS can ameliorate gut dysbiosis-induced pro-inflammatory effects, and VNS may rescue decreased gut mucosal integrity by upregulating enteric glial cells, reducing systemic proinflammatory cytokines, promoting recovery as well as modulating immune functions118,119. With regard to the effect of VNS/ta-VNS on the GM profile, there is only the paper by Haney et al. www.nature.com/scientificreports/ and although their results were neutral, the authors concluded that VNS remains a promising experimental and therapeutic modality for manipulating GM communities120. Here, we hypothesize that VNS/ta-VNS can play a positive role in the treat- ment of PCOS by maintaining the GM homeostasis, and this is worth studying in the future. Restoring the sympatho‑vagal balance to improve CVD outcomes. CVD is one of the important long-term complications in PCOS patients and is a general clustering of cardiac risk factors. Multiple studies have indicated the relationship between PCOS and an increased risk of CVD121. Hudecova et al. found that middle-aged patients with PCOS showed more pronounced endothelial dysfunction in comparison to their age- matched controls122. Another 21-year follow-up study indicated that women with PCOS had a higher prevalence of hypertension and increased triglyceride levels during their postmenopausal period than controls123. Mani et al. reported a high incidence and age-group-specific prevalence of T2DM, myocardial infarction, and angina among women with PCOS, and over a quarter of them had myocardial infarction or angina when they were older than 65 years124. The underlying links between PCOS and CVD are complex and involve various inter- acting cardiovascular and metabolic factors. These pathophysiological processes include visceral obesity, IR, impaired glucose and lipid homeostasis, HA, and chronic low-grade inflammatory status, all of which appear to responsible for making women with PCOS more prone to developing CVD125,126. Consequently, all women with PCOS should be assessed for cardiovascular risk factors. Lifestyle management is recommended as the first-line therapy to prevent primary CVD, and insulin sensitizers, cholesterol-lowering drugs, and other drugs should be administered if dyslipidemia or other risk factors persist127.h y p p The ANS plays a pivotal role in the onset and progression of CVD, including hypertension, arrhythmias, coronary artery disease, and heart failure128. On the one hand, an increase in cardiovascular sympathetic modula- tion is associated with poor clinical outcomes and serious complications129, while on the other hand the cardiac parasympathetic system appears to be protective against CVD and related mortality both in normal subjects and PCOS patients130. Thus, both increased sympathetic and decreased vagal activity may predict abnormal cardiovascular parameters. A growing number of studies have attempted to restore sympatho-vagal balance for the treatment of CVD, and current therapies mainly focus on reducing sympathetic overactivity. www.nature.com/scientificreports/ to successful treatment. In contrast, targeted stimulation of the parasympathetic branch of the ANS through VNS has shown remarkable effects as an alternative therapeutic method for treating CVD. In the study by Huang et al. the results showed that ta-VNS significantly decreased systolic blood pressure over time compared to sham ta-VNS92. Sclocco et al. found that mid-intensity respiratory-gated auricular vagal afferent nerve stimulation (RAVANS) could increase the cardiovagal tone and reduce the sympathetic tone during a paced breathing task, which suggested that RAVANS could be used as a non-invasive and inexpensive therapeutic intervention for hypertension131. In a prospective study, 24 patients with diastolic dysfunction and preserved left ventricular ejection fraction received two separate 1 h sessions, at least 1 day apart, of active low-level transcutaneous vagus nerve stimulation at the tragus (LLTS) or sham stimulation, and active LLTS treatment acutely improved left ventricular function and favorably altered the heart rate variability frequency domain components132. Moreover, the antiarrhythmic properties of ta-VNS on atrial fibrillation were also reported in human studies133,134. Given the impressive results of ta-VNS in the treatment of CVD, interventions against the cardiovascular complica- tions of PCOS with non-invasive stimulation approaches such as ta-VNS may lead to a significant improvement in patients’ long-term health. Effectively modulating mental disorders. Depression is a common disorder that negatively impairs psychological function and reduces quality of life. A large number of studies have shown that PCOS patients suffers from increased incidence of psychological distress, such as depression14,135–137. A recent meta-analysis covering more than 3000 subjects from 10 different countries reported that women with PCOS have twice the prevalence of depression compared to controls (36% vs. 14%), and women with PCOS showed moderate to severe depressive symptoms with an odds ratio of 4.18 compared to women without PCOS138. The clear etiol- ogy of PCOS-associated depression has not yet been described. The various pathophysiological mechanisms that contribute to depression include IR, disturbance in the HPA axis, androgen excess, inflammation, and the clinical presentations of obesity, hirsutism, and infertility139,140. The growing attention to depression has been recognized, and international guidelines now recommend that all women with PCOS should be screened and managed for depression141. Weight loss and dietary changes are important measures to prevent PCOS-associated depression. www.nature.com/scientificreports/ The treatment of PCOS-induced depression is similar to that for depression resulting from other causes, including lifestyle modifications, cognitive behavioral therapy, and pharmacological agents like oral con- traceptive pills, metformin, spironolactone, and other antidepressants139. In the past 20 years, a growing number of studies have suggested VNS as a treatment for depression142–144. In 2005, VNS was acknowledged by the FDA as an alternative treatment for difficult-to-treat depression in patients more than 18 years old who do not respond to four or more antidepressant treatment protocols145. In 2016, with the further development of medical research, the Canadian Network for Mood and Anxiety Treatments recommended VNS as the third-line therapy for drug-resistant depression146. Data showed that VNS treatment could improve depression scores by about 25–35%147. However, psychiatrists generally do not consider surgical therapy to treat mental illness, even when such therapy is minimally invasive and reversible. Recently, other non-invasive methods, especially ta-VNS, have shown strong therapeutic potential. Numerous animal studies in rat models of stress have suggested a promising role for ta-VNS in alleviating depressive disorder compared to sham ta-VNS96,148–150. A recent meta-analysis including four clinical studies with 222 individuals prelimi- narily demonstrated that ta-VNS could effectively decrease 24-item Hamilton Depression Rating Scale scores, Self-Rating Depression Scale scores, Beck Depression Inventory scores, and Self-Rating Anxiety Scale scores and concluded that ta-VNS was an effective and safe therapy for major depressive disorder151. With respect to the potential mechanisms for ta-VNS in the treatment of depression, several hypotheses were put forward, including directly and indirectly modulating the activity and connectivity of key brain regions involved in the pathophysiology of depression, inhibiting neuro-inflammatory sensitization, modulating hippocampal neuro- genesis, and regulating the GM-brain-gut axis152. Thus, we hypothesize that direct stimulation of the afferent nerve fibers of the ear through ta-VNS produces a similar effect as classic VNS in order to treat PCOS patients with depressive symptoms. www.nature.com/scientificreports/ However, the appearance of drug resistance and the invasive nature of some surgical procedures have become major challenges https://doi.org/10.1038/s41598-023-34746-z Scientific Reports \| (2023) 13:7721 \| www.nature.com/scientificreports/ Conclusion and future direction ANS dysfunction plays a nonnegligible role in the development of PCOS. The VN belongs to the ANS, and the participation of the VN in regulating ovarian functions has been suggested as an innovative point of departure for studying PCOS. Based on the clinical and experimental findings mentioned above, the enhancement of vagal activity by VNS/ta-VNS may lead to improvement in the various symptoms and complications associated with PCOS, including obesity, IR, T2DM, inflammation, microbiome dysregulation, CVD, and depression. Therefore, we propose a model focusing on ta-VNS that can act on multiple pathways that may treat PCOS (as shown in Table 1 and Fig. 2), including (1) regulating energy metabolism via bidirectional vagal signaling; (2) reversing IR via its antidiabetic effects; (3) activating anti-inflammatory responses through the HPAA, ChAP, and SSAP; (4) restoring homeostasis of the microbiota-gut-brain axis; (5) restoring the sympatho-vagal balance to improve CVD outcomes; and (6) modulating mental disorders. Thus, ta-VNS may be a promising novel therapeutic approach for the treatment of PCOS. The research results for VNS/ta-VNS and for PCOS point to each other; however, there is a lack of relevant research combining the two. Exploring ta-VNS in the management of PCOS is pioneering work, and further studies are needed to dig into the underlying therapeutic mechanisms. Clinical trials with large sample sizes need to be conducted to verify the real effect and long-term safety of ta-VNS in women with PCOS before ta-VNS can be applied in the clinic. https://doi.org/10.1038/s41598-023-34746-z Scientific Reports \| (2023) 13:7721 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. Potential pathways and action mechanisms for the therapeutic effects of ta-VNS on PCOS. ta-VNS transcutaneous auricular vagal nerve stimulation, PCOS Polycystic ovary syndrome, IR insulin resistance, T2DM type 2 diabetes mellitus, HPAA hypothalamic–pituitary–adrenal axis, ChAP cholinergic anti- inflammatory pathway, SSAP splenic sympathetic anti-inflammatory pathway, GM gut microbiota, CVD cardiovascular diseases. Data availabilityh Data availability The datasets used in the current study are available from the corresponding author on reasonable reque Received: 30 November 2022; Accepted: 6 May 2023 Conclusion and future direction Potential pathways Action mechanisms PCOS-associated complications Regulation of energy metabolism via bidirectional vagal signaling Reduce food intake Accelerate energy expenditure Obesity Reversing IR via antidiabetic effect Increase insulin secretion Suppress glucose production IR and T2DM Activation of the anti-inflammatory pathways HPAA pathway ChAP pathway SSAP pathway Inflammation Restoring homeostasis of the Microbiota-Gut-Brain Axis Upregulate enteric glial cells Reduce proinflammatory cytokines Promote recovery Modulate immune functions Modulate GM composition GM dysregulation Restoring the sympatho-vagal balance to improve CVD outcomes Increase the cardiovagal tone Reduce the sympathetic tone CVD Effectively modulating mental disorders Modulate the activity and connectivity of key brain regions Inhibit neuro-inflammatory sensitization Modulate hippocampal neurogenesis Regulate the GM-brain-gut axis Depression Table 1. Potential pathways and action mechanisms for the therapeutic effects of ta-VNS on PCOS. ta-VNS transcutaneous auricular vagal nerve stimulation, PCOS Polycystic ovary syndrome, IR insulin resistance, T2DM type 2 diabetes mellitus, HPAA hypothalamic–pituitary–adrenal axis, ChAP cholinergic anti- inflammatory pathway, SSAP splenic sympathetic anti-inflammatory pathway, GM gut microbiota, CVD cardiovascular diseases. Table 1. Potential pathways and action mechanisms for the therapeutic effects of ta-VNS on PCOS. ta-VNS transcutaneous auricular vagal nerve stimulation, PCOS Polycystic ovary syndrome, IR insulin resistance, T2DM type 2 diabetes mellitus, HPAA hypothalamic–pituitary–adrenal axis, ChAP cholinergic anti- inflammatory pathway, SSAP splenic sympathetic anti-inflammatory pathway, GM gut microbiota, CVD cardiovascular diseases. Figure 2. Possible pathways and action mechanisms of ta-VNS in the treatment of PCOS. ta-VNS transcutaneous auricular vagal nerve stimulation, PCOS Polycystic ovary syndrome, IR insulin resistance, T2DM type 2 diabetes mellitus, HPAA hypothalamic–pituitary–adrenal axis, ChAP cholinergic anti- inflammatory pathway, SSAP splenic sympathetic anti-inflammatory pathway, GM gut microbiota, CVD cardiovascular diseases. Figure 2. Possible pathways and action mechanisms of ta-VNS in the treatment of PCOS. ta-VNS transcutaneous auricular vagal nerve stimulation, PCOS Polycystic ovary syndrome, IR insulin resistance, T2DM type 2 diabetes mellitus, HPAA hypothalamic–pituitary–adrenal axis, ChAP cholinergic anti- inflammatory pathway, SSAP splenic sympathetic anti-inflammatory pathway, GM gut microbiota, CVD cardiovascular diseases. www.nature.com/scientificreports/ 26 V l i D & Si b i S S th l i b l d h i l i iti t i k d t g y p p y y y y J y ( ) (2015). 26. Velusami, D. & Sivasubramanian, S. 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Psychiatry 9, 20 (2018). Author contributions Conceptualization: S.K.Z. and H.H.; Methodology: S.K.Z.; Investigation: X.W. and X.F.L.; Writing-Original Draft Preparation: S.K.Z. and Y.W.; Writing-Review and Editing: H.H.. All authors have read and approved the final manuscript. Competing interests h p g The authors declare no competing interests. www.nature.com/scientificreports/ Pharm. Des. 19(32), 5764–5774 (2013). 127. Wild, R. A. et al. Assessment of cardiovascular risk and prevention of cardiovascular disease in women with the polycystic ovary syndrome: A consensus statement by the Androgen Excess and Polycystic Ovary Syndrome (AE-PCOS) Society. J. Clin. Endocrinol. Metab. 95(5), 2038–2049 (2010). d d ll d l f d l d h l ( ) ( ) ( ) 128. Hadaya, J. & Ardell, J. L. Autonomic modulation for cardiovascular disease. Front Physiol. 11, 617459 (2020). 129 Grassi G Seravalle G & Mancia G Sympathetic activation in cardiovascular disease: Evidence clinical impact and therapeutic 128. Hadaya, J. & Ardell, J. L. Autonomic modulation for cardiovascular disease. Front Physiol. 11, 617459 (2020). 128. Hadaya, J. & Ardell, J. L. Autonomic modulation for cardio 128. Hadaya, J. & Ardell, J. L. Autonomic modulation for cardiovascular disease. Front Physiol. 11, 617459 (2020). 129. Grassi, G., Seravalle, G. & Mancia, G. Sympathetic activation in cardiovascular disease: Evidence, clinical impact and therap implications. Eur. J. Clin. Invest. 45(12), 1367–1375 (2015). https://doi.org/10.1038/s41598-023-34746-z https://doi.org/10.1038/s41598-023-34746-z Scientific Reports \| (2023) 13:7721 \| Funding g Financial support for this study was provided by (1) the Scientific Research Fund of Heilongjiang University of Chinese Medicine (No. 2019BS09); (2) the Chinese Medicine research project of Heilongjiang Provincial Administration of Chinese Medicine (ZHY2022-124); and (3) The 2022–2024 Youth Talent Lifting Project of Heilongjiang Chinese Medicine Association (2022–QNRC1–02). 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https://openalex.org/W2084203472	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0000914&type=printable	English	null	Metagenomics of the Deep Mediterranean, a Warm Bathypelagic Habitat	PloS one	2,007	cc-by	12,149	INTRODUCTION microbes, thus sidestepping the need for culturing or isolation [5– 7]. Except for a few small-scale analyses of genome fragments from archaea and bacteria from mesopelagic waters [8–10], metage- nomic studies in the ocean have been also primarily devoted to surface waters (e.g. [11,12], including recent large-scale compar- ative studies along surface transects [13]. So far, the only large- scale metagenomic analysis of deep-sea communities correspond to a comparative study that DeLong and co-workers carried out at different depths in the water column at the North-Pacific Subtropical Gyre ALOHA station, ranging from 10 to 4,000 m The deep ocean is one of the most important and less understood microbial-driven ecosystems on Earth. Since the recognition of the essential role of microbes on the ocean water column [1], most marine microbiology studies have been devoted to the photic zone, where microbial cell density and activity are high and most primary production occurs. Microbial communities in deeper oceanic layers, particularly below 1,000 m (bathypelagic and abyssal waters), have low cell densities and low metabolic activities partially due to the extreme reigning conditions. Not only light is absent but deep waters are most often oligotrophic, pressure increases and temperature decreases very rapidly to reach average values around 2uC in the open ocean. Despite so, given the vast dimensions of the deep ocean, occupying nearly two thirds of the planet’s surface and reaching an average depth of 3,800 m, the microbial community of this ecosystem becomes fundamental for global biogeochemical cycling. Studying the microbial communi- ties in offshore deep marine locations has always been difficult and demanding. Pure culture approaches are very difficult to apply, and relevant (not opportunistic) microbes are extremely difficult to isolate. Molecular approaches based on the amplification of small subunit ribosomal RNA genes improved the situation markedly [2–4], but still leave the deep water mass of most oceans under- sampled and provide no functional information about lineages that rarely have close cultured relatives. Metagenomics, the study of genetic and genomic information from whole environmental communities, has brought some hope to get insights about the metabolic potential and evolutionary history of uncultured marine Academic Editor: Niyaz Ahmed, Centre for DNA Fingerprinting and Diagnostics, India Received July 31, 2007; Accepted August 27, 2007; Published September 19, 2007 Copyright: 2007 MARTIN-CUADRADO et al. Metagenomics of the Deep Mediterranean, a Warm Bathypelagic Habitat Ana-Belen Martı´n-Cuadrado1., Purificacio´n Lo´pez-Garcı´a2., Juan-Carlos Alba1, David Moreira2, Luis Monticelli3, Axel Strittmatter4, Gerhard Gottschalk4, Francisco Rodrı´guez-Valera1* 1 Divisio´n de Microbiologı´a, Universidad Miguel Herna´ndez, San Juan de Alicante, Spain, 2 Unite´ d’Ecologie, Syste´matique et Evolution, Centre National de la Recherche Scientifique, Universite´ Paris-Sud, Orsay, France, 3 Istituto per l’Ambiente Marino Costiero, Consiglio Nazionale delle Ricerche, Sezione di Messina, Messina, Italy, 4 Laboratorium fu¨r Genomanalyse, Institut fu¨r Mikrobiologie und Genetik, Georg-August-University Go¨ttingen, Go¨ttingen, Germany Background. Metagenomics is emerging as a powerful method to study the function and physiology of the unexplored microbial biosphere, and is causing us to re-evaluate basic precepts of microbial ecology and evolution. Most marine metagenomic analyses have been nearly exclusively devoted to photic waters. Methodology/Principal Findings. We constructed a metagenomic fosmid library from 3,000 m-deep Mediterranean plankton, which is much warmer (,14uC) than waters of similar depth in open oceans (,2uC). We analyzed the library both by phylogenetic screening based on 16S rRNA gene amplification from clone pools and by sequencing both insert extremities of ca. 5,000 fosmids. Genome recruitment strategies showed that the majority of high scoring pairs corresponded to genomes from Rhizobiales within the Alphaproteobacteria, Cenarchaeum symbiosum, Planctomycetes, Acidobacteria, Chloroflexi and Gammaproteobacteria. We have found a community structure similar to that found in the aphotic zone of the Pacific. However, the similarities were significantly higher to the mesopelagic (500–700 m deep) in the Pacific than to the single 4000 m deep sample studied at this location. Metabolic genes were mostly related to catabolism, transport and degradation of complex organic molecules, in agreement with a prevalent heterotrophic lifestyle for deep-sea microbes. However, we observed a high percentage of genes encoding dehydrogenases and, among them, cox genes, suggesting that aerobic carbon monoxide oxidation may be important in the deep ocean as an additional energy source. Conclusions/Significance. The comparison of metagenomic libraries from the deep Mediterranean and the Pacific ALOHA water column showed that bathypelagic Mediterranean communities resemble more mesopelagic communities in the Pacific, and suggests that, in the absence of light, temperature is a major stratifying factor in the oceanic water column, overriding pressure at least over 4000 m deep. Several chemolithotrophic metabolic pathways could supplement organic matter degradation in this most depleted habitat. Citation: Martı´n-Cuadrado A-B, Lo´pez-Garcı´a P, Alba J-C, Moreira D, Monticelli L, et al (2007) Metagenomics of the Deep Mediterranean, a Warm Bathypelagic Habitat. PLoS ONE 2(9): e914. doi:10.1371/journal.pone.0000914 PLoS ONE \| www.plosone.org INTRODUCTION 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 work was supported by the GEMINI (QLK3-CT-2002-02056) project of the European Commission, and MEC (Spain) (CTM2005-04564) to FRV and by an ATIP project of the French Centre National de la Recherche Scientifique (CNRS) to PLG. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: frvalera@umh.es * To whom correspondence should be addressed. E-mail: frvalera@umh.es . These authors contributed equally to this work. . These authors contributed equally to this work. PLoS ONE \| www.plosone.org September 2007 \| Issue 9 \| e914 1 Mediterranean Metagenomics Mediterranean Metagenomics depth [14]. Extending metagenomic analyses to other deep-sea communities would help unravel important questions about metabolism and lifestyle of deep-sea microbes. For instance, although the deep ocean is generally considered a metabolic sink for the organic matter produced in the photic zone, autotrophic archaea (crenarchaeota), possibly ammonia-oxidizers [15,16], are mostly abundant in deep waters [17]. a model for a deep relatively warm bathypelagic habitat. The Ionian Sea at the South East of Sicily possesses very pristine and stable deep waters. On this ground, the Ionian station Km3 has been extensively studied as a candidate site for a neutrino telescope (NEMO) (http://nemoweb.lns.infn.it/publication.htm). The pro- karyotic diversity of a 3,000 m deep sample from the station Km3 was studied recently by analyzing 16S rRNA gene libraries, which revealed a wide variety of prokaryotic lineages [4]. This prompted us to construct a metagenomic fosmid library from the same sample. Pressure is thought to have a significant influence in deep-sea stratification, as piezophilic (barophilic) species have been isolated [18], and specific adaptations such as pressure regulated operons are present in some deep-sea bacteria [19,20]. However, along with high pressure, low temperatures also characterize deep-sea waters. They limit growth rate through its slowing-down effect on metabolic chemical reactions and, consequently, psycrophilic organisms develop particular adaptations tending to increase protein flexibility and reactivity [21]. A few exceptional locations provide the chance to study microbial communities at high oceanic depth without being affected by near zero temperatures, therefore providing the opportunity to assess the relative importance of pressure and temperature in microbial adaptation at genomic level in the natural environment. RESULTS The Km3 metagenomic library was constructed from planktonic fractions smaller than 5 mm of seawater collected at 3,010 m depth that was at a temperature of nearly 14uC and 38.7% salinity (see Methods). It contained ca. 20,000 fosmid clones, from which approximately one-fourth was subjected to bi-directional end sequencing, yielding 7.2 Mbp of DNA sequence from the approximately 725 Mbp total archive (Table 1). This represents raw sequence of approximately 2 prokaryotic genome equivalents (considering an average genome size of 3.5 Mbp). Consistently Table 1. General features of the Km3 metagenome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major feature Subcategory Value Number of fosmid clones 20,767 Total archived sequence 725 Mbpa Number of 16S rRNA genes amplified by PCR Archaeal 28 Bacterial b 16 Number of high-quality fosmid-end sequences 9,048 Average length of sequence reads 794 bp Total generated sequence length 7,184 kbp Average GC content 50.1% Fosmid-end sequences belonging to defined taxonomic groups (,1e-50) 23.4% of ambiguous taxonomic ascription (1e-7 to 1e-50) 53.7% with homologues only in GOSc 11.4% without homologues in any database 11.4% Distribution of sequences in major COG categories Metabolism 50.4% Information storage and processing 17.1% Cellular Processes and Signaling 16.0% Poorly characterized 16.5% Distribution of sequences in major KEGG categories Metabolism 70.6% Genetic Information Processing 17.4% Environmental Information Processes 10.2% Cellular Processes 1.7% BLAST cut-off values used in each case are shown in brackets. aConsidering average insert sizes of 35 kbp bOnly fosmids containing bacterial ITS regions different in size of that from E. coli were identified. INTRODUCTION The largest and most ecologically relevant is the Mediterranean Sea. Although it has an exceptionally deep basin for a basically landlocked sea, reaching 5,000 m at its deepest Eastern end and with an average depth of 2,000 m, the Mediterranean is free of cold polar water that cannot get over the sill of the Gibraltar Strait [22]. The deep Mediterranean water mass never gets below 13.5uC, providing Here we report the construction and analysis of such a fosmid metagenomic library from 3,000 m deep Km3 small plankton (0.2–5 mm fraction). The gene repertoire identified supports a predominant heterotrophic lifestyle for this deep Mediterranean community and suggests versatility in energy-gaining mechanisms, including a widespread use of CO oxidation. Community composition y p The analysis of metagenomic libraries can complement diversity studies based on 16S rRNA gene PCR amplification, since they are not subjected to the same biases. We used a double approach to estimate the prokaryotic diversity in the Km3 library, PCR amplification of 16S rRNA genes in pooled clones of the whole library, and phylogenetic assignment of fosmid insert terminal sequences from approximately 5,000 clones. In the case of archaea, we used different primer combinations to recover a maximal variety of archaeal genes. We detected a total of 28 archaeal fosmid containing 16S rRNA genes (Table 1 and Fig. 1). Eighteen out of the 28 archaeal clones were crenarchaeota, most of them members of the bona fide marine Group I crenarchaeota, and one of them belonging to the recently identified pSL12-related cluster or group 1A [14,24]. The remaining 11 clones belonged to the Euryarchaeota of the marine Group II (9 clones) and Group III (2 clones) (Fig. 2A). A similar trend could be observed from fosmid-end phylogenetic ascription (Fig. 1). We applied expecta- tion cut-off values of 1e-50 for binning our fosmid sequences within known taxa with sequences in databases; approximately 23% (Table 1) fulfilled this condition. Although this proportion might seem limited, these sequences represent a random sample from the total diversity, hence providing a reasonably proxy to the prokaryotic census in the deep Mediterranean. Based on fosmid- ends, about 9% of the microbial diversity in Km3 would be archaea, which is in the same order of magnitude that the 15% estimated by archaeal 16S rRNA gene-containing fosmids in the g ) According to fosmid-end taxon-binning, the bacterial compo- nent in the Km3 metagenomic library was dominated by the Proteobacteria, and within them, by the Alphaproteobacteria, followed by Gamma-, Delta- and Betaproteobacteria (Fig. 1). Gram positive bacteria, both Actinobacteria and Firmicutes were also relatively abundant. There might be a slight bias in the proportion of these phyla due to the fact that, in spite of an increasing effort to widen the taxonomic spectra of sequenced genomes, there are many more genomes available from proteo- bacteria and Gram positive bacteria than from other taxa, which might result in a slight overrepresentation of these lineages. However, other taxonomic groups appeared also very abundant, namely Planctomycetales, Chloroflexi, Bacteroidetes and Acid- obacteria (Fig. 1). The presence of Acidobacteria was also patent by amplification of 16S rRNA genes from pooled clones. RESULTS cGOS, Global Ocean Surveyor database {Rusch, 2007 #16} doi:10.1371/journal.pone.0000914.t001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. General features of the Km3 metagenome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subcategory BLAST cut-off values used in each case are shown in brackets. aConsidering average insert sizes of 35 kbp bOnly fosmids containing bacterial ITS regions different in size of that from E. coli were identified. cGOS, Global Ocean Surveyor database {Rusch, 2007 #16} doi:10.1371/journal.pone.0000914.t001 . . . . . . . . . . . . . September 2007 \| Issue 9 \| e914 2 PLoS ONE \| www.plosone.org Mediterranean Metagenomics with this estimation we retrieved a single hit to recA [23] a gene that has been used to establish the number of genome (or cell) equivalents in a metagenomic library [23]. metagenomic library. Likewise, crenarchaeota were also found in equivalent relative proportions (,60%) compared to euryarch- aeota (,40%) in the metagenomic library (Fig. 1). Our results confirm and extend previous studies showing that crenarchaeota increase their relative abundance at high depth [14,25], although euryarchaeota still keep significant levels (see Fig.S5 in Ref.14 and Fig. 2B). Community composition Acidobacterial 16S rRNA gene sequences were also detected in deep sea waters in DeLong et al.’s study [14]. Even with the highly limited approach used here to detect bacterial ribosomal operons by PCR (see methods), 8 out of 18 16S rRNA genes detected here belonged to Acidobacteria (Fig. 3A) Similarly, although we detected Gemmatimonadetes 16S rRNA genes in the Km3 metagenomic library, fosmid-end sequences corresponding to this group could not be recognized in the absence of available complete genome sequences. Sequences belonging to groups for Figure 1. Prokaryotic taxa identified in Km3 3,000 m-deep plankton inferred from fosmid-ends and 16S rRNA detection in environmental and metagenomic libraries. Relative abundances of PCR-amplified 16S rRNA genes in environmental libraries are from Ref.4. For bacteria in the Km3 metagenomic library (central panel), only 16S rRNA genes whose adjacent ITS can be distinguished in size from that of E. coli were detected. doi:10.1371/journal.pone.0000914.g001 Figure 1. Prokaryotic taxa identified in Km3 3,000 m-deep plankton inferred from fosmid-ends and 16S rRNA detection in environmental and metagenomic libraries. Relative abundances of PCR-amplified 16S rRNA genes in environmental libraries are from Ref.4. For bacteria in the Km3 metagenomic library (central panel), only 16S rRNA genes whose adjacent ITS can be distinguished in size from that of E. coli were detected. doi:10.1371/journal.pone.0000914.g001 September 2007 \| Issue 9 \| e914 PLoS ONE \| www.plosone.org 3 Mediterranean Metagenomics Figure 2. Taxonomy of archaeal fosmids. A, Phylogenetic tree of 16S rRNA genes amplified from the metagenomic library Km3. The tree was constructed by maximum likelihood using PhyML and a total of 704 non ambiguously aligned positions. Non-parametric bootstrapping was performed upon 1,000 replicates. Only bootstrap values above 50 are shown. Km3 and ALOHA water column sequences are indicated in red and blue, respectively. B, Comparative taxonomic distribution obtained by best BLAST hit (see methods) of archaeal fosmid-ends in KM3 and ALOHA deep-sea libraries [14]. Marine group I and environmental samples are non-taxonomic designations as used in databases. doi:10.1371/journal.pone.0000914.g002 Fi T f h l f id A Ph l i f S R Figure 2. Taxonomy of archaeal fosmids. A, Phylogenetic tree of 16S rRNA genes amplified from the metagenomic library Km3. The tree was constructed by maximum likelihood using PhyML and a total of 704 non ambiguously aligned positions. Non-parametric bootstrapping was performed upon 1,000 replicates. Only bootstrap values above 50 are shown. Community composition Km3 and ALOHA water column sequences are indicated in red and blue, respectively. B, Comparative taxonomic distribution obtained by best BLAST hit (see methods) of archaeal fosmid-ends in KM3 and ALOHA deep-sea libraries [14]. Marine group I and environmental samples are non-taxonomic designations as used in databases. doi:10.1371/journal.pone.0000914.g002 collectively. Also within the Alphaproteobacteria, the two strains of Pelagibacter ubique (84 hits) were among the most frequently matched by our sequences, in agreement with the highly recorded prevalence in oceans [27]. Genomes from other Proteobacteria (Gammaproteobacteria, followed by Beta- and Deltaproteobac- teria) were also, though to a much lesser extent, well represented. Similarly, the genome of the archaeon Cenarchaeum symbiosum A recruited a large number of hits, supporting a relative high proportion of related crenarchaeota in the deep-sea (Fig. 4). The lack of euryarchaeotal Group II and III genomes prevents a similar comparison, although the relative high number of hits against the few available small environmental Group II genome fragments advances a similar situation to that of Group I crenarchaeota and the C. symbiosum genome (Fig. 2B). Planctomycetes followed in relative abundance, with Blastopirellula marina and Rhodopirellula baltica as frequently hit genomes, confirming the dominant role played by these organisms in most open ocean oligotrophic waters [28]. Candidatus Kuenenia stuttgartiensis, which lives in extremely organic-rich environments oxidizing ammonium anaerobically [29] receives also some genome hits (Table 2). Such anammox bacteria could live associated to sinking particles with anoxic niches. If this is confirmed, it would imply that ammonium oxidation, either aerobic or anaerobic, is a very important process in the deep ocean, and key to the nitrogen cycle. Less predictable but also supported by recent metagenomic and 16S rRNA-based studies is the abundance of Acidobacteria, represented by the genomes of Solibacter usitatus Ellin 6077 and the Bacterium which representative genome sequences are still missing may fall in the category of unknown proteins (Table 1) if they contain so far non-described proteins, which appear to be numerous according to recent massive sequence analyses [26]. Proteins with known homologues may also be of ambiguous taxonomic classification or artificially placed in another phylum with relatively low scores as a result of insufficiently close relative genomes in databases. Roughly 50% of the fosmid-end sequences were included in this category (Table 1). Consequently, the description of the pro- karyotic diversity by this approach needs to be taken with caution. Community composition Despite so, the diversity pattern observed in deep-sea Km3 waters by this approach does not differ significantly from that observed in the deep ALOHA water column, being especially similar to the 770 m depth sample (Fig. 3B and Ref.14). PLoS ONE \| www.plosone.org Prevailing organismal genomes The large sequencing effort of prokaryotic genomes, many of them marine, carried out during the last few years allows a direct comparison of marine metagenomic sequences with complete genomes with a reasonable chance of meaningful results. BLASTX HSPs with scores smaller than 1e-50 allowed assigning with confidence fosmid-end sequences to a taxonomic category containing the hit genome. Genomes recovering at least 10 high- score Km3 hits are indicated in Table 2. The most striking observation was the consistent recruitment of alphaproteobacterial genomes, particularly from Rhizobiales, which accounted for the largest number of hits considered September 2007 \| Issue 9 \| e914 Issue 9 \| e914 4 Mediterranean Metagenomics Figure 3. Taxonomy of bacterial fosmids. A, Phylogenetic tree of 16S rRNA genes amplified from the metagenomic library Km3 whose adjacent intergenic spacers differ in size from those of Escherichia coli. The tree was constructed by maximum likelihood using PhyML and a total of 1128 non ambiguously aligned positions. Non-parametric bootstrapping was performed upon 1,000 replicates. Only bootstrap values above 50 are shown. Km3 and ALOHA water column sequences are indicated in red and blue, respectively. Unc., uncultured. [], AA0D00000000 genome sequence underway. B, Comparative taxonomic distribution of bacterial fosmid-ends by best BLAST hits in KM3 and ALOHA deep-sea libraries [14]. doi:10.1371/journal.pone.0000914.g003 Figure 3. Taxonomy of bacterial fosmids. A, Phylogenetic tree of 16S rRNA genes amplified from the metagenomic library Km3 whose adjacent intergenic spacers differ in size from those of Escherichia coli. The tree was constructed by maximum likelihood using PhyML and a total of 1128 non ambiguously aligned positions. Non-parametric bootstrapping was performed upon 1,000 replicates. Only bootstrap values above 50 are shown. Km3 and ALOHA water column sequences are indicated in red and blue, respectively. Unc., uncultured. [], AA0D00000000 genome sequence underway. B, Comparative taxonomic distribution of bacterial fosmid-ends by best BLAST hits in KM3 and ALOHA deep-sea libraries [14]. doi:10.1371/journal.pone.0000914.g003 Ellin345. Chloroflexi were also abundantly represented through the genomes of Roseiflexus RS1 and Dehalococcoides ethenogenes. The concordance of the two end hits with the expected distance found in the corresponding genome was only found in few cases, among them is that of Alteromonas macleodii DE, an isolate obtained from a location not very distant from the sampling site [30], P. ubique HTCC 1062, Cellulophaga sp. MED134 and Magnetospirillum magnetotacticum MS-1. a carbon and nitrogen storage resource [31]. Prevailing organismal genomes Genes involved in rhizopine degradation were also identified in rhizobia that lack nodulation genes and apparent symbiotic behavior [32]. Similarly, Acidobacteria, another taxonomic group widely distributed in soils, appears fairly abundant. The possibility that Acidobacteria and Rhizobiales are contaminants from the bottom sediment is unlikely, as water samples were collected more than 200 m above the sea floor. Interestingly, Acidobacteria and rhizobia have exchanged genes by horizontal gene transfer as revealed by soil metagenomic analysis [33], suggesting that they might entertain some kind of interaction in habitats where they co-exist. The observation of high abundances of Rhizobiales and Acidobacteria in the deep Mediterranean extends knowledge about the natural habitats of both bacterial groups. Among the organisms whose genomes had more HSPs with Km3 sequences, aerobic heterotrophic metabolism seems to prevail as a lifestyle, but additional patterns arise. Many retrieved genomes correspond to marine oligotrophs or to bacteria having diverse degradative potential including xenobiotics and/or re- calcitrant organic compounds and polymers. This would tend to support a dominant role for heterotrophy, and particularly the degradation of complex organic molecules. The relatively large number of hits to some bacterial taxa seems particularly remark- able, for example the Rhizobiales. These are typical soil inhabitants that may perform symbiotic nitrogen fixation in association with some plants. Accordingly, one of the genes identified is involved in rhizopine catabolism (42% similarity to mocD). Rhizopine is produced by some rhizobia and provides a competitive advantage in nodulating symbioses, perhaps as PLoS ONE \| www.plosone.org Gene content and metabolic potential p We classified Km3 fosmid-end identified ORFs in functional classes according to the Cluster of Orthologous Groups (COG) database [34] and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/). The metabolic potential of microorganisms thriving in the deep Mediterranean can be assessed to some extent from the type of metabolic genes encountered. Most of the genes, nearly 50% to 70% according PLoS ONE \| www.plosone.org 5 September 2007 \| Issue 9 \| e914 Mediterranean Metagenomics Table 2. Hallmark genomes recruited by Km3 fosmid-end sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taxonomic group Species genome Number of hits* % identity %GC genome %GC Km3 seq. O2 requirement Habitat Metabolism Alphaproteobacteria Rhizobiales Rhizobiales (19 genomes) 112 56.53 _ _ aerobic soil heterotrophy Rhodopseudomonas sp. (5 genomes) 27 60.54 _ _ aerobic Parvibaculum lavamentivorans DS-1 23 56.31 62.3 53.08 aerobic Crenarchaeota Cenarchaeum symbiosum 102 64.47 57.4 36.06 aerobic marine autotrophy? Alphaproteobacteria Rickettsiales Pelagibacter ubique (2 genomes) 84 69.81 29.8/29.7 34.16/33.64 aerobic marine oligotrophy Planctomycetales Blastopirellula marina DSM 3645 78 57.23 57 58.71 aerobic marine oligotrophy Planctomycetales Rhodopirellula baltica SH 1 58 56.65 55.4 58.22 aerobic marine heterotrophy Acidobacteria Solibacter usitatus Ellin6076 56 55.67 61.9 54.64 aerobic soil oligotrophy Chloroflexi Dehalococcoides sp. (3 genomes) 42 56.58 _ _ facultative an. Gene content and metabolic potential chemolithotrophy Alphaproteobacteria Rhodospirillales Magnetospirillum (2 genomes) 36 61.81 65.1/66.4 55.44/56.88 aerobic heterotrophy Gammaproteobacteria Pseudomonadales Pseudomonas sp. (13 genomes) 36 58.56 _ _ aerobic heterotrophy Alphaproteobacteria Rhodobacterales Roseovarius (3 genomes) 33 61.31 _ _ aerobic heterotrophy Acidobacteria Bacterium Ellin345 32 57.59 58.4 52.43 aerobic soil oligotrophy Betaproteobacteria Burkholderiales Burkholderia sp. (13 genomes) 32 52.33 _ _ aerobic heterotrophy Chloroflexi Roseiflexus sp. RS-1 26 52.86 60.4 50.61 facultative an. photoautotrophy Deltaproteobacteria Desulfuromonadales Geobacter sp. (4 genomes) 25 55.73 _ _ anaerobic soil / sediment chemolitotrophy Actinobacteria Rubrobacter xylanophilus DSM 9941 23 56.54 70.5 53.21 aerobic soil heterotrophy Gammaproteobacteria gamma proteobacterium KT 71 21 59.61 48.61 aerobic marine oligotrophy Gammaproteobacteria Chromatiales Alkalilimnicola ehrlichei MLHE-1 19 62.71 67.5 52.94 anaerobic chemolithotrophy Bacteroidetes Sphingobacteria Salinibacter ruber DSM 13855 18 51.55 66.1 44.88 aerobic hypersaline heterotrophy Gammaproteobacteria Oceanospirillales Alcanivorax borkumensis SK2 16 67.65 54.7 52.68 aerobic heterotrophy Gammaproteobacteria Chromatiales Nitrosococcus oceani ATCC 19707 16 62.2 50.3 50.25 aerobic marine chemolithotrophy Betaproteobacteria Burkholderiales Ralstonia sp. (5 genomes) 16 62.11 _ _ aerobic soil heterotrophy Firmicutes Clostridia Carboxydothermus hydrogenoformans Z-2901 16 53.94 42 51.88 facultative an. chemolithotrophy Alphaproteobacteria Rhodospirillales Rhodospirillum rubrum ATCC 11170 15 64.42 65.4 55.33 facultative an. Gammaproteobacteria Methylococcales Methylococcus capsulatus str. Bath 15 62.49 63.6 52 aerobic methylotrophy Deltaproteobacteria Myxococcales Anaeromyxobacter dehalogenans 2CP-C 15 57 74.9 56.86 facultative an. soil / sediments heterotrophy Gammaproteobacteria Alteromonadales Marinobacter aquaeolei VT8 13 62.63 56.9 48.38 aerobic marine heterotrophy Bacteroidetes Flavobacteria Flavobacteriales bacterium HTCC2170 13 59.93 37 48.23 aerobic marine heterotrophy Gammaproteobacteria Chromatiales Nitrococcus mobilis Nb-231 13 57.72 60 51 aerobic chemolithotrophy Actinobacteria Streptomyces sp. (4 genomes) 13 55.84 _ _ aerobic soil heterotrophy Gammaproteobacteria marine gamma proteobacterium HTCC2207 12 60.98 49.4 49.25 aerobic marine heterotrophy Cyanobacteria Chroococcales Synechococcus sp. (5 genomes) 11 61.28 _ _ photoautotrophy Deltaproteobacteria delta proteobacterium MLMS-1 11 58.1 60.1 52.54 anaerobic chemolithotrophy Gammaproteobacteria Alteromonadales Alteromonas macleodii ‘Deep ecotype’ 10 69.33 44.9 42.3 aerobic marine heterotrophy Betaproteobacteria Burkholderiales Bordetella sp. (5 genomes) 10 51.66 _ _ aerobic heterotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene content and metabolic potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mediterranean Metagenom Table 2. Hallmark genomes recruited by Km3 fosmid-end sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Mediterranean Metagenomics Figure 4. Genome recruitment by Cenarchaeum symbiosum A. Individual fosmid-end sequences were aligned to the sequenced strain genome and the alignment-sequence conservation visualized in the form of percent identity plot. Each dot of the graph represents an individual fosmid-end sequence aligned along its homologous region in C. symbiosum A genome. Y axis reflects its nucleotide percent identity to the syntenic region. Both Km3 and ALOHA water column datasets were used. doi:10.1371/journal.pone.0000914.g004 Figure 4. Genome recruitment by Cenarchaeum symbiosum A. Individual fosmid-end sequences were aligned to the sequenced strain genome and the alignment-sequence conservation visualized in the form of percent identity plot. Each dot of the graph represents an individual fosmid-end sequence aligned along its homologous region in C. symbiosum A genome. Y axis reflects its nucleotide percent identity to the syntenic region. Both Km3 and ALOHA water column datasets were used. doi:10.1371/journal.pone.0000914.g004 September 2007 \| Issue 9 \| e914 PLoS ONE \| www.plosone.org 7 Mediterranean Metagenomics Table 3. Hallmark proteins in the Km3 metagenome compared to the deep ALOHA water column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene content and metabolic potential . . . . . . . . . . . . . . . . . . . . . . . BLASTX cut- off value Protein / Protein class Number of gene/protein per Mbp 500 770 4000 Km3 1e-50 RecA 0.3 0.3 0.3 0.1 DnaK 0.5 0.7 0.6 1.5 RpoB 0.3 0.3 0.3 0.7 Pgm 0.0 0.2 0.1 0.4 PycA 0.5 0.6 1.0 0.7 GyrB 0.4 0.7 0.7 0.5 Mdh 1.1 1.0 1.0 1.1 1e-20 Dehydrogenases 40.3 38.1 43.7 59.7 Carbon-monoxide- dehydrogenase (cox) 1.0 1.7 1.0 2.5 Luciferase-like genes 0.4 1.4 1.5 2.9 Transposase 1.6 1.7 8.1 4.6 Phage-related 2.0 1.3 2.5 3.6 Chaperones 2.1 2.4 2.5 3.7 Dehalogenases 0.5 0.2 0.6 1.4 doi:10.1371/journal.pone.0000914.t003 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . recalcitrant pools of organic matter. Similarly to previous observations in the ALOHA water column, the enrichment of genes related to pilus, polysaccharide and antibiotic synthesis genes observed might suggest a potential role for a surface- attached lifestyle. Among enzyme-coding genes, oxidases, reduc- tases and oxidoreductases were relatively numerous, followed by carboxylases and decarboxylases. However, by far, the most abundant enzymatic class was that of the dehydrogenases (Table 3). Notably, among the most represented genes of this class were those encoding the different subunits (CoxL, CoxM, CoxS) of carbon monoxide dehydrogenase (CODH) (Table 3). We detected confidently (1e-50) up to 5 coxL genes, and this, for about two genome equivalents of Km3 sequence. In addition, at least 10 additional coxL sequences were detected by phylogenetic analyses from putative cox genes with BLASTX scores .1e-50 (data not shown). In the Sargasso Sea metagenome there was only one coxL per 11 genome equivalents [12]. coxL was more abundant in aphotic waters in the ALOHA water column, ranging from 1 to 5 copies in the photic region, and from 7 to 10 copies in the aphotic waters column for an average of 3–4 genome equivalents at each depth [14]. Gene content and metabolic potential The best represented pathways were oxidative phosphorylation (131 hits) followed by carbon fixation pathways (129 hits) and nitrogen metabolism (80 hits). In agreement with the deep origin of the sample, genes related to photosynthesis were minoritary. By contrast, genes involved in the degradation of different kinds of compounds, including notably biopolymers and xenobiotics, and catabolic pathways in general were fairly abundant. Among them were several acetone decarboxylases and dehalogenases and KEGG-pathways associated with the degradation of limonene and pinene, 1-and 2-methylnaphthalene, glycosaminoglycan, benzoate, 1,2-dichloroethane, nitrobenzene, gamma-hexachloro- cyclohexane, ethylbenzene and fluorene. This reinforces the idea that microorganisms living at this depth are adapted to degrade to the COG or the KEGG classification, respectively, were related to metabolism and transport, whereas only 17% corresponded to housekeeping genes involved in information-related processes (Table 1 and 3). The most abundant functional family was that of transporters, and within it, amino acid transporters (Figs. 5 and 6). Transporter systems can tell about nutrient pools or substrates that are present in the environment and that the organisms use. The largest group of transporters in Km3 consisted of the multi- subunit ABC family (64,4% of the identified transporters) and the most represented of this family were transporters for dipeptides/ oligopeptides and branched chain amino acids (11,2% and 4.3% sequences, respectively). TRAP transporters, which allow substrate accumulation using an electrochemical ion gradient rather that ATP hydrolysis, were also frequently encountered as well as transporters for carboxylic acids (6.5%), while only 5.7% were sugar/polymer transporters (Fig. 6). The relative high number of peptide and branched chain amino acid transporters suggests that proteins, perhaps associated with sinking marine snow, are an important carbon source for deep-sea microbes. By contrast to shallow waters, with sugars forming an easily accessible labile pool of organic matter, recalcitrant forms of dissolved organic carbon to biological degradation such amides predominate in deep waters [35]. A i l f h id ifi bl l d Other genes in relative high numbers in the Km3 metagenome were luciferase-like genes (Table 3), which likely encode mono- oxygenases. However, it is difficult to advance a function for most of them, since only a few were clearly related to luxA (encoding the luciferase alpha subunit and therefore directly involved in bioluminescence). Gene content and metabolic potential The capacity to oxidize CO aerobically without a direct link to autotrophy has been recently identified in several bacteria. For instance, Silicibacter pomeroyi, a marine bacterium of the Roseobacter clade whose genome has been sequenced [23] possess these genes but lacks autotrophic carbon fixation path- ways. Its strategy consists of supplementing heterotrophy with the use of inorganic compounds (CO and sulfide). The capacity to use simultaneously CO and organic substrates is known for several other bacteria, including marine genera such as Stappia [36]. Most interestingly, cox genes are also present in the genome sequences of the acidobacteria Solibacter usitatus and the Acidobacteria Bacteri- um Ellin345 [37]. Phylogenetic analyses of Km3 coxL showed that some of them were clearly related to Alphaproteobacteria, Actinobacteria and Chloroflexi homologues, but not to Solibacter coxL (data not shown). Nonetheless, since Km3 Acidobacteria were very diverse (Fig. 3A), it might be possible that some of the phylogenetically unclassified Km3 coxL belong to this phylum. Table 3. Hallmark proteins in the Km3 metagenome compared to the deep ALOHA water column. doi:10.1371/journal.pone.0000914.t003 to the COG or the KEGG classification, respectively, were related to metabolism and transport, whereas only 17% corresponded to housekeeping genes involved in information-related processes (Table 1 and 3). The most abundant functional family was that of transporters, and within it, amino acid transporters (Figs. 5 and 6). Transporter systems can tell about nutrient pools or substrates that are present in the environment and that the organisms use. The largest group of transporters in Km3 consisted of the multi- subunit ABC family (64,4% of the identified transporters) and the most represented of this family were transporters for dipeptides/ oligopeptides and branched chain amino acids (11,2% and 4.3% sequences, respectively). TRAP transporters, which allow substrate accumulation using an electrochemical ion gradient rather that ATP hydrolysis, were also frequently encountered as well as transporters for carboxylic acids (6.5%), while only 5.7% were sugar/polymer transporters (Fig. 6). The relative high number of peptide and branched chain amino acid transporters suggests that proteins, perhaps associated with sinking marine snow, are an important carbon source for deep-sea microbes. By contrast to shallow waters, with sugars forming an easily accessible labile pool of organic matter, recalcitrant forms of dissolved organic carbon to biological degradation such amides predominate in deep waters [35]. Approximately 10% of the identifiable Km3 genes were related to energy production and conversion (Figs. 5 and 6). PLoS ONE \| www.plosone.org Metagenomic comparison of deep Mediterranean samples and the water column at ALOHA station samples and the water column at ALOHA station The availability of metagenomic sequences from different depths at the Pacific ALOHA station makes it possible to test whether warmer Mediterranean temperatures at similar high depth (3,000– 4,000 m) can affect community structure significantly. Of course other environmental parameters, such as salinity or biogeography may affect it as well. However, despite slight differences between the aphotic ALOHA and the deep Km3 metagenomic libraries, the equivalent sequence volume produced per library together with the overall similar general patterns of taxa found (Figs. 2 and 3), gene content (Figs. 5 and 6) and aminoacid usage (data not shown) allows a reasonable comparison between them. We made TBLASTX searches of our Km3 sequences against each one of the depth-related metagenomic libraries in the ALOHA water column to construct a similarity matrix that was analyzed by neighbour- joining (Fig. 7A). The sequence datasets were additionally used to construct coverage maximal unique matches (MUMs) plots (Fig. 7B). The 3,000 m deep Km3 metagenome recruited most Approximately 10% of the identifiable Km3 genes were related to energy production and conversion (Figs. 5 and 6). The best represented pathways were oxidative phosphorylation (131 hits) followed by carbon fixation pathways (129 hits) and nitrogen metabolism (80 hits). In agreement with the deep origin of the sample, genes related to photosynthesis were minoritary. By contrast, genes involved in the degradation of different kinds of compounds, including notably biopolymers and xenobiotics, and catabolic pathways in general were fairly abundant. Among them were several acetone decarboxylases and dehalogenases and KEGG-pathways associated with the degradation of limonene and pinene, 1-and 2-methylnaphthalene, glycosaminoglycan, benzoate, 1,2-dichloroethane, nitrobenzene, gamma-hexachloro- cyclohexane, ethylbenzene and fluorene. This reinforces the idea that microorganisms living at this depth are adapted to degrade PLoS ONE \| www.plosone.org September 2007 \| Issue 9 \| e914 September 2007 \| Issue 9 \| e914 8 Mediterranean Metagenomics Figure 5. Comparison of COG distribution of fosmid-ends in Km3 and ALOHA water column. Fosmid-ends were classified according to the COG database both Km3 and ALOHA [14] datasets were analyzed (see methods). doi:10.1371/journal.pone.0000914.g005 Figure 5. Comparison of COG distribution of fosmid-ends in Km3 and ALOHA water column. Fosmid-ends were classified according to the COG database both Km3 and ALOHA [14] datasets were analyzed (see methods). doi:10.1371/journal.pone.0000914.g005 pressure, appears to be the major stratifying factor for microbial communities. Metagenomic comparison of deep Mediterranean samples and the water column at ALOHA station Other factors might also have an effect, such as the limited transport of deep-ocean microbes to the Mediterranean through the Gibraltar sill. However, since identical o nearly identical sequences for conserved (16S rRNA gene) and, furthermore, variable (16S-23S rRNA intergenic spacer) markers have been retrieved from the deep Mediterranean and open oceans [8,38], the colonization of the Mediterranean by deep oceanic microorganisms is possible. Therefore, temperature rather than other physic-chemical or biogeographical parameters seems the most influential stratification factor in these waters. hits from the ALOHA aphotic water column, as expected. Surprisingly, contrary to the initial expectation of a higher similarity with the 4,000 m deep sample, enduring analogous pressures, the most abundant and highest MUMs were observed with intermediate deep-water libraries, particularly that of 770 m. This relationship was not limited to sequence similarity, but also gene types (metabolism and cell physiology) and even the frequency of mobile elements (IS, phage related, integrons) in the Mediterranean 3,000 m-deep sample seemed more alike to the upper section of the aphotic zone in the Pacific. We interpret this result as being essentially the consequence of the warmer temperature (13.9uC) of deep Mediterranean waters. Most other biologically relevant parameters are remarkably similar (Table 4). As a matter of fact, although much deeper, the Km3 sample has in common with the ALOHA 500 and 770 m deep libraries intermediate temperatures, 4.8 to 7.2uC, instead of the 1.4uC characterizing the 4,000 m-deep library. The 200 m deep ALOHA library, although having also a relatively similar temperature (18uC) to Km3 shares many features and genes with the photic region, and falls apart in neighbour-joining analyses (Fig. 7A). Therefore, in the absence of light, temperature, and not hits from the ALOHA aphotic water column, as expected. Surprisingly, contrary to the initial expectation of a higher similarity with the 4,000 m deep sample, enduring analogous pressures, the most abundant and highest MUMs were observed with intermediate deep-water libraries, particularly that of 770 m. This relationship was not limited to sequence similarity, but also gene types (metabolism and cell physiology) and even the frequency of mobile elements (IS, phage related, integrons) in the Mediterranean 3,000 m-deep sample seemed more alike to the upper section of the aphotic zone in the Pacific. We interpret this result as being essentially the consequence of the warmer temperature (13.9uC) of deep Mediterranean waters. Metagenomic comparison of deep Mediterranean samples and the water column at ALOHA station Most other biologically relevant parameters are remarkably similar (Table 4). As a matter of fact, although much deeper, the Km3 sample has in common with the ALOHA 500 and 770 m deep libraries intermediate temperatures, 4.8 to 7.2uC, instead of the 1.4uC characterizing the 4,000 m-deep library. The 200 m deep ALOHA library, although having also a relatively similar temperature (18uC) to Km3 shares many features and genes with the photic region, and falls apart in neighbour-joining analyses (Fig. 7A). Therefore, in the absence of light, temperature, and not DISCUSSION This is the second large sequencing effort carried out in the deep ocean, the only precedent being the central Pacific gyre water column study at the ALOHA station [14]. In our case a single depth (3,000 m) was sampled at very pristine waters in the Ionian Sea, and its study adds interesting complementary information to the previous work in two ways. First, it provides a second geographic location belonging to a very different water mass, not only distant from the Pacific sampling site but also differing in PLoS ONE \| www.plosone.o PLoS ONE \| www.plosone.org September 2007 \| Issue 9 \| e914 9 Mediterranean Metagenomics Figure 6. Distribution of Km3 fosmid-ends in KEGG categories. A, Detailed KEGG categories. B, Major KEGG categories and classification by type of substrate of Km3 fosmid-ends identified as transporters. * Other transporters. doi:10.1371/journal.pone.0000914.g006 6. Distribution of Km3 fosmid-ends in KEGG categories. A, Detailed KEGG categories. B, Major KEGG categories and classification by type of te of Km3 fosmid-ends identified as transporters * Other transporters Figure 6. Distribution of Km3 fosmid-ends in KEGG categories. A, Detailed KEGG categories. B, Major KEGG categories and classification by type of substrate of Km3 fosmid-ends identified as transporters. * Other transporters. doi:10.1371/journal.pone.0000914.g006 PLoS ONE \| www.plosone.org September 2007 \| Issue 9 \| e914 10 Mediterranean Metagenomics Figure 7. Normalized metagenome comparison of 3000 m-deep Mediterranean Km3 and Pacific ALOHA water column. For normalization, of 6,853 sequences (size of the smallest library compared, ALOHA 130 m) from each library were randomly selected and compared. A, Neig joining analysis of fosmid-end sequences in Km3 and different depths in ALOHA. Temperature, salinity, and the total number of sequences ava for each library are shown on the right; Jackknife values, at nodes. B, Normalized MUMmer plots showing the number of maximal unique ma (MUM ) h d b th 3000 d K 3 d th diff t ALOHA t i lib i MUM di t ib t d f ti f th i id Figure 7. Normalized metagenome comparison of 3000 m-deep Mediterranean Km3 and Pacific ALOHA water column. For normalization, a total of 6,853 sequences (size of the smallest library compared, ALOHA 130 m) from each library were randomly selected and compared. A, Neighbour joining analysis of fosmid-end sequences in Km3 and different depths in ALOHA. PLoS ONE \| www.plosone.org DISCUSSION Temperature, salinity, and the total number of sequences available for each library are shown on the right; Jackknife values, at nodes. B, Normalized MUMmer plots showing the number of maximal unique matches (MUMs) shared by the 3000 m deep Km3 and the different ALOHA metagenomic libraries. MUMs are distributed as a function of their identity (ordinates) and the type of COG to which they belong (abscises). Average identity values are indicated for each pair of libraries compared. The number of MUMs having more that 80% identity are given to the right of each panel. doi:10.1371/journal.pone.0000914.g007 Figure 7. Normalized metagenome comparison of 3000 m-deep Mediterranean Km3 and Pacific ALOHA water column. For normalization, a total of 6,853 sequences (size of the smallest library compared, ALOHA 130 m) from each library were randomly selected and compared. A, Neighbour joining analysis of fosmid-end sequences in Km3 and different depths in ALOHA. Temperature, salinity, and the total number of sequences available for each library are shown on the right; Jackknife values, at nodes. B, Normalized MUMmer plots showing the number of maximal unique matches (MUMs) shared by the 3000 m deep Km3 and the different ALOHA metagenomic libraries. MUMs are distributed as a function of their identity (ordinates) and the type of COG to which they belong (abscises). Average identity values are indicated for each pair of libraries compared. The number of MUMs having more that 80% identity are given to the right of each panel. doi:10.1371/journal.pone.0000914.g007 notable difference of the deep Mediterranean (Table 4) is the lower concentrations of inorganic nutrients N and P (about an order of magnitude lower) and, contrastingly, a higher biomass density in the Mediterranean that seems to be at least as active as in the deep global ocean. Thus, these nutrients do not seem to be limiting. Actually various fundamental parameters, notably temperature. Further- more the Gibraltar sill also precludes deep ocean currents from reaching the Deep Med and would isolate this habitat from the input of psycrophiles from Antarctic waters which relatives seem to populate the global ocean bathypelagic regions [39]. Another PL September 2007 \| Issue 9 \| e914 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 11 Mediterranean Metagenomics Table 4. ALOHA and Km3 oceanographic data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . North Pacific ALOHA Mediterranean Km3 coordinates 22u459N, 158uW 36u309N, 15u409E max.depth (m) 4800 3243 depth (m) 10 70 130 200 500 770 4000 3010 Sampling Time Oct. 7th 2002 Oct. 7th 2002 Oct. 6th 2002 Oct. 6th 2002 Oct. 6th 2002 Dec. 21st 2003 Dec. 21st 2003 Nov. 17th 2004 Temp. (uC) 26.40 (24.8361.27)* 24.93 (23.5861.00)* 22.19 (21.3760.96)* 18.53 (18.3961.29)* 7.25 (7.2260.44)* 4.78 (4.8660.21)* 1.46 (1.4660.01)* 13.93 (13.8060.05)* Salinity (PSU) 35.08 (35.0560.21)* 35.21 (35.1760.16)* 35.31 (35.2060.10)* 35.04 (34.9660.18)* 34.07 (34.0660.03)* 34.32 (34.3260.04)* 34.69 (34.6960.00)* 38.74 (38.6960.03)* Chl (mg/Kg) 0.08 (0.0860.03)* 0.18 (0.1560.05)* 0.10 (0.1560.06)* 0.02 (0.0260.02)* ND ND ND ND DOC (mM/Kg) 78 (90.6614.3)* 79 (81.4611.3)* 69 (75.269.1)* 63 (60.469.8)* 47 (47.866.3)* 39.9 (41.564.4)* 37.5 (42.364.9)* 54.265.85* Oxygen (mM/Kg) 204.6 (209.364.5)* 217.4 (215.865.4)* 204.9 (206.666.2)* 198.8 (197.667.1)* 118.0 (120.5618.3)* 32.2 (27.964.1)* 147.8 (147.861.3)* 203.7 (202.6661.2)* DIP (nmol/Kg) 41.0 (56.0633.7)* 16 (43.1625.1)* 66.2 (106.0649.7)* 274.26109.1* 2153 (20516175.7)* 3070 (3000647.1)* 2558 (2507619)* 159.0622.6* N+N (nmol/Kg) 1.0 (2.663.7)* 1.3 (14.7660.3)* 284.8 (282.96270.2)* 1161.96762.5* 28850 (2846062210)* 41890 (409406500)* 36560 (359706290)* 47066133.3* SLCA (ml/Kg) 1.3060.37* 1.3460.37* 1.7260.56* 5.3160.74* 45.3765.75* 92.0664.08* 160* 8.3260.24* HPP (cell6104 ml21) 30.2616.2* 25.269.9* 19.966.9* 13.0362.5* 5.1961.5* 3.1560.7* 0.5560.06* 3.161.73* POC (mM C / Kg) 2.1660.54* 1.9760.35* 1.2960.36* 0.5560.15* 0.3960.13* 0.3060.13* - 1.92560.56* Values shown are those from the same CTD casts as the samples (DeLong et al. 2006 and this work). DISCUSSION *Archival data are from ALOHA HOT-DOGS database (http://hahana.soest.hawaii.edu/hot/hot-dogs/) or in the case of Km3 from the ICES oceanographic database (http://www.ices.dk/ocean) and correspond to several datasets collected at the depth and approximate location (less than 50 NM away) as the samples. Values in parentheses are the average value6standard deviation. Abbreviations are Temp, Temperature; Chl, chlorophyll; DOC, dissolved organic carbon; DIP, dissolved inorganic phosphate; N+N, nitrate plus nitrite; SLCA, silicate; HPP, heterotrophic picoplankton (DAPI counts); POC, particulate organic carbon. d i 10 1371/j l 0000914 t004 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 PLoS ONE \| www.plosone.org September 2007 \| Issue 9 \| e914 PLoS ONE \| www.plosone.org 12 Mediterranean Metagenomics or some cases of sharp ecotype differentiation [38,44] In this sense, temperature might be the second most relevant environmental factor operating in the global open ocean, the first being accessibility to light as energy source, while pressure would seem less critical in determining community structure and lifestyles in the deep ocean, at least down to about 4,000 m. This does not imply that piezophilic microbes are not important but are probably much more relevant in very deep trenches. By contrast, temperature is a crucial parameter that requires specific long- studied molecular adaptations [21,45,46]. Low temperatures in most bathypelagic habitats prevent the metabolism of many microbes beyond a certain threshold. By contrast, in the Mediterranean, the persistence of warm temperatures down to bathypelagic waters would allow the persistence of mesopelagic microbial communities adapted to aphotic regions but unable to cope with near-zero temperatures where more psychrophilic organisms dominate. archival data of biomass estimators, like temperature, are more similar to the mesopelagic global ocean than the deep. Hence, common observations in deep Mediterranean and Pacific metagen- omes may reasonably be taken as general deep ocean traits. y y g p We found that the assignment of fosmid-ends to already sequenced microbial genomes, something similar to the ‘‘genome fragment recruitment’’ used recently by Rusch et al. DNA extraction and fosmid library construction DNA extraction and fosmid library construction Sample filters were thawed on ice and then treated with 1mg/ml lysozyme and 0.21 mg/ml proteinase K (final concentrations). Nucleic acids were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1), and then concentrated with sterile water using a microconcentrator (Cen- tricon 100, Amicon). DNA integrity was checked by agarose gel electrophoresis. A fosmid genomic library was constructed from approximately 1.1 mg of DNA from the 0.2–5 mm plankton fraction using the CopyControlTM Fosmid Library Production Kit (Epicentre) as described by the manufacturer’s instructions. A total of 20,767 fosmid clones were obtained, which corresponds to ca. 725–830 Mpb environmental DNA assuming an average insert size of 35–40 Kbp. At any rate, our vision of the deep ocean ecosystem functioning is changing. Not only chemolithoautotrophy (crenarchaeota) appears to be significant, but also mixed strategies including lithoheterotrophy may be as important as pure heterotrophy. Thus, we detected a considerable number of cox genes encoding different subunits of the carbon monoxide dehydrogenase, CODH, responsible for the aerobic oxidation of CO. Initially thought to be exclusive of autotrophs, CO oxidation is being discovered in a plethora of organisms, including members of the marine Roseobacter clade [23,36,37]. Though present in photic layers, CODH genes are more abundant in deep layers of the ALOHA water column [14]. This strongly suggests that deep-sea microorganisms oxidize carbon monoxide (and perhaps other reduced substrates) released from tectonically active areas or anaerobic microenvironments, as alternative or complementary energy sources to heterotrophy. This energy metabolism versatility would be advantageous in this highly depleted environment, where secondary production might be boosted by chemolithotrophy much in the way that phototrophy helps heterotrophy at the surface [43]. DISCUSSION to analyze metagenomic libraries from the Global Ocean Sampling transect [13], was very useful to analyze our data and predict microbial lifestyles. However, these analyses are to be taken cautiously as many of our sequences did not match known genes or could not be confidently assigned to defined taxa (Table 1). Certainly, many more marine genomes covering the whole phylogenetic spectrum would be needed to have a picture of better resolution. Yet from the taxa and gene functional categories identified in this way, some conclusions can be drawn about the lifestyle and ecosystem functioning in the deep Mediterranean. Except for the crenarch- aeota, most of the remaining lineages in deep Mediterranean waters are likely heterotrophs, as confirmed by the classification of gene functions and metabolic pathways (Table 1, Figs. 5 and 6), including transporters, particularly for amino acids and carboxylic acids, and catabolic routes involved in complex organic degrada- tion, e.g. xenobiotics. This is in agreement with an essential role of the heterotrophic deep-sea microbes in the mineralization of organic carbon [40,41]. In many ways, they are to the marine phytoplankton what the soil microbiota are to the forest. The presence of microbial groups typically found in soil (Rhizobiales, Actinobacteria, Acidobacteria) would indeed be consistent with the ecological role of the deep ocean as an ‘‘invisible soil’’ for the ‘‘invisible forest’’ [40]. Our own analysis show that these groups appear also at the ALOHA aphotic zone samples (Figs. 2 and 3). These lineages might live attached to sinking particles. The presence of planctomycetes, often associated to sinking particles [42], also points in this direction. In most marine metagenomic studies carried out do date, particles were excluded by restrictive pre-filtration (0.8 mm), partly with the objective of excluding eukaryotic (including picoeukaryotic) cells [12,13]. As we used 5 mm pore-sized filters for the pre-filtration step, it might be possible that we captured more biomass from marine snow particles. PLoS ONE \| www.plosone.org Sample collection 250 l of seawater were collected by using Niskin bottles mounted on a General Oceanics rosette from a depth of 3,010 m (sea- bottom at 3,243 m depth) at the Ionian Km3 station (36u299980N, 15u399970E) in November 17th 2004 during a cruise of the R/V Urania. Water temperature was 13.93uC and salinity 38.75 PSU. Seawater was sequentially filtered through a 5 mm pore size poly- carbonate filter and the filtrate passed through 0.22 mm pore size Sterivex filters (Durapore, Millipore) using a peristaltic pumping system. Sterivex filters were filled with lysis buffer (40 mM EDTA, 50 mM Tris/ HCl, 0.75M sucrose) and stored at 220uC, until DNA extraction. ACKNOWLEDGMENTS Data deposition: The sequences reported in this paper have been submitted to GenBank, accession numbers EF597680-EF597722 and EI942868-EI951915. Screening and fosmid-end sequencing g q g The library was pooled in groups of 96 clones. DNA from pooled cultures was extracted using the QIAprep Spin Miniprep Kit (Qiagen) and then PCR-screened for the presence of archaeal and bacterial 16S rRNA genes. Different primer combinations were used for archaeal 16S rRNA gene amplification using 21F (59- TTCCGGTTGATCCTGCCGGA), Ar109 (59-AC(G/T)GCTG- CTCAGTAACACGT), ANMEF (59-GGCTCAGTAACACGT- GGA) and 1492R (59-GGTTACCTTGTTACGACTT). In the case of bacteria, we amplified 16S rRNA gene together with the adjacent intergenic spacer (ITS) using 27F (59-AGAGTTT- GATCCTGGCTCAG) and 23S1R (59-GGGTTTCCCCATT- CGGAAATC). In this way, only bacterial fosmids containing ITSs of different size to that of Escherichia coli were detected. PCR reactions were carried out under the following standard condi- tions: 35 cycles (denaturation at 94uC for 15 s, annealing at 50uC The comparison of our deep Mediterranean data with those obtained in the Pacific ALOHA water column suggests that, in the absence of light, temperature becomes the major stratifying factor for community structure (Fig. 7). This effect of temperature seen with depth corroborates analogous temperature-dependent pat- terns in surface waters in the recent GOS metagenomic study [13] PLoS ONE \| www.plosone.org September 2007 \| Issue 9 \| e914 13 Mediterranean Metagenomics for 30 s, extension at 72uC for 2 min) preceded by 2 min denaturation at 94uC and followed by 7 min extension at 72uC. Twenty-eight 16S rRNA gene-containing archaeal clones and sixteen bacterial clones were detected in the library. The genes were sequenced (Genome Express, Meylan, France) and the closest relatives in databases searched using BLAST [47]. In parallel, the insert terminal sequences of ca. 5,000 fosmid clones were sequenced at the Go¨ttingen Genomics Laboratory, Germany (http://www.g2l.bio.uni-goettingen.de). A total of 9,048 high quality sequence reads were obtained (average length 794 bp), which implies approximately 7.2 Mbp sequence, i.e. roughly two prokaryotic genome equivalents. uio.no/ahoammer/past) by cluster analysis. Node support was assessed by Jackknife matrix resampling [51]. Cumulative pairwise bitscore values were normalized by dividing each one by the cumulative bistscore value derived from the TBLASTX of one dataset versus itself and the number of HSPs of each case. Fosmid-end sequence analysis q y Fosmid end sequences were revised and cleaned of vector contaminant sequences using Sequencher 4.1.4 software (Gene Codes Corp.). For taxonomic binning, sequences were queried against the NCBI non-redundant (nr) protein database using BLASTX using a cut-off value of ,1e-50. Top BLAST high- scoring pairs (HSPs) were tabulated according to the NCBI taxonomic identifier for each sequence. For COG assignments, sequences were compared to the cluster of orthologous genes (COG) databases using BLAST (rpsblast (-p F)) using a cut-off value of 1e-7. Also, sequences were compared to the KEGG database using BLASTX. Results were tabulated, and used to determine the proportion of sequences contained in each COG category or KEGG pathway. To calculate the amino acid usage pattern, ORFs for each data sample were identified using the automated genome annotation software Glimmer 2.02 [48]. The frequency of each amino acid was represented as suggested by [49]. Author Contributions Conceived and designed the experiments: FR PL DM. Performed the experiments: AM JA. Analyzed the data: FR AM PL DM. Contributed reagents/materials/analysis tools: FR AM. Wrote the paper: FR PL. Other: Provided sequencing: GG AS. Conceived and designed the experiments: FR PL DM. Performed the experiments: AM JA. Analyzed the data: FR AM PL DM. Contributed reagents/materials/analysis tools: FR AM. Wrote the paper: FR PL. Other: Provided sequencing: GG AS. Comparative analysis of marine metagenomic libraries In order to compare the Km3 library with that of the Sargasso Sea [12] and the North-Pacific Subtropical Gyre ALOHA station [14], coverage plots were generated by using the Promer program implemented in MUMmer 3.18, using the ‘‘maxmatch’’ option [50] and visualized using the MUMmer-plot program (http:// mummer.sourceforge.net/). For sequence analysis, resulting delta files were converted into coordinate files and sequence analysis by using the ‘show-coords’ option. To estimate cumulative protein sequence differences in Km3 and the water column at the ALOHA station, we made TBLASTX searches of the complete set of sequences from every single library versus all the others. The bitscores of the top HSPs from every single sequence from one set versus another were summed to yield a cumulative pairwise bitscore value that was normalized and used to construct a distance matrix. The matrix was analyzed using PAST software (v. 1.58) (http://folk. Phylogenetic analyses y g y Archaeal 16S rRNA gene sequences detected in Km3 fosmids were aligned using ClustalX [52] with those from the ALOHA water column and those from available Group I crenarchaeotal genomes and selected Group I and II archaeal genome fragments. We then made a preliminary neighbour-joining tree with the 244 used sequences in order to make a selection of representative sequences to be included in a maximum likelihood tree. In the case of bacteria, we included the closest relatives to the identified Km3 sequences by BLAST, as well as representative members of the detected bacterial phyla. Sequences were aligned using Clustal X, and the alignment manually edited using the ED program of the MUST package [53]. Gaps and ambiguously aligned positions were excluded from our analyses. Maximum likelihood trees were reconstructed using PhyML [54] applying a general time reversible model of sequence evolution (GTR), and taking among-site rate variation into account by using a six-category discrete approximation of a distribution and a proportion of invariable sites. ML bootstrap proportions were inferred using 1000 replicates. For phylogenetic analysis of the Cox proteins, TBLASTX searches were carried out, the corresponding sequences recovered from GenBank and a multiple alignment generated using Clustal X and manually refined as mentioned above. Maximum likelihood trees were reconstructed using PhyML [54] with the JTT model of sequence evolution and taking among-site rate variation into account by using a six- category discrete approximation of a distribution and a proportion of invariable sites. ML bootstrap proportions were inferred using 1000 replicates. Cox phylogenetic trees are available upon request. Phylogenetic trees were viewed using the program TREEVIEW [55]. 9. Moreira D, Rodriguez-Valera F, Lopez-Garcia P (2004) Analysis of a genome fragment of a deep-sea uncultivated Group II euryarchaeote containing 16S rDNA, a spectinomycin-like operon and several energy metabolism genes. Environ Microbiol 6: 959–969. 10. Moreira D, Rodrı´guez-Valera P, Lo´pez-Garcı´a P (2006) Genome fragments from mesopelagic Antarctic waters reveal a novel deltaproteobacterial group related to the myxobacteria. Microbiology 152: 505–517. 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https://openalex.org/W2744101564	http://www.scielo.br/pdf/sa/v74n6/0103-9016-sa-74-06-0469.pdf	English	null	Use of the software Seed Vigor Imaging System (SVIS®) for assessing vigor of carrot seeds	Scientia Agrícola	2,017	cc-by	4,214	469 469 Scientia Agricola Scientia Agricola http://dx.doi.org/10.1590/1678-992X-2016-0220 Scientia Agricola Introduction were conducted using this technique and successfully evaluated seed vigor, for crops such as soybean seeds (Hoffmaster et al., 2003), corn (Hoffmaster et al., 2005), melon (Marcos-Filho et al., 2006), peanut (Marchi et al., 2011), cucumber (Chiquito et al., 2012) and okra (Kikuti and Marcos Filho, 2013). Carrot seed quality depends on the growth stage and the general condition of the plant. The vegetative and flowering periods for carrot plants are usually very long. This leads to the formation of seeds with different levels of quality, leading to difficulties in standard estab lishment and growth, contributing to excessive use of seeds, which raises production costs. Therefore, fast and accurate procedures for the assessment of seed quality are needed. The aim of this study was to verify the possibility of using the computerized system of seedling analysis (SVIS®) to detect differences in vigor between lots of car rot seeds when compared to the information provided by traditionally used vigor tests. Seed vigor can be evaluated by the physiological, biochemical and stress tolerance tests which are com monly used for carrot and other species (Pereira et al., 2008; Chiquito et al., 2012; Kikuti and Marcos Filho, 2013). Computerized image analysis of seedlings has been used for seed vigor evaluation in several species; these are non-destructive methods and can provide ob jective information in a short period of time with less human interference (McCormac et al., 1990; Marcos Filho et al., 2006). Use of the software Seed Vigor Imaging System (SVIS®) for assessing vigor of carrot José Luís de Marchi1, Silvio Moure Cicero1 ABSTRACT: Seed vigor has traditionally been evaluated by physiological, biochemical and stress tolerance tests. More recently, with the use of computerized image analysis, objective informa tion has become accessible in a relatively short period of time, with less human interference. The aim of this study was to verify the efficiency of computerized seedling image analysis by Seed Vigor Imaging System (SVIS®) to detect differences in vigor between carrot (Daucus carota L.) seed lots as compared to those provided by traditional vigor tests. Seeds from seven lots from the Brasilia cultivar were subjected to a germination test, first count of germination, speed of germination, accelerated aging with saline solution and seedling emergence; furthermore, a vig or index, growth index and uniformity index were determined by the Seed Vigor Imaging System (SVIS®) during four evaluation periods. The results obtained by the computerized seedling analy sis (vigor index and growth index) show that SVIS® is efficient in assessing carrot seed vigor. Keywords: Daucus carota L., Daucus carota subsp. Sativus, quality control, physiological poten tial, seed analysis 1University of São Paulo/ESALQ − Crop Production Dept., Av. Pádua Dias, 11 − 13419-900 − Piracicaba, SP − Brazil. Corresponding author <jlmarchi@usp.br> Edited by: Paulo Cesar Sentelhas 1University of São Paulo/ESALQ − Crop Production Dept., Av. Pádua Dias, 11 − 13419-900 − Piracicaba, SP − Brazil. Corresponding author <jlmarchi@usp.br> Edited by: Paulo Cesar Sentelhas Received June 02, 2016 Accepted November 28, 2016 Materials and Methods The study was carried out in Piracicaba, in the state of São Paulo, Brazil (22°46’24” S, 47°36’33” W, 582 m above sea level). The experiment was conducted on carrot seeds (Daucus carota L. ‘Brasilia’) provided by a seed company, produced and harvested under the same environmental conditions in the region of Bage, in the state of Rio Grande do Sul. During processing, the seed company separated the seeds into 7 lots presenting dif ferences in physical purity and germination (Table 1). They were examined over 4 evaluation periods, with 3 months separating the 1st, 2nd, and 3rd periods, and 6 months between the 3rd and 4th periods. Seeds were packed into polyethylene bags and stored under con trolled ambient conditions (30 % U.R. and 10 °C). The seeds were subjected to the following tests. The feasibility of using computerized image anal ysis was first demonstrated by McCormac et al. (1990) who determined the average length of the primary roots of tomato, lettuce and cauliflower seedlings. Simi larly, in researches conducted by Geneve and Kester (2001) on cauliflower, tomato and impatiens seeds, and Tohidloo and Kruse (2009) on radish seeds using seed lings imaging analysis systems, were able to determine correlations between seedling growth and seedling emergence. Germination speed The germination speed index was determined alongside the standard germination test (MAPA, 2009); evaluations of normal seedlings were performed daily until 14 days after sowing. Only seeds that produced a normal seedling were counted as germinated. The ger mination speed index was calculated according to the formula proposed by Maguire (1962). Accelerated aging Following the methodology described by Marcos Filho (2006), a single layer of seeds was distributed on wire mesh attached to plastic boxes (11 × 11 × 3 cm) filled with 40 mL saturated NaCl solution and kept at 41 °C in a chamber. After treatment, 4 replicates of 50 seeds were used to test for germination as described. Table 2 presents results obtained from traditional vigor tests. The germination test showed differences be tween lots in all the evaluation periods, wherein lots 1 and 2 showed the highest percentage germination as op posed to lots 6 and 7 showing the lowest. Furthermore, the vigor tests done in the 1st period indicated lots 1 and 2 as having higher vigor than lots 6 and 7. The other lots were rated as intermediate in physiological quality. This classification could not be verified in the 2nd pe riod of evaluation, except for the accelerated aging test which classified only the first lot as having the best per formance. Seedling vigor imaging system (SVIS®) Table 1 − Lots from carrot seeds (Daucus carota L. ‘Brasilia’) used in the study and their differences in terms of germination (G) and physical purity (P) as informed by the seed company who supplied the seeds for this research g g g g y ( ) Four replicates of 50 seeds per lot were processed as follows: two rows of 25 seeds each were distributed in plastic boxes (15 × 23 × 4 cm) on two blue blotter papers moistened with distilled water at a ratio of 2.8 times the paper’s dry mass (this ratio was specifically ad justed for this research by previous tests) and inserted in a germination chamber at 20 ± 1 °C at an inclination of 25 degrees in relation to the base of the chamber, in the absence of light, in order that the seedlings grew parallel to the blotter. Seedlings were scanned 6 days after sow ing. The images were captured by a scanner operated by Photosmart software with a resolution of 100 dpi. The captured images were analyzed by SVIS® software. The program generates vigor, growth of seedlings and uni formity of development indexes, as described by Sako et al. (2001). Germination test Four replications of 50 seeds for each lot were dis tributed in plastic boxes on filter paper moistened with distilled water at a ratio of 2.5 times the paper’s dry mass, and maintained at a constant temperature of 20 °C. Evaluations were made at 7 and 14 days after sow ing, according to the criteria established by the Rules for Seed Analysis for this species (MAPA, 2009). The results were expressed in terms of percent of normal seedlings for each lot. The vigor index values (0 to 1000) are based on the speed and uniformity of seedling development in rela tion to the maximum possible values for 6 day old car rot seedlings. The growth index (0 to 1000) is calculated by the software based on the length of the hypocotyl - radicle axis. The uniformity index values (0 to 1000) are based on deviations from the standard seedling develop ment set in software. The experimental design was completely random ized, and the means compared by the Scott-Knott test at 5 % probability. Subsequently Pearson correlation coeffi cients (r) between the results from the SVIS® test and the results from the other vigor tests were calculated. The significance of the r values was verified by the t test at 5 % and 1 % probability. Germination first count The counts were performed simultaneously with the germination test and the percent of normal seedlings were evaluated 7 days after sowing. Results The moisture content of the seeds varied between lots and times of analysis, from 6.1 % to 7.0 % (Table 2). This small variation in moisture content is an important fact to be taken into consideration in the evaluation of physiological potential, since variations greater than 1 % between samples may affect the vigor test results (Mar cos Filho, 2006; Tekrony, 2003). Seed moisture content In this context, Sako et al. (2001) developed an au tomated system for assessing the vigor of lettuce seeds called the Seed Vigor Imaging Sistem (SVIS®). The pro cess involves scanning the seedlings and then generating vigor, growth and uniformity indexes. Further studies Determined at 105 ± 3 °C for 24 h in duplicate samples of intact seeds as recommended by the Rules for Seed Analysis (MAPA, 2009). The results were expressed in terms of percentage water content (fresh weight ba sis). Sci. Agric. v.74, n.6, p.469-473, November/December 2017 470 Imaging techniques for carrot seeds Marchi & Cicero Table 1 − Lots from carrot seeds (Daucus carota L. ‘Brasilia’) used in the study and their differences in terms of germination (G) and physical purity (P) as informed by the seed company who supplied the seeds for this research. Seed Lot G P ------------------------------------------------ % ------------------------------------------------ 1 86 100 2 86 99.1 3 85 100 4 84 99.6 5 83 100 6 83 100 7 82 99.8 Seedling vigor imaging system (SVIS®) Imaging techniques for carrot seeds Imaging techniques for carrot seeds Marchi & Cicero Table 2 − Total germination (G), seed water content (W), seedling emergence (E), accelerated ageing with saturated salt solution (EA), first count of germination (1st GC), germination velocity index (GVI), emergence velocity index (EVI) of seven lots of carrot seeds, cv. Brasília, obtained in the first evaluation period (before storage), in the 2nd evaluation period (after 3 months storage), in the 3rd evaluation period (after 6 months storage) and in the 4th evaluation period (after 12 months storage). i p g , p g , p storage) and in the 4th evaluation period (after 12 months storage). Imaging techniques for carrot seeds Evaluation Period Lots G W E EA 1st GC GVI EVI ----------------------------------------------------------------------------------------------------------------------------------- % ----------------------------------------------------------------------------------------------------------------------------------- 1 87 a 6.3 75 a 58 a 66 a 6.8 a 6.9 a 2 86 a 6.4 77 a 55 a 62 abc 6.5 a 7.1 a 3 85 a 6.4 74 ab 52 ab 65 ab 6.3 ab 6.8 a 1st 4 82 ab 6.4 69 abc 42 ab 70 a 6.7 a 6.4 ab 5 84 a 6.1 66 abc 38 b 59 abc 6.4 ab 5.9 ab 6 80 b 6.2 63 bc 34 b 46 c 5.7 b 5.6 b 7 81 b 6.5 61 c 33 b 50 bc 5.8 b 5.6 b CV (%) 5.9 1.4 7.2 20.1 13.5 8.1 7.1 1 86 a 6.8 80 a 51 a 51 a 6.5 a 6.9 a 2 82 a 6.8 75 ab 46 b 49 a 6.2 a 6.3 a 3 74 b 6.9 66 b 43 b 41 ab 5.5 b 6.1 ab 2nd 4 66 bc 6.7 56 c 37 c 35 b 4.8 bc 5.1 b 5 67 bc 6.5 56 c 33 c 35 b 4.8 bc 5.0 b 6 60 c 6.8 50 cd 25 d 31 b 4.2 c 4.3 bc 7 63 c 7.0 48 d 23 d 32 b 4.6 c 4.1 c CV(%) 6.5 1.5 7.6 21.2 20.5 8.2 7.3 1 86 a 6.4 63 a 55 a 44 a 6.0 a 4.7 a 2 86 a 6.5 62 a 54 a 38 a 5.8 a 4.7 a 3 78 b 6.4 62 a 53 ab 36 ab 5.3 a 4.7 a 3rd 4 74 b 6.3 61 ab 52 b 37 ab 5.1 b 4.5 ab 5 72 c 6.2 61 ab 45 c 37 ab 5.0 bc 4.4 ab 6 77 bc 6.5 51 c 36 d 30 c 4.5 c 3.9 b 7 67 c 6.5 47 c 33 d 33 c 4.1 c 4.0 b CV(%) 6.1 1.2 6.8 19.6 12.5 8.3 6.9 1 74 a 6.6 57 a 62 a 39 a 5.2 a 4.6 a 2 72 a 6.5 50 a 60 a 34 a 5.1 a 4.6 a 3 62 a 6.8 47 a 46 b 32 a 4.9 a 3.9 ab 4th 4 71 b 6.8 46 ab 45 b 35 a 5.1 b 4.4 ab 5 65 b 6.3 47 b 52 bc 32 ab 4.7 c 3.9 b 6 59 b 6.5 42 c 40 c 30 b 4.1 c 3.8 c 7 60 c 6.7 41 c 45 c 31 c 3.8 c 3.7 c CV(%) 5.9 2.9 6.0 16.1 9.5 8.5 6.7 Different letters indicate a statistically significant difference (p < 0.05) according to Tukey’s test. Seedling emergence in the greenhouse Four replicates of 50 seeds per lot were distributed in multi-cell styrofoam trays containing Plantmax HT substrate. The trays were kept in a greenhouse equipped with an intermittent fogging system. The number of normal seedlings was evaluated daily, at the same hour (10h00), between the 1st and 14th day after sowing to es tablish the emergence speed index, which, in addition to the germination speed index, was calculated according to the formula proposed by Maguire (1962). In the 3rd evaluation period, the vigor tests showed that lots 1, 2 and 3 returned a higher performance than the others, and that lots 6 and 7 again showed a lower performance. Sci. Agric. v.74, n.6, p.469-473, November/December 2017 471 Sci. Agric. v.74, n.6, p.469-473, November/December 2017 Imaging techniques for carrot seeds Brasília, obtained in the 1st evaluation period (before storage), on the 2nd evaluation period (after 3 months storage), in the 3rd evaluation period (after 6 months storage) and in the 4th evaluation period (after 12 months storage). Table 3 − Vigor index (V), growth (Gr) and uniformity (U) of carrot seedlings obtained by software SVIS® analysis of seven lots of carrot seeds, cv. Brasília, obtained in the 1st evaluation period (before storage), in the 2nd evaluation period (after 3 months storage), in the 3rd evaluation period (after 6 months storage) and in the 4th evaluation period (after 12 months storage). Table 3 − Vigor index (V), growth (Gr) and uniformity (U) of carrot seedlings obtained by software SVIS® analysis of seven lots of carrot seeds, cv. Brasília, obtained in the 1st evaluation period (before storage), in the 2nd evaluation period (after 3 months storage), in the 3rd evaluation period (after 6 months storage) and in the 4th evaluation period (after 12 months storage). Evaluation period Lots V Gr U 1 476 a 354 a 762 a 2 464 a 337 ab 764 a 3 462 a 331 ab 770 a 1st 4 441 a 294 ab 784 a 5 468 a 341 a 781 a 6 356 b 187 c 754 a 7 312 ab 154 bc 782 a CV (%) 6.7 12.1 5.5 1 521 a 411 ab 827 a 2 488 ab 378 bc 752 b 3 469 ab 368 bc 801 a 2nd 4 482 ab 351 bc 811 a 5 377 a 285 a 795 a 6 368 b 206 c 772 a 7 384 b 258 c 787 a CV(%) 10.1 13.2 8.2 1 456 a 315 a 788 a 2 432 a 243 a 795 a 3 421 a 263 a 802 a 3rd 4 425 a 250 ab 817 a 5 420 a 240 b 849 a 6 381 b 199 b 815 a 7 406 b 236 b 799 a CV(%) 5.8 11.4 5.2 1 352 a 145 a 719 a 2 341 a 162 a 756 a 3 368 a 178 a 735 a 4th 4 382 ab 228 a 742 a 5 375 b 267 a 767 a 6 373 b 211 b 753 a 7 353 b 200 b 716 a CV(%) 7.57 10.9 5.6 Different letters indicate a statistically significant difference (p < 0.05) by Tukey’s test. storage). Imaging techniques for carrot seeds periods are summarized in Table 3. The data show that lots 6 and 7 could be classified as having lower levels of vigor and growth as compared to the others. Lots 1 and 2 ranked among those with higher levels of vigor and growth across all 4 evaluation periods. Statistical analysis of the uniformity index data did not depict any significant results in any of the four evaluation periods. Similarly, analysis of the 4th period showed lots 1, 2 and 3 as having the highest vigor except for the accelerated aging test which indicated only lots 1 and 2 had the best performance. Moreover, lots 6 and 7 had the lowest level of vigor except for the first count of germination which found only lot 7 with the lowest vigor. The results reinforce the higher vigor of lots 1 and 2 over that of 6 and 7. Thus, lots 1 and 2 stood out from the rest and lots 6 and 7 showed an all-round lower performance. It is notable that although the germination test indicated high levels of physiologi cal potential in the lots, the vigor tests identified lots with significant differences in performance, thereby providing important additional information regarding seed quality (Marcos Filho, 2005). Table 4 presents correlation analysis between the traditionally used tests (Table 2) against the re sults from the imaging analysis test (Table 3). The data shows that the vigor tests presented positive correla tion coefficients (p < 0.01 and 0.01 ≤ p < 0.05) with the variables’ vigor index and growth index and non- significant correlation coefficients (p ≥ 0.05) with the variable uniformity index across all 4 evaluation pe riods. Statistical analyses for vigor index, growth and uniformity index generated by SVIS® in all evaluation Sci. Agric. v.74, n.6, p.469-473, November/December 2017 472 Imaging techniques for carrot seeds Marchi & Cicero Table 4 − Correlation coefficients (r) between the averages of the results from total germination (G), seedling emergence (E), accelerated ageing with saturated salt solution (EA), 1st count of germination (1st GC), germination velocity index (GVI), emergence velocity index (EVI) and vigor index (V), growth (Gr) and uniformity (U) of carrot seedlings obtained by software SVIS® analysis of seven lots of carrot seeds, cv. Imaging techniques for carrot seeds Evaluation period Variables V Gr U G 0.9583 0.8681* 0.4827 NS E 0.8652* 0.8725* -0.5209 NS 1st EA 0.7824 0.9899** -0.6932 NS 1st GC 0.9053* 0.9490** -0.7627 NS GVI -0.8314NS 0.9323* -0.6015 NS EVI -0.7981 NS 0.9009* -0.7194 NS V G U G 0.9645* 0.9490* 0.4618 NS E 0.8665* 0.8798* 0.7948 NS 2nd EA 0.9040* 0.9770 * 0.7287 NS 1st GC 0.8665* 0.8948 * 0.8184 NS GVI 0.8532* 0.9289* -0.6006 NS EVI 0.9333* 0.9218* 0.7407 NS V G U G 0.8850* 0.9048* 0.6555 NS E 0.8108 NS 0.9827* 0.8280 NS 3rd EA 0.8658* 0.9899* -0.7702 NS 1st GC 0.8667* 0.8575* -0.6555 NS GVI 0.6610 NS 0.9141* 0.7774 NS EVI 0.7756 NS 0.9284* 0.7152 NS V G U G 1,0000 0.9999 -0.8289 NS E 0.9562* 0.9497* 0.7756 NS 4th EA 0.9880 0.8101 NS 0.6146 NS 1st GC 0.9827 0.8594 * 0.5187 NS GVI 0.9527* 0.8594* 0.7248 NS EVI 0.9439* 0.8763* 0.7264 NS Significant by t test at 5 % probability; *Significant according to t test at 1 % probability; NS = not significant according to t test. Acknowledgments Ministério da Agricultura, Pecuária e Abastecimento [MAPA]. 2009. Rules for Seed Analysis = Regras para Análise de Sementes. Secretaria de Defesa Agropecuária, Brasília, DF, Brazil (in Portuguese). To São Paulo State Foundation for Research Sup port (FAPESP) for the founding and resources to conduct the research. Pereira, R.S.; Nascimento, W.N.; Vieira, J.V. 2008. Carrot seed germination and vigor in response to temperature and umbel orders. Scientia Agricola 65: 145-150. Conclusions Computerized image analysis of seedlings using the software SVIS® was effective in determining the vigor of carrot seeds and has a level of sensitivity comparable to traditional vigor tests. Marcos Filho, J. 2005. Cultivated Plants Seed Physiology = Fisiologia de Sementes de Plantas Cultivadas. FEALQ, Piracicaba, SP, Brazil (in Portuguese). McCormac, A.C.; Keefe, P.D.; Draper, S.R. 1990. Automated vigor testing of field vegetables using image analysis. Seed Science and Technology 18: 103-112. Imaging techniques for carrot seeds Marchi & Cicero ing to the authors, this behavior was due to uniform germination of the seeds which is related to the high vigor of the same. The variable uniformity index (Table 4) shows non-significant coefficients with the other vigor tests (p ≥ 0.05) throughout all 4 evaluation periods. This can be attributed to similarity in physiological potential of seed lots assessed in the present research work. Gomes Júnior, F.G.; Chamma, H.M.C.P.; Cicero, S.M. 2014. Automated image analysis of seedlings for vigor evaluation of common bean seeds. Acta Scientiarum 36: 195-200. Hoffmaster, A.L.; Fujimura, K.; McDonald, M.B.; Bennett, M.A. 2003. An automated system for vigour testing three-day-old soybean seedlings. Seed Science and Technology 31: 701-713. Hoffmaster, A.L.; Fujimura, K.; McDonald, M.B.; Bennett, M.A.; Evans, A.F. 2005. The Ohio State University Seed Vigor Imaging System (SVIS) for soybean and corn seedlings. Seed Technology 27: 7-24. Other authors have successfully verified signifi cant coefficients between computerized image analysis results and those obtained by traditional vigor tests for various species such as melon (Marcos Filho et al., 2006) soybean (Marcos Filho et al., 2006), peanut (Marchi et al., 2011) and bean (Gomes Júnior et al., 2009). How ever, significant correlation values were not observed for rapeseed (Tohildloo and Kruse, 2009) nor sunflower seeds (Caldeira et al., 2014). Kikuti, A.L.P.; Marcos Filho, J. 2013. Seedling imaging analysis and traditional tests to assess okra seed vigor. Journal of Seed Science 35: 443-448. Maguire, J.D. 1962. Speed of germination-aid in selection and evaluation for seedling emergence and vigor. Crop Science 2: 176-177. Marchi, J.L.; Cicero, S.M.; Gomes Junior, F.G. 2011. Using computerized analysis of seedlings to evaluate the physiological potential of peanut seeds treated with fungicide and insecticide. Revista Brasileira de Sementes 33: 652-662 (in Portuguese, with abstract in English). Thus, with the results obtained in this research, it was possible to conclude that data obtained with the SVIS® software (vigor index and growth index), provided similar results to the recommended vigor tests, provid ing sensitivity in the evaluation of carrot seed vigor in a shorter period of time and with less human interference. Marcos Filho, J.; Bennett, M.A.; McDonald, M.B.; Evans, A.F.; Grassbaugh, E.M. 2006. Assessment of melon seed vigour by an automated computer imaging system compared to traditional procedures. Seed Science and Technology 34: 485-497. Sci. Agric. v.74, n.6, p.469-473, November/December 2017 Discussion In addition, the data from the correlation analysis (Table 4) shows that the vigor index and the growth in dex can be successfully used to access carrot seed vigor. Germination, germination velocity index, first count of germination, seedling emergence, emergence velocity index and accelerated ageing with saturated salt solu tion (EA) presented significant coefficients (p < 0.01 and 0.01 ≤ p < 0.05) with the variables vigor index and the growth index, generated by the SVIS® software, across all 4 evaluation periods, with few exceptions. Seedling image analysis permitted the identifica tion of physiological differences between seed lots in all evaluation periods. However, the vigor index and seedling growth index enabled the separation of lots at 3 levels of vigor, as did the traditional vigor tests, identifying lots 1 and 2 as having the highest performance, lots 6 and 7 with the lowest, and the remaining lots as having intermedi ate vigor between these levels. Using SVIS®, similar results were obtained with seeds of other species, which present ed an association between lots showing different vigor lev els and the traditional vigor tests such as lettuce (Sako et al., 2001), corn (Otoni and McDonald, 2005), melon (Mar cos Filho et al., 2006) and bean (Gomes Júnior et al., 2014). However, uniformity of seedling development did not differ between seed lots for all evaluation periods (Table 3). Similar behavior was observed by Kikuti and Marcos Filho (2013) and Marcos Filho et al., 2006 study ing okra seeds and soybean seeds, respectively. Accord Sci. Agric. v.74, n.6, p.469-473, November/December 2017 473 References Caldeira, C.M.; Carvalho, M.L.M.; Oliveira, J.A.; Coelho, S.V.B.; Kataoka, V.Y. 2014. Sunflower seed vigor determined by computerized seedling analysis. Científica 42: 346-353 (in Portuguese, with abstract in English). Sako, Y.; McDonald, M.B.; Fujimura, K.; Evans, A.F.; Bennett, M.A. 2001. A system for automated seed vigour assessment. Seed Science and Technology 29: 625-636. Tohidloo, G.; Kruse, M. 2009. Development of an image analysis aided seedling growth test for winter oilseed rape and verification as a vigour test. Seed Science and Technology 37: 98-109. Chiquito, A.A.; Gomes Júnior, F.G.; Marcos Filho, J. 2012. Assessment of physiological potential of cucumber seeds using the software seedling vigor imaging system (SVIS). Revista Brasileira de Sementes 34: 255-263. Gomes Júnior, F.G.; Mondo, V.H.V.; McDonald, M.B.; Bennett, M.A. 2009. Evaluation of priming effects on sweet corn seeds by SVIS. Seed Technology Journal 33: 95-100.
https://openalex.org/W4396545456	https://link.springer.com/content/pdf/10.1007/s40544-023-0840-9.pdf	English	null	Core-rim structured MXene@SiO2 composites as oil-based additives for enhanced tribological properties	Friction	2,024	cc-by	7,908	Core-rim structured MXene@SiO2 composites as oil-based additives for enhanced tribological properties Yuhong CUI1, Shenghua XUE1, Tiantian WANG1, Shujuan LIU1, Qian YE1,, Feng ZHOU1,2,, Weimin LIU1,2 1 State Key Laboratory of Solidification Processing, Center of Advanced Lubrication and Seal Materials, School of Materials Science and Engineering, Northwestern Polytechnical University, Xi'an 710072, China 2 State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China Received: 08 August 2023 / Revised: 18 September 2023 / Accepted: 16 October 2023 © The author(s) 2023. Abstract: Herein, we have prepared SiO2 particles uploaded MXene nanosheets via in-situ hydrolysis of tetraetholothosilicate. Due to the large number of groups at the edges of MXene, SiO2 grows at the edges first, forming MXene@SiO2 composites with a unique core-rim structure. The tribological properties of MXene@SiO2 as lubricating additive in 500 SN are evaluated by SRV-5. The results show that MXene@SiO2 can reduce the friction coefficient of 500 SN from 0.572 to 0.108, the wear volume is reduced by 73.7%, and the load capacity is increased to 800 N. The superior lubricity of MXene@SiO2 is attributed to the synergistic effect of MXene and SiO2. The rolling friction caused by SiO2 not only improves the bearing capacity but also increases the interlayer distance of MXene, avoiding accumulation and making it more prone to interlayer slip. MXene@SiO2 is adsorbed on the friction interface to form a physical adsorption film and isolate the friction pair. In addition, the high temperature and high load induce the tribochemical reaction and form a chemical protection film during in the friction process. Ultimately, the presence of these protective films results in MXene@SiO2 having good lubricating properties. Keywords: Ti3C2Tx MXene; SiO2 nanoparticles; lubricating additives; protective film Keywords: Ti3C2Tx MXene; SiO2 nanoparticles; lubricating additives; protective film use of different nano-additives can improve the lubrication effect and carrying capacity of the base oil [8]. Therefore, it is particularly important to develop a series of efficient and green oil-based lubricating additives. ISSN 2223-7690 CN 10-1237/TH ISSN 2223-7690 CN 10-1237/TH Friction 12(8): 1728–1740 (2024) https://doi.org/10.1007/s40544-023-0840-9 * Corresponding authors: Qian YE, E-mail: yeqian213@nwpu.edu.cn; Feng ZHOU, E-mail: zhouf@licp.cas.cn Yuhong CUI1, Shenghua XUE1, Tiantian WANG1, Shujuan LIU1, Qian YE1,, Feng ZHOU1,2,, Weimin LIU1,2 1 State Key Laboratory of Solidification Processing, Center of Advanced Lubrication and Seal Materials, School of Materials Science and Engineering, Northwestern Polytechnical University, Xi'an 710072, China 2 State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China Received: 08 August 2023 / Revised: 18 September 2023 / Accepted: 16 October 2023 © The author(s) 2023. 2.1 Materials TEOS (>99%) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Hydrochloric acid (HCl) and ethyl alcohol absolute were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. Lithium fluoride (LiF) and ammonium hydroxide (NH3·H2O) were provided by Energy Chemical. Ti3AlC2 MAX came from Jinzhou Haixin Metal Materials Co., Ltd. All reagents are of analytical grade and can be used directly without further purification. In addition to 2D materials, spherical nanoparticles are also widely used as high-performance additives, such as copper nanoparticles [25], liquid metals [26], carbon nanospheres [27], etc. SiO2 is a lubricating material with excellent mechanical properties due to its high hardness and thermal stability. The approximately spherical SiO2 can be used as a rolling bearing to roll into the high-pressure contact area of the friction pair, thereby improving the extreme pressure characteristics of the lubricating oil. In addition, the surface of nano SiO2 particles contains a large amount of hydroxyl groups and unsaturated residual bonds, which can form a solid adsorption film on the surface of the friction pair, significantly improving the anti-friction and anti-wear performance of the lubricating oil [28, 29]. For example, Peng et al. [30] studied the effect of different sizes of SiO2 on the tribological properties of liquid paraffin. It was found that the smaller the size, the greater the load-bearing capacity and wear resistance of liquid paraffin. 1 Introduction The ideal combination of MXene and HS produced synergistic lubrication effects through physical interactions and tribochemical reactions. properties of MXene can be traced to their unique layered structure with weak van der Waals forces between layers, which provide a fairly low shear strength for layering [18, 19]. In addition, 2D MXene is prone to adhesion to different surfaces, passivating the contact points and forming a beneficial protective film. However, the most common drawback of 2D MXene as lubricating materials is their inability to work under high contact pressure and sliding speed [20, 21]. In addition, when MXene is used as a lubricating additive in base oil, the hydrophilic groups on the polar surface limit their dispersion stability and cause aggregation in non-polar media. Fortunately, the lipophilicity can be adjusted by functionalization and recombination through the rich functional groups on the surface of MXene [22]. For example, Guo et al. [23] used poly [2-(Perfluorooctyl) ethyl methacrylate)] to modify the MXene and as a lubricant additive, the fluorinated MXene significantly improved the friction reduction and wear resistance of perfluoropolyether. Zhou et al. [24] successfully prepared MXene/HS composites with self-dispersion ability in polyalphaolefin (PAO) 10 through hydrothermal reaction. The ideal combination of MXene and HS produced synergistic lubrication effects through physical interactions and tribochemical reactions. Razavi et al. [31] found that adding SiO2 nanoparticles to lithium-based lubricating grease can increase the stiffness of the oil film, reduce direct contact of the friction surface, and improve the tribological and rheological properties of lubricating grease. In this work, a novel layered MXene@SiO2 nanocomposite was prepared by in-situ growing SiO2 nanoparticles on MXene nanosheets through controlled hydrolysis of tetraethylorthosilicate (TEOS). Due to the large number of functional groups at the edge of MXene, SiO2 first grew at the edge of MXene to maintain structural stability, forming a unique non-uniform core-rim structure. The structure evolution and tribological properties of MXene@SiO2 with different SiO2 loadings in base oil 500 SN were investigated, and the lubrication mechanism was also discussed in-detail. 1 Introduction Friction, as one of the most common phenomena, often occurs on the surfaces of two objects with relative motion. Although friction plays a positive role in situations such as vehicle movement and human walking, the energy loss caused by friction is inevitable [1–3]. According to statistics, energy losses caused by friction and wear account for about one-third of global energy consumption [4]. To solve this problem, the liquid lubricants have been applied to various mechanical equipment to reduce direct contact between solids, resulting in low friction and low wear [5]. However, the performance of traditional base oils has always been limited, resulting in poor lubrication performance and low film forming ability [6, 7]. The Over the past two decades, various two-dimensional (2D) materials have been used in tribological research, including MoS2 [9], black phosphorus [10], and graphene and its derivatives [11, 12], etc. Since Gogotsi et al. found MXene in 2011, the family of 2D nanomaterials has been significantly expanded, and broken new ground in tribology [13]. The chemical formula of MXene is Mn+1XnTx (n = 1, 2, 3, 4), where M represents the transition metal (Ti, Nb, Mo, etc.), X represents the C or N element, and Tx represents the end functional group [14–17]. The excellent tribological Friction 12(8): 1728–1740 (2024) 1729 properties of MXene can be traced to their unique layered structure with weak van der Waals forces between layers, which provide a fairly low shear strength for layering [18, 19]. In addition, 2D MXene is prone to adhesion to different surfaces, passivating the contact points and forming a beneficial protective film. However, the most common drawback of 2D MXene as lubricating materials is their inability to work under high contact pressure and sliding speed [20, 21]. In addition, when MXene is used as a lubricating additive in base oil, the hydrophilic groups on the polar surface limit their dispersion stability and cause aggregation in non-polar media. Fortunately, the lipophilicity can be adjusted by functionalization and recombination through the rich functional groups on the surface of MXene [22]. For example, Guo et al. [23] used poly [2-(Perfluorooctyl) ethyl methacrylate)] to modify the MXene and as a lubricant additive, the fluorinated MXene significantly improved the friction reduction and wear resistance of perfluoropolyether. Zhou et al. [24] successfully prepared MXene/HS composites with self-dispersion ability in polyalphaolefin (PAO) 10 through hydrothermal reaction. 2.3 Preparation of MXene@SiO2 composites SiO2 nanoparticles were in-situ grown on MXene nanosheets by hydrolysis of TEOS [33]. Typically, 10 mL of MXene nanosheet dispersion was added to a mixture of 100 mL ethanol and water (1:1) and stirred for 30 min. Then, 6 mL NH3·H2O was added into the above solution, and TEOS with different weights were slowly dropped into the solution, and stirred at room temperature for 6 h to obtain MXene@SiO2 composites with different SiO2 loadings. After the reaction, in order to remove the residual solvent, the product was centrifuged and washed with deionized water. Finally, MXene@SiO2 composite materials were collected by freeze-drying method. The products with TEOS additions of 0.2, 0.4, and 0.6 mL were named MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3, respectively. 2.2 Fabrication of single layer or few layer MXene nanosheets Based on our previous work, single-layer or few-layer dispersions of Ti3C2Tx MXene nanosheets were prepared [32]. First, 1 g of LiF and 20 mL of HCl (9 M) were magnetically stirred in a reaction vessel of polytetrafluoroethylene for 10 min to form a uniform solution. Then, 1 g of Ti3AlC2 MAX powder was slowly added to the mixture and magnetically stirred at 35 °C for 24 h. After the reaction, the mixture was washed by centrifugation using deionized water. First, the solution was washed several times by centrifugation at 4,000 rpm until the upper solution turned black. Then increased the speed to 8,000 rpm, continue www.Springer.com/journal/40544 \| Friction www.Springer.com/journal/40544 \| Friction 1730 Friction 12(8): 1728–1740 (2024) tester (SRV-5). The friction pair consists of a steel ball and a steel disc. The ball (ø 10 mm, hardness: 60 ± 2 HRC, mean roughness: 20 nm) and disc (ø 24 mm × 7.9 mm, hardness: 62 ± 2 HRC) were all made of AISI 52100 steel. Variable load test: under the conditions of amplitude of 1 mm, temperature of 50 °C, and frequency of 25 Hz, the load started at 50 N and increased by 50 N every 5 min. Frequency conversion test: under the condition of amplitude of 1 mm, temperature of 50 °C and load of 150 N. The frequency increased from 5 to 50 Hz, increasing by 5 Hz every 5 min. Variable temperature test: under the condition of amplitude of 1 mm, frequency of 25 Hz and load of 150 N. The temperature rose from 40 to 120 °C, increasing by 10 °C every 5 min. centrifugal washing, and finally increased the speed to 10,000 rpm, continue centrifugal washing until the upper solution turned black and viscous. After centrifugation, the upper layer solution was taken for ultrasonic treatment, and the MXene nanosheets separated from the upper layer were collected by centrifugation. 3 Results and discussion The preparation process of MXene@SiO2 composites was shown in Scheme 1. Ti3C2Tx MXene nanosheets were obtained by selective removal of the Al atomic layer from Ti3AlC2 MAX by in-situ formation of HF etching from a mixed HCl–LiF solution. Afterwards, 2D MXene with single or fewer layers can be obtained by ultrasonication. The H, O, and F atoms in the etching medium bond with the surface unsaturated Ti atoms, forming surface end groups such as –OH, –O, or –F on the surface of MXene, resulting in the final formation of MXene materials with mixed groups on the surface. As shown in Figs. 1(a)–1(d), due to the high number of functional groups at the edge of MXene, it provided a large number of nucleation sites for TEOS hydrolysis. Therefore, when a small amount of TEOS was added, SiO2 preferentially grew at the edge of the MXene layer, forming a unique core-rim structure. As TEOS increased, SiO2 gradually diffused from the edge of the MXene to the surface. \| https://mc03.manuscriptcentral.com/friction 2.4 Characterization Morphological characteristics of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 were characterized by transmission electron microscope (TEM, Talos F200X) and scanning electron microscopy (SEM, Tescan Clara GMH). Fourier infrared spectra (FTIR, Bruker, TENSOR II) was used to measure the surface functional groups of different samples. X-ray diffraction (XRD, Thermo Scientific 7000) patterns were conducted in the range of 5°–90°, and the scanning speed was 5°/min. X-ray photoelectron spectroscopy (XPS, PHI 5000 VersaProbe III) were used to analyze the surface chemical state. The full XPS spectrum range was 0–1,100 eV and the step size was 1 eV. For the narrow spectrum, each element was scanned 5 times and cycled twice, and the step size was 0.125 eV. Using three-dimensional (3D) surface profiler to observe the wear status of scratches. The morphologies of different samples were observed by SEM and TEM. The original precursor of Ti3AlC2 MAX was an irregularly layered bulk material (Fig. S1 in the Electronic Supplementary Material (ESM)) [34]. MXene nanosheets were obtained by sonication after removing the Al layer with HCl–LiF solution (Fig. 1(e)). The TEM image in Fig. 1(i) showed the transparent state of MXene, which indicated that the prepared MXene had a monolayer or few-layer The tribological properties of different additives were studied using a micro-action friction and wear 1731 Friction 12(8): 1728–1740 (2024) Scheme 1 Schematic diagram of the preparation process of MXene@SiO2. Fig. 1 (a–d) Structural changes of MXene@SiO2 with different levels of TEOS. SEM and TEM images of (e, i) M (f, j) MXene@SiO2-1, (g, k) MXene@SiO2-2, and (h, l) MXene@SiO2-3. High angle annular dark field (HAADF) images and e mapping analysis of Ti and Si of (m) MXene@SiO2-1, (n) MXene@SiO2-2, and (o) MXene@SiO2-3. Scheme 1 Schematic diagram of the preparation process of MXene@SiO2. Scheme 1 Schematic diagram of the preparation process of MXene@SiO2. Fig. 1 (a–d) Structural changes of MXene@SiO2 with different levels of TEOS. SEM and TEM images of (e, i (f, j) MXene@SiO2-1, (g, k) MXene@SiO2-2, and (h, l) MXene@SiO2-3. High angle annular dark field (HAADF) images a mapping analysis of Ti and Si of (m) MXene@SiO2-1, (n) MXene@SiO2-2, and (o) MXene@SiO2-3. Fig. 1 (a–d) Structural changes of MXene@SiO2 with different levels of TEOS. SEM and TEM images of (e, i) MXene, (f, j) MXene@SiO2-1, (g, k) MXene@SiO2-2, and (h, l) MXene@SiO2-3. 2.4 Characterization High angle annular dark field (HAADF) images and element mapping analysis of Ti and Si of (m) MXene@SiO2-1, (n) MXene@SiO2-2, and (o) MXene@SiO2-3. www.Springer.com/journal/40544 \| Friction 1732 Friction 12(8): 1728–1740 (2024) structure. The hexagonal crystal structure of MXene was confirmed by selected area electron diffraction [35]. Figures 1(f)–1(h) showed the SEM images of MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 at TEOS addition amounts of 0.2, 0.4, and 0.6 mL. It can be seen that all samples maintained a lamellar structure. As can be seen from the TEM images, the edges of MXene@SiO2-1 and MXene@SiO2-2 were covered with particles and became rough when a small amount of TEOS was added, which indicated that SiO2 nanoparticles first grew at the edge of the MXene nanosheets (Figs. 1(j) and 1(k)). The elemental mapping analysis in Fig. S2 in the ESM determined the elemental composition of MXene@SiO2, including Si, Ti, O, and C elements. Meanwhile, the Ti element was surrounded by Si element, which proved its unique core-rim structure (Figs. 1(m) and 1(n)). Compared with MXene@SiO2-1 and MXene@SiO2-2, the SiO2 nanoparticles in MXene@SiO2-3 were uniformly distributed on the edge and surface of the MXene and their sizes gradually increased. near 3,451.4 cm-1 corresponding to the stretching vibration of –OH. The peaks at 1,626.2 and 561.2 cm-1 were due to C=C and Ti–O bonds, respectively. In addition, the peaks of MXene@SiO2 at 1,089.4 and 466.7 cm-1 were caused by asymmetric or bending vibrations of Si–O–Si and tensile vibrations of Si–O [36, 37]. As the loading amount of SiO2 increased, the peak value of Si–O and Si–O–Si bonds became larger and larger. Figure 2(b) showed the XRD patterns of the different samples, from which it can be seen that all the samples had a distinct characteristic peak below 10°, corresponding to the (002) crystal plane of MXene. After loading SiO2, the position of the (002) peak was gradually shifted to a smaller angle, which was manifested as an increase in the interlayer spacing [38]. The surface chemical states of the MXene@SiO2 composites were further explored by XPS spectroscopy. As shown in Fig. 2(c), the XPS full spectra of MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 showed the appearance of additional Si 2p peaks compared to MXene, which indicated the successful complexation of MXene with SiO2. In Figs. \| https://mc03.manuscriptcentral.com/friction 2.4 Characterization At the same time, the stroke of the steel ball on the steel plate increased sharply, and after reaching stability, the stroke remained at 1 mm (Fig. S6 in the ESM). The COF curves of MXene and MXene@SiO2-1 were similar, with a sudden surge at the end of preloading, but compared to 500 SN, the average COF decreased from 0.180 to 0.140 and 0.141. This was because when the amount of TEOS added was relatively small, only a small amount of SiO2 nanoparticles were present at the edge of MXene. Although it improved the lipophilicity of MXene to some extent, due to its small size and insufficient quantity, MXene still exhibited partial stacking phenomenon. In contrast, MXene@SiO2-2 showed good friction reduction performance, not only avoiding friction peaks, but also maintaining a stable COF during operation, with an average COF of 0.108. The addition of MXene@SiO2-3 also avoided the initial friction peak, but the average COF rose to 0.127 compared to MXene@SiO2-2. This implied that too little or too much SiO2 is not conducive to improving lubrication performance (Fig. 3(c)). As shown in Fig. S4 in the ESM, the dispersion stability of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 in 500 SN were investigated. First, Ti3C2Tx MXene, MXene@SiO2-1, MXene@ SiO2-2, and MXene@SiO2-3 were fully dispersed in the base oil 500 SN through ultrasonic treatment to obtain a stable black mixture, and the concentration of all mixtures was 2.4 wt%. After that, the samples were left to stand to observe the precipitation state. From Fig. S4 in the ESM, we can see that after ultrasonic treatment, all additives can be dispersed in 500 SN to form a uniform mixture. When the samples were left for five days, the color of the upper solution became lighter, indicating that all samples had a small amount of precipitation. After eight days, the upper solution in 500 SN containing MXene turned yellow and a large amount of precipitation appeared, which was due to the large number of hydrophilic groups on the surface of MXene. Compared with MXene@SiO2-1 and MXene@SiO2-2, 500 SN containing MXene@SiO2-3 had more precipitation. This showed that excessive SiO2 will increase the weight of MXene nanosheets, and appropriate SiO2 will occupy part of the hydrophilic groups on the surface of MXene, resulting in an increase in lipophilicity. 2.4 Characterization 2(d) and 2(e), the gray line with circle icon represented the raw measured data, the red line represented the peak fitted data, and the areas filled with different colors The surface functional groups of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 were measured by FTIR spectroscopy, and the results were shown in Fig. 2(a). All samples showed a peak Fig. 2 (a) FTIR spectra, (b) XRD patterns, and (c) XPS full spectra of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3. High-resolution XPS spectra of MXene@SiO2-2: (d) Ti 2p, (e) O 1s, and (f) Si 2p. Fig. 2 (a) FTIR spectra, (b) XRD patterns, and (c) XPS full spectra of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3. High-resolution XPS spectra of MXene@SiO2-2: (d) Ti 2p, (e) O 1s, and (f) Si 2p. \| https://mc03.manuscriptcentral.com/friction \| https://mc03.manuscriptcentral.com/friction 1733 Friction 12(8): 1728–1740 (2024) in the concentration range of 1.2 wt%–2.8 wt%. As shown in Fig. 3(a), when the concentration was too low or high, the COF stabilized and tended to a straight line at the beginning of the friction, whereas the COF gradually rose in the later part of the run, and the average COF reached the lowest value at 2.4 wt% (Fig. S5 in the ESM). This was because when the concentration was too low, a continuous and stable protective film cannot be formed, while when the concentration was too high, excessive sample deposition prevented the formation of a friction film and increased wear on the contact surfaces [41, 42]. To further compare the tribological properties of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3, the COF versus over time were measured at a concentration of 2.4 wt% (Fig. 3(b)). The COF of the base oil 500 SN sharply increased within 100 s, generated a large friction noise and seizing failure occurred. At the same time, the stroke of the steel ball on the steel plate increased sharply, and after reaching stability, the stroke remained at 1 mm (Fig. S6 in the ESM). The COF curves of MXene and MXene@SiO2-1 were similar, with a sudden surge at the end of preloading, but compared to 500 SN, the average COF decreased from 0.180 to 0.140 and 0.141. This was because when the amount of TEOS added was relatively small, only a small amount of SiO2 nanoparticles were present at the edge of MXene. 2.4 Characterization Although it improved the lipophilicity of MXene to some extent, due to its small size and insufficient quantity, MXene still exhibited partial stacking phenomenon. In contrast, MXene@SiO2-2 showed good friction reduction performance, not only avoiding friction peaks, but also maintaining a stable COF during operation, with an average COF of 0.108. The addition of MXene@SiO2-3 also avoided the initial friction peak, but the average COF rose to 0.127 compared to MXene@SiO2-2. This implied that too little or too much SiO2 is not conducive to improving lubrication performance (Fig. 3(c)). Figures 3(d)–3(f) compared the lubrication performance of different samples under extreme represented different peak information. The high- resolution Ti 2p spectrum of MXene@SiO2-2 can accumulate five characteristic peaks corresponding to C–Ti (457.8 eV), Ti 2p3/2 (459.1 eV), Ti–O (461.5 eV), and Ti 2p1/2 (463.9 eV) (Fig. 2(d)). In the O 1s spectrum, the peaks located at 527.6, 530.5, 531.3, and 532.1 eV were caused by lattice O, Ti–O, C–Ti–(OH)x, and Si–O, respectively (Fig. 2(e)). The peaks at 101.5 and 102.1 eV in the high-resolution Si 2p spectrum were attributed to Si–C and Si–O (Fig. 2(f)). As shown in Fig. S3 in the ESM, the peak of C 1 s spectrum consisted of C–Ti (281.9 eV), C–C (284.6 eV), C–OH (286.1 eV), and C=O bonds (288.4 eV) [39, 40]. in the concentration range of 1.2 wt%–2.8 wt%. As shown in Fig. 3(a), when the concentration was too low or high, the COF stabilized and tended to a straight line at the beginning of the friction, whereas the COF gradually rose in the later part of the run, and the average COF reached the lowest value at 2.4 wt% (Fig. S5 in the ESM). This was because when the concentration was too low, a continuous and stable protective film cannot be formed, while when the concentration was too high, excessive sample deposition prevented the formation of a friction film and increased wear on the contact surfaces [41, 42]. To further compare the tribological properties of MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3, the COF versus over time were measured at a concentration of 2.4 wt% (Fig. 3(b)). The COF of the base oil 500 SN sharply increased within 100 s, generated a large friction noise and seizing failure occurred. 2.4 Characterization The coefficient of friction (COF) and wear volume were used to evaluate the anti-friction and anti-wear performance of different samples. The COF curves of all samples over time were measured using SRV-5, and the results were shown in Fig. 3. The concentration of additives significantly affected the lubrication performance of lubricating oil. Therefore, we measured the COF of MXene@SiO2-2 at 150 N, 25 Hz, and 50 °C Figures 3(d)–3(f) compared the lubrication performance of different samples under extreme conditions. Figure 3(d) showed lubrication failure of base oil 500 SN at 100 N. The addition of MXene and MXene@SiO2-1 can increase the carrying capacity of 500 SN to 200 N. This indicated that when there were www.Springer.com/journal/40544 \| Friction Friction 12(8): 1728–1740 (2024) 1734 Fig. 3 (a) COF curves of MXene@SiO2-2 at different concentrations. (b) COF curves and (c) average COF of 500 SN, MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 (2.4 wt%). COF curves of all additives with the change of (d) load, (e) frequency, and (f) temperature. Fig. 3 (a) COF curves of MXene@SiO2-2 at different concentrations. (b) COF curves and (c) average COF of 500 SN, MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3 (2.4 wt%). COF curves of all additives with the change of (d) load, (e) frequency, and (f) temperature. of friction reduction, at a concentration of 2.4 wt%, the wear depth and wear volume of MXene@SiO2-2 were the smallest (Figs. S7–S9 in the ESM). Figure 4 compared the morphology of wear and scratch marks after lubricated by 500 SN, MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3. The wear scratches after lubricated by 500 SN showed wide and deep grooves, and the maximum wear depth was 2.357 μm. The corresponding SEM images clearly showed a large number of grooves and uneven particles on the worn surface. These uneven particles further accelerated the wear of the interface (Figs. 4(a1)–4(a4)). As shown in Figs. 4(b1)–4(b4), after adding MXene to 500 SN, the wear depth decreased to 2.095 μm. However, SEM images showed that there were still quite a few grooves on the surface. The addition of MXene@SiO2-1 slightly increased the wear depth, but the grooves on the wear surface became shallower (Figs. 4(c1)–4(c4)). \| https://mc03.manuscriptcentral.com/friction 2.4 Characterization Compared with 500 SN, the addition of MXene@SiO2-2 resulted in a significant reduction in the wear width and wear depth, with the maximum wear depth decreasing to 1.098 μm, while the wear surface became relatively smooth, with very few and shallow grooves appearing (Figs. 4(d1)–4(d4)). After fewer SiO2 particles, it was difficult to form a friction film that supported the load, so the load resistance will not be significantly improved. The load carrying capacity of 500 SN reached 800 N with the addition of MXene@SiO2-2. While with the addition of MXene@SiO2-3, the load carrying capacity dropped to 400 N. This indicated that appropriate SiO2 can serve as a rolling bearing to form a protective film that can support the load to compensate for the low loading capacity of MXene. When SiO2 was excessive or large in size, it was not conducive to continuous entry into the contact area of the friction pair, resulting in a decrease in load carrying capacity. In different frequency and temperature tests, MXene@SiO2-2 also exhibited the best tribological performance, with the lowest COF and maintaining stability (Figs. 3(e) and 3(f)). This showed that MXene@SiO2-2 can adapt to heavy-duty, high-speed, and high-temperature operating conditions and maintain excellent lubrication performance. In order to further evaluate the wear resistance of different samples, the wear scratches on the lower disk of the friction pair were characterized by 3D profilometer and SEM. Consistent with the results Friction 12(8): 1728–1740 (2024) 1735 Fig. 4 3D images, wear depth, and SEM images on the wear scar of the disk lubricated by (a1–a4) 500 SN, (b1–b4) MXene, (c1–c4) MXene@SiO2-1, (d1–d4) MXene@SiO2-2, and (e1–e4) MXene@SiO2-3. Fig. 4 3D images, wear depth, and SEM images on the wear scar of the disk lubricated by (a1–a4) 500 SN, (b1–b4) MXene, (c1–c4) MXene@SiO2-1, (d1–d4) MXene@SiO2-2, and (e1–e4) MXene@SiO2-3. and 57.1%. Loading appropriate SiO2 can significantly improve the wear resistance of the sample. Compared with 500 SN, after adding MXene@SiO2-2, the wear volume decreased to 1.41×105 μm3, reduced by 73.7%. Figure 5(b) summarized the friction reduction and anti-wear properties of all the samples, and MXene@SiO2-2 outperformed the other samples in all the evaluated metrics (average COF, loading capacity, wear volume, and wear depth), reflecting the superiority of its tribological properties. adding MXene@SiO2-3, the maximum wear depth decreased to 1.382 μm and grooves still appeared on the wear surface (Figs. 4(e1)–4(e4)). 2.4 Characterization In addition, the element mapping analysis of the worn surface after MXene@SiO2-2 lubrication was shown in Fig. S10 in the ESM. C, Ti, Si, O, and Fe elements were uniformly distributed on the worn surface. This indicated that appropriate interaction between SiO2 and MXene can easily form a friction film on the contact surface of steel, promoting sliding by reducing friction, thereby reducing wear trajectory [43]. XPS spectra of wear surface after MXene@SiO2-2 lubrication was investigated to further analyze the chemical composition of the friction protection film and the lubrication process. The full XPS spectrum of Fig. 6(a) showed not only major Fe and O elements, but also the presence of Ti, C, and Si elements, which was consistent with the results of elemental mapping analysis in Fig. S10 in the ESM. As shown in Fig. 6(b), two characteristic peaks of Fe3O4 and Fe2O3 can be The wear volume of the lower disk of the friction pair after lubrication of different samples was shown in Fig. 5(a). The wear volume after base oil lubrication was as high as 5.36×105 μm3. With the addition of MXene, the wear volume dropped to 3.01×105 μm3, reduced by 43.8%. When loading a small or excessive amount of SiO2, the wear volume after MXene@SiO2-1 and MXene@SiO2-3 lubrication was decreased by 44.7% www.Springer.com/journal/40544 \| Friction Friction 12(8): 1728–1740 (2024) 1736 Fig. 5 (a) Wear volume of wear scar on lower disk lubricated by 500 SN, MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3. (b) Comparison of tribological properties of different samples. Fig. 5 (a) Wear volume of wear scar on lower disk lubricated by 500 SN, MXene, MXene@SiO2-1, MXene@SiO2-2, and MXene@SiO2-3. (b) Comparison of tribological properties of different samples. Fig. 6 (a) XPS full spectrum and high-resolution XPS spectra of (b) Fe 2p, (c) O 1s, (d) Ti 2p, (e) C 1s, and (f) Si 2p on the wear scar of the disk. Fig. 6 (a) XPS full spectrum and high-resolution XPS spectra of (b) Fe 2p, (c) O 1s, (d) Ti 2p, (e) C 1s, and (f) Si 2p on the wear scar of the disk. The C1s peak was composed of C–C, C–OH, and C=O bonds (Fig. 6(e)). The peaks of Si 2p at 101.4 and 102.2 eV were caused by Si–C and Si–O (Fig. 6(f)) [43, 44]. 2.4 Characterization Therefore, it can be inferred from the above XPS analysis that MXene@SiO2 can be deposited on the wear surface to form a physical adsorbed film. Due to the high load and high temperature at the sliding interface, MXene@SiO2 induced frictional chemical reaction, forming a chemical protective film mainly composed of TiO2, Fe2O3, and Fe3O4. The physical and chemical film adsorbed on the worn surface and fitted in the Fe 2p peak, which indicated that part of the Fe element was oxidized during the friction process. In the O 1s spectrum, the peaks located at 530.2, 531.9, and 532.6 eV were attributed to Ti–O, Si–O, and Fe–O bonds, respectively. The Fe–O bond corresponded to the oxide of Fe in Fe 2p, and the Ti–O and Si–O peaks originated from MXene and SiO2, respectively (Fig. 6(c)). The XPS spectrum of Ti 2p in Fig. 6(d) consisted of four characteristic peaks, corresponding to Ti–O (457.9 eV), Ti2+ (458.8 eV), Ti 2p1/2 (463.7 eV), and TiO2-xFx (464.9 eV), respectively. \| https://mc03.manuscriptcentral.com/friction Friction 12(8): 1728–1740 (2024) 1737 played an effective protective role on the friction interface [45]. 4 Conclusions In this work, SiO2 modified MXene nanocomposite materials were synthesized via a simple self-growth method. The growth position of SiO2 can be controlled by controlling the addition of tetraethylorthosilicate (TEOS), resulting in the formation of MXene@SiO2 composites with a unique non-uniform core-rim structure. The prepared MXene@SiO2 had good dispersion stability in base oil 500 SN and thus exhibited excellent tribological properties. When added at a concentration of 2.4 wt%, the coefficient of friction (COF) of 500 SN reduced from 0.572 to 0.108, the wear volume decreased from 5.36×105 to 1.41×105 μm3, and the load capacity increased to 800 N. Its excellent tribological properties were due to the synergistic effect of MXene and SiO2. The laminar structure of MXene facilitated interlayer slip during friction, and the spherical structure of SiO2 can be used as a rolling bearing to enhance the load carrying capacity. In addition, the good dispersion stability of MXene@SiO2 ensured a continuous supply of samples to form a stable and continuous protection film to isolate the contact surface of the friction partner. This study enriched the application of MXene-based nanocomposites as lubrication additives and had great potential in mechanical lubrication system. The lubrication mechanism of MXene@SiO2 as an additive in 500 SN was shown in Scheme 2. Because SiO2 improved the lipophilicity of MXene, MXene@SiO2 had good dispersion stability in the base oil 500 SN. Therefore, in the process of friction, MXene@SiO2 can enter the contact area of the friction pair with the flow of the base oil and adsorbed on the wear surface. Under the bidirectional action of pressure and shear force, SiO2 widened the interlayer distance between MXene nanosheets, making it more prone to interlayer slip and fully utilizing the interlayer shear mechanism. SiO2 with high hardness and thermal stability can be rolled into the high-pressure contact area as a rolling bearing to enhance bearing capacity. In addition, MXene@SiO2 broke into smaller fragments to fill in the pits and valleys of the friction vice, smoothing the contact surface and acting as a repair. In the rapid reciprocating operation during friction, the high temperature and high load induced friction chemical reaction to occur, forming a chemical reaction film dominated by Fe oxide and Ti oxide to improve the interaction at the interface. In summary, the good lubricating properties of MXene@SiO2 were attributed to the effect of synergistic lubrication. Scheme 2 Anti-friction and anti-wear mechanism of MXene@SiO2 composites. Declaration of competing interest [5] Zhai W Z, Zhou K. Nanomaterials in superlubricity. Adv Funct Materials 29(28): 1806395 (2019) The authors have no competing interests to declare that are relevant to the content of this article. The authors Feng ZHOU and Weimin LIU are the Editorial Board Members of this journal. [6] Jin B, Chen G Y, Zhao J, He Y Y, Huang Y Y, Luo J B. Improvement of the lubrication properties of grease with Mn3O4/graphene (Mn3O4#G) nanocomposite additive. 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She is currently a doctoral student in the Center of Advanced Lubrication and Seal Materials at School of Materials Science and Engineering, Northwestern Polytechnical University. Her research interest is MXene nanosheets as lubricant additives. (2010). He spent one year (2013–2014) at Université catholique de Louvain as a research associate. His research interests focus on functional nanomaterials, surface modification, lubricant coating, and anti-fouling materials. His work has been published in more than 80 peer-reviewed papers with a current h-index of 32. Qian YE. He is an associate professor at Northwestern Polytechnical University. He received his B.S. degree in Lanzhou University (2005), and got his Ph.D. degree in organic chemistry at Lanzhou University the high-index of 88. His research interests include the bioinspired tribology, biomimic surfaces/ interfaces of soft matters, drag-reduction and antibiofouling, and functional coatings. He has gained a number of awards including “Outstanding Youth Award” of International Society of Bionic Engineering, 2013, and one National Award for Natural Sciences (the second class). References Adv Colloid Interface Sci 309: 102792 (2022) [23] Guo J L, Wu P X, Zeng C, Wu W, Zhao X Y, Liu G Q, Zhou F, Liu W M. Fluoropolymer grafted Ti3C2Tx MXene as an efficient lubricant additive for fluorine-containing lubricating oil. Tribol Int 170: 107500 (2022) [35] Cui Y H, Yang K, Wang J Q, Shah T, Zhang Q Y, Zhang B L. Preparation of pleated RGO/MXene/Fe3O4 microsphere and its absorption properties for electromagnetic wave. Carbon 172: 1–14 (2021) [24] Zhou C Z, Li Z P, Liu S W, Ma L M, Zhan T R, Wang J Q. Synthesis of MXene-based self-dispersing additives for enhanced tribological properties. Tribol Lett 70(2): 1–13 (2022) [36] Gong K L, Yin L, Shi C L, Qian X D, Zhou K Q. Dual char-forming strategy driven MXene-based fire-proofing epoxy resin coupled with good toughness. 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Compos Sci Technol 169: 52–59 (2019) www.Springer.com/journal/40544 \| Friction 1740 Friction 12(8): 1728–1740 (2024) [39] Zhang Y Y, Chen P, Wang Q Y, Wang Q A, Zhu K, Ye K, Wang G L, Cao D X, Yan J, Zhang Q A. High-capacity and kinetically accelerated lithium storage in MoO3 enabled by oxygen vacancies and heterostructure. Adv Energy Mater 11(31): 2101712 (2021) [42] Xie H J, Wang Y X, Wang P, Liu S J, Ye Q, Liu W M. Poly(tannic acid)-functionalized onion-like carbon nanoparticles derived from candle soot serving as potent lubricant additives. \| https://mc03.manuscriptcentral.com/friction References He serves as an editorial board member of Tribology International, Journal of Fiber Bioengineering and Informatics, and Friction, etc. Feng ZHOU. He is a full professor in Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, China, and the director of State Key Laboratory of Solid Lubrication. He gained his Ph.D. degree in 2004 and spent three years (2005–2008) in the Department of Chemistry, University of Cambridge as a research associate. He has published more than 300 journal papers that received more than 25,000 citations and has \| https://mc03.manuscriptcentral.com/friction
https://openalex.org/W3011698992	https://www.mdpi.com/2071-1050/12/6/2244/pdf	English	null	Consumers’ Perceptions and Attitudes toward Products Preventing Microfiber Pollution in Aquatic Environments as a Result of the Domestic Washing of Synthetic Clothes	Sustainability	2,020	cc-by	9,742	Article Article Received: 30 January 2020; Accepted: 11 March 2020; Published: 13 March 2020 Abstract: Microﬁbers released by synthetic clothes have a signiﬁcant negative eﬀect on the environment. Several solutions have been proposed and evaluated for their eﬀectiveness, but studies have failed to address the human-centered aspects of these products. In this research, the possibilities and needs from a consumer perspective for a new ﬁltering system for domestic washing machines were examined. First, a quantitative (questionnaire) and a qualitative (interviews and observations) exploration were done to understand the desired requirements from a user perspective. Next, the acceptance of various existing solutions for microﬁber catching was investigated. To verify these requirements, a new concept was designed and evaluated with a questionnaire. The results were analyzed using descriptive statistics. It can be concluded that the problem of microﬁbers is not well known, and the impact of people’s washing behavior is underestimated. Since microﬁbers are almost invisible, the eﬀectiveness needed to be proven. Eﬀectiveness is seen as the most important characteristic of a product that captures microﬁbers, followed by durability. Both factors ensure long-term usage. However, changing washing habits is not evident, and usage should be straightforward and user-friendly to save time, especially considering the new cleaning actions, which should be clear and unambiguous. Abstract: Microﬁbers released by synthetic clothes have a signiﬁcant negative eﬀect on the environment. Several solutions have been proposed and evaluated for their eﬀectiveness, but studies have failed to address the human-centered aspects of these products. In this research, the possibilities and needs from a consumer perspective for a new ﬁltering system for domestic washing machines were examined. First, a quantitative (questionnaire) and a qualitative (interviews and observations) exploration were done to understand the desired requirements from a user perspective. N t th t f i i ti l ti f i ﬁb t hi i ti t d T if Keywords: microﬁbers; plastic pollution; laundry and maintenance; awareness; microﬁber ﬁlter; consumer perception Els Du Bois 1,* 1 Department of Product Development, Faculty of Design Sciences, University of Antwerp, 2000 Antwerpen, Belgium; laure.herweyers@uantwerpen.be (L.H.); linda.scheelen@uantwerpen.be (L.S.); regan.watts@uantwerpen.be (R.W.) 1 Department of Product Development, Faculty of Design Sciences, University of Antwerp, 2000 Antwerpen, Belgium; laure.herweyers@uantwerpen.be (L.H.); linda.scheelen@uantwerpen.be (L.S.); regan.watts@uantwerpen.be (R.W.) 1 Department of Product Development, Faculty of Design Sciences, University of Antwerp, 2000 Antwerpen, Belgium; laure.herweyers@uantwerpen.be (L.H.); linda.scheelen@uantwerpen.be (L. regan.watts@uantwerpen.be (R.W.) g p ( ) 2 Department of Biology, Faculty of Sciences, University of Antwerp, 2000 Antwerpen, Belgium; camilla.catarcicarteny@uantwerpen.be 2 Department of Biology, Faculty of Sciences, University of Antwerp, 2000 Antwerpen, Belgium; camilla.catarcicarteny@uantwerpen.be y p * Correspondence: els.dubois@uantwerpen.be; Tel.: +32-32658893 Sustainability 2020, 12, 2244; doi:10.3390/su12062244 sustainability sustainability sustainability sustainability Consumers’ Perceptions and Attitudes toward Products Preventing Microﬁber Pollution in Aquatic Environments as a Result of the Domestic Washing of Synthetic Clothes Laure Herweyers 1 , Camilla Catarci Carteny 2 , Linda Scheelen 1, Regan Watts 1 and Els Du Bois 1,* 1. Introduction Plastics are the primary source of litter found in oceans and inland waters [1]. Besides large plastic items that mostly ﬂoat on the surface, microscopic plastic particles (<5 mm in their largest dimension) cause considerable environmental problems to the oceans [2]. One speciﬁc type of microplastics are microﬁbers released from the washing of synthetic fabrics [3], which are nonrenewable and nonbiodegradable and take up more than 60% of global ﬁber consumption [4,5]. In this paper, “microﬁbers” are ﬁbers from a synthetic source. Currently, most wastewater treatment plants are not equipped with tertiary treatment and ﬁlter out only around 50% of microﬁbers [6]. Even the most advanced wastewater treatment plants are not able to capture all the microﬁbers [7,8]. Consequently, signiﬁcant amounts of microﬁbers end up in the oceans [2]. In this research, the possibilities and www.mdpi.com/journal/sustainability Sustainability 2020, 12, 2244; doi:10.3390/su12062244 www.mdpi.com/journal/sustainability 2 of 14 Sustainability 2020, 12, 2244 needs for a ﬁltering device for domestic washing machines aiming to prevent microﬁbers released by synthetic clothes from reaching the environment are examined. 1.2. Tackling Microﬁber Pollution Removing the ﬁbers from contaminated sources is a very expensive and diﬃcult task [4], thus tackling this problem at the source is desirable. Microﬁber pollution can be stopped at multiple levels: the production of textiles, the fabrication, usage, and washing of synthetic garments, and wastewater treatment plants among others [4]. The most advanced wastewater treatment plants (WWTPs) stop more or less 90% of the microplastics [7,8,11], although none of the WWTPs that exist now are speciﬁcally designed to capture microplastics [16]. On top of that, most WWTPs are not equipped with a tertiary treatment, which means they stop only around 50% of all passing microﬁbers [6]. At the moment, the WWTP in Flanders, which is taken as our ﬁeld of investigation, is not adjusted to the removal of microplastics from wastewater [17]. In developing countries, with less infrastructure, it is common that wastewater is not collected and treated [18]. Even if the WWTP were completely eﬀective in removing all microﬁbers from the wastewater, the extracted microplastics often still enter the environment through the sludge being employed as soil fertilizer [5,11,19,20]. Concentrations of microplastics in the seabed where sewage sludge was dumped until 1998 in the UK were three times higher than at other reference sites [3]. In conclusion, this means that WWTPs, as well as untreated sewage, septic tanks, or greywater, can be side-pathways into the environment for microﬁbers [7,21]. On the European scale, the inﬂuence of industrial laundry is smaller than the sum of laundry done in all households [8]. Thus, we made the choice to focus on intervening at the level of domestic washing. Furthermore, this solution can increase the microplastic issue awareness among consumers, in contrast with solutions focusing on industrial laundry or wastewater treatment plants. 1.1. Microﬁbers The most common synthetic microﬁber materials are polyamide 6, polyethylene terephthalate (polyester), and polypropylene [4,9]. Polyester is the dominant microﬁber pollutant, with an annual production of 70 million tons in 2018 [4]. The main sources of microﬁber pollution are domestic laundry, the textile industry, landﬁlls, domestic drainages, and dumping of synthetic clothes in rivers or oceans [4]. This research is focused on household laundry. It is known that polyester, polyamide, and acrylic garments release the most microﬁbers. [10] In a typical washing load, the number of released ﬁbers can vary greatly, depending on the content of the load [8,11]. Fleece fabrics (also polyester) release up to two million ﬁbers [8]. Although synthetic–natural blends might release fewer ﬁbers than pure polyester [12], due to their combined properties [11], mixing materials is not desirable for future recycling possibilities [13]. The known textile parameters inﬂuencing microﬁber shedding are ﬁber type (diameter, length, tensile strength), yarn type (spinning type, yarn structure), fabric type (composition, structure, weight) and the chemical or mechanical ﬁnishing [5,8]. The construction quality of the garments is an additional important factor, having fewer ﬁbers released when the quality is higher [5,14,15]. Although natural materials (such as cotton) and artiﬁcial materials from a natural source (e.g., viscose) degrade in natural water and are not as harmful as synthetics, they can contain toxic additives and dyes that may be harmful to the environment [5,14,16]. Since cotton and other non-synthetic ﬁbers often release even more ﬁbers than synthetics during a typical washing cycle [10,16], it is interesting to also consider capturing other types of ﬁbers. 1.4. Outline 1.4. Outline The research aims to investigate the possibilities and needs from a consumer perspective of a ﬁltering device for domestic washing machines. This paper reports upon the criteria for the adoption and acceptance by potential consumers of a new design concept for catching microﬁbers in a domestic context. Both qualitative and quantitative research was done to understand the critical aspects of the design and the attitude of people toward the idea of the product, as well as the evaluation of existing products. Evaluating these products from a consumer and behavioral perspective is important to explore the likelihood of adoption, thus the potential success of the proposed solutions [26]. In summary, the questions that we aim to answer are: How aware are people of the microﬁber problem? What action do people already take? How do people critique existing solutions? What do people consider the most relevant properties concerning a new design? What variables can convince people to buy a product? We initiated a Design Inclusive Research process in order to translate the resulting insights into a new design and test this concept against the nuances of our understandings. The goal of including design into the research process is to create new opportunities for generating knowledge [27]. Design Inclusive Research combines analytic research methods with synthetic/constructive design methods. Consequently, the research approach is composed of three phases as visualized in Figure 1: (i) explorative research actions (analysis), (ii) creative design actions (synthesis), and (iii) evaluative research actions (evaluation). The research aims to investigate the possibilities and needs from a consumer perspective of a filtering device for domestic washing machines. This paper reports upon the criteria for the adoption and acceptance by potential consumers of a new design concept for catching microfibers in a domestic context. Both qualitative and quantitative research was done to understand the critical aspects of the design and the attitude of people toward the idea of the product, as well as the evaluation of existing products. Evaluating these products from a consumer and behavioral perspective is important to explore the likelihood of adoption, thus the potential success of the proposed solutions [26]. In summary, the questions that we aim to answer are: How aware are people of the microfiber problem? What action do people already take? How do people critique existing solutions? What do people consider the most relevant properties concerning a new design? 1.3. Inﬂuence of Household Laundry Washing machine conditions have a considerable inﬂuence on the number of ﬁbers released. Synthetic fabrics become soiled faster, and are therefore washed more frequently [22]. The mass of recovered ﬁbers is signiﬁcantly greater for top-loader than front-loader machines. While top-loader machines are most common in North America, the front loader machine is dominant in Europe [21], where 87% of European citizens own a washing machine and 3% go to a laundromat. In developing 3 of 14 3 of 14 Sustainability 2020, 12, 2244 Sustainability 2020, 12, x FO countries, such as Brazil, China, and India, over 30% of residents use hand washing. However, a growth of washing machine usage is predicted in the near future [23]. growth of washing machine usage is predicted in the near future [23]. During domestic washing, both mechanical and chemical factors can influence fiber shedding: mechanical influential factors are temperature cycle axis direction rotations per minute the During domestic washing, both mechanical and chemical factors can inﬂuence ﬁber shedding: mechanical inﬂuential factors are temperature, cycle, axis direction, rotations per minute, the quantity of water, and capacity of the machine. Chemical inﬂuencers are the type and quantity of detergent, the use of fabric softener, and the presence of other products [5,8,24]. Most ﬁbers are released during the ﬁrst washing of the garment. After that, the ﬁber shedding decreases and stabilizes [8,11,14,25]. When the piece starts to wear down, the ﬁber release will start increasing again [15,21]. mechanical influential factors are temperature, cycle, axis direction, rotations per minute, the quantity of water, and capacity of the machine. Chemical influencers are the type and quantity of detergent, the use of fabric softener, and the presence of other products [5,8,24]. Most fibers are released during the first washing of the garment. After that, the fiber shedding decreases and stabilizes [8,11,14,25]. When the piece starts to wear down, the fiber release will start increasing again [15,21]. 2.1.1. Quantitative Study with Survey A structured survey was done using the Qualtrics platform (Utah, USA). In total, we received 434 answers, of which 411 were valid. Invitations to participate to the survey were sent out in Flanders (Belgium) by email or social media with a link to the questionnaire. The sampled population consists of Flemish respondents of all ages. The survey was deliberately not distributed among product developers nor design engineering practitioners and students since they could inﬂuence the validity of the sample, as it is assumed that designers have a diﬀerent view and approach toward product semantics [28]. Snowball sampling [29] was used to recruit a large number of participants at low cost. In order to reach a broad range of socio-economic groups, people of diﬀerent education and income classes were asked to distribute the survey further among their acquaintances. However, we are aware of the fact that, when using this technique sample, representativeness is not ensured and should be discussed further to deﬁne what level of generalization can be done to which population with any level of conﬁdence [30]. Within the respondents, 69% was female and 31% male; 87% of the female respondents do laundry, while among the men, only 55% do their own laundry. This variation is comparable to existing data on washing behavior as reported by Nielsen (2016) [23]. Between age 26 and 40, 98% of women and 86% of men do their laundry themselves, while within the age group of 41–60 years old, 60% of men and 99% of women do their own laundry. The survey was divided into two main sections. In the ﬁrst section, the aim was to get insights in consumer awareness on the problem of microﬁber pollution and the impact of their washing habits. At ﬁrst, general demographic multiple-choice questions were asked—i.e., age and gender—to monitor equal distribution. Next, several questions were asked about laundry habits and ecological behavior regarding the washing of clothes—for example, about washing temperatures and the frequency people run their washing machine on a non-full load. Furthermore, the knowledge and awareness of participants about the plastic soup and microﬁber pollution were investigated, asking how important they considered these problems. In previous studies, it is shown that better knowledge of environmental issues can, although in a limited way, inﬂuence people’s ecological buying behavior [31–33]. Consequently, we were interested in discovering how these understandings would inﬂuence people’s washing behavior. 1.4. Outline 1.4. Outline What variables can convince people to buy a product? We initiated a Design Inclusive Research process in order to translate the resulting insights into a new design and test this concept against the nuances of our understandings. The goal of including design into the research process is to create new opportunities for generating knowledge [27]. Design Inclusive Research combines analytic research methods with synthetic/constructive design methods. Consequently, the research approach is composed of three phases as visualized in Figure 1: (i) explorative research actions (analysis), (ii) creative design actions (synthesis), and (iii) evaluative research actions (evaluation). Figure 1. Outline of the research approach. Figure 1. Outline of the research approach. Figure 1. Outline of the research approach Figure 1. Outline of the research approach. 4 of 14 Sustainability 2020, 12, 2244 This research was part of a larger project in which, in addition to this behavior study, research was done to investigate the technical possibilities to catch microﬁbers when washing clothes. Both research actions happened simultaneously and supported the ﬁnal design of the new concept. 2.1.1. Quantitative Study with Survey In the second section, the aim was to investigate the critical aspects for ﬁber capturing products to change the attitude of people. Therefore, the participants were asked to give their thoughts on existing solutions for microﬁber pollution from domestic laundry. The questions were mostly multiple choice (single answer or multiple answer), with two Likert scales and one rank order. Based on these questions, the aim was to consider from a designerly perspective what the most ideal (i.e., user friendly and eﬀective) solutions are for catching microﬁbers in domestic washing. 2.1.2. Products Included in This Research 2.1. Explorative Research Approach The executed research is twofold. First, a quantitative study was executed. Next, an in-depth qualitative study was done to understand the reasonings and details behind the ﬁndings of the ﬁrst study. 2.1.2. Products Included in This Research Several novel solutions for ﬁltering out microplastics in domestic washing machines have been developed in the last few years. Three varying products that were on the market during the time of 5 of 14 Sustainability 2020, 12, 2244 this research were used in the last section of the survey: Cora Ball [34], Guppyfriend [35], and Filtrol 160 [36]. Besides this, more products that tackle this problem already existed but were not on the market yet and thus not included in the research. Examples are XFiltra [37], PlanetCare [38], and FiberFree [39]. this research were used in the last section of the survey: Cora Ball [34], Guppyfriend [35], and Filtrol 160 [36]. Besides this, more products that tackle this problem already existed but were not on the market yet and thus not included in the research. Examples are XFiltra [37], PlanetCare [38], and FiberFree [39]. • The Cora Ball (Vermont, USA) is inspired by the ability of corals to capture tiny particles from the water. It is placed in the washing machine tub with the clothes, where it captures released ﬁbers. After washing, the ﬁbers must be removed from the Cora Ball by hand. The products costs 29.99 euros, and independent research concluded it captures on average 26% of microﬁbers [26]. • The Guppyfriend (Germany) is a laundry bag in which the synthetic clothes are put during washing. A ﬁne polyamide mesh captures the ﬁbers. The ﬁbers must be removed by hand after every wash cycle. The product costs around 30 euros. • The Filtrol 160 (Maine, USA) is an external ﬁlter located at the drain of the washing machine. Every 8 to 15 washing cycles the ﬁlter bag must be cleaned by turning the bag upside down, after the residue has had time to dry ﬁrst. The manual suggests emptying the bag outside, thus spreading the microplastics into the environment. The bag will last 1–2 years. The complete product with one ﬁlter bag costs 120 euros and is only available in the United States of America. The main purpose of the Filtrol 160 is protecting the septic tank: capturing microﬁbers is an extra feature. Since the Filtrol 160 was only available in the United States of America and the Cora Ball had a production delay at the time, only the Guppyfriend was included in the qualitative research part. 2.1.3. Qualitative Observation and In-Depth Interview 2.1.3. Qualitative Observation and In-Depth Interview In the second phase of the research, qualitative research was done in order to get more in-depth answers to the following questions: (R1) Who is aware of the microﬁber problem and to what extent? (R2) What is the diﬀerence in the perception of people who already act sustainably in their daily lives versus people who do not? (R3) What are the reasons to (not) be willing to use a product that tackles microﬁber pollution? (R4) What are the main problems with the Guppyfriend (a washing bag for synthetic clothes) regarding user experience, eﬀectiveness, price-quality, and time consumption? (R5) What are the relevant properties the new product should have and is the most eﬀective way to convince people to use the new product? A semi-structured interview was constructed to ask people about their awareness of the problem, their knowledge of the Guppyfriend and whether they considered it a good solution. It consisted of a ﬁxed order of (open) questions, so the interviewees could make suggestions and comments, or explain themselves further. The test subjects were asked to test the Guppyfriend in a real-life situation with their own standard washing load and to ﬁll in a task sheet; in the meantime, they were observed, and comments were noted. After the test was ﬁnished, they were interviewed further to evaluate their experience with the Guppyfriend. The testing experience ended with general questions about what they would expect from the new product and how they could be convinced to buy it. In total, eight observations plus interviews were done, with a gender distribution of ﬁve women and three men and an age variation of 18–25 (2), 25–60 (3), 60+ (3). Before use, ﬁve out of eight people were not aware of the microﬁber problem, which is comparable with the results of the quantitative survey. None of the respondents had ever heard of the Guppyfriend or knew its function. Most of them thought it was a washing bag for delicate clothes. This increases the validity of the observations and interviews as people had no previous experience. The “grounded theory” [40] was applied to analyze the results; in this way, results could be analyzed in a reliable way. The answers were coded and put into categories. This process consists of three phases—open coding, axial coding, and selective coding. Atlas was used to create the codes. The structured questions (mostly using the Likert scale) were analyzed semi-quantitatively. 2.3. Evaluation The developed concept was used in a ﬁnal evaluative survey and distributed among a wide range of people. The survey was distributed in three languages: Dutch, English, and French. The aim of the survey was to identify what people think of the new product concept. What is the degree of acceptance? Who would buy the product at the predetermined price? What payment formula is the most desirable? Price was used as a variable to get insights into people’s willingness, as it was mentioned as a crucial factor during the exploration. A structured interview was made in Qualtrics. The survey was distributed online and received 227 valid answers. The questions were mostly multiple choice (single) and two sliders. The sample consists of Belgian and international respondents of all ages. The ﬁrst blocks asked for demographic data and the eco-friendly behavior level of the respondents. Next, the product was presented and ﬁnally, the respondents were asked whether they would buy it or not. 3. Results 3.1. Explorative Quantitative Study 3.1.1. Consumer Awareness Regarding Microﬁber Pollution and the Impact of Their Washing Habits Concerning the respondents’ washing behavior, most people (55,4%) wash mostly at 40 ◦C; 37% wash at 30 ◦C; and 7% at 60 ◦C. Only 0.6 % wash regularly at 90 ◦C, and 57% of respondents always wash a full machine. Regarding the awareness of the plastic soup and microﬁber pollution issues, 62% of people over 60 years old are not aware, while 73% of respondents aged between 25 and 40 are aware. This means people over 60 are the least aware compared to other age groups, and people between 25 and 40 are most aware. This is in line with other studies [41]. In total, 68% of the respondents know of the plastic soup problem. In contrast, only 37% know about the existence of microﬁbers, and its relation to the problem. Respondents under the age of 25 are the least aware of the synthetic ﬁber problem (29%). People age 41–60 and above 60 are most informed about the problem, but still, less than half of the respondents in those age categories know that synthetic ﬁbers can cause a problem (43%). 2.2. Design and Synthesis Based on the ﬁndings of the explorative phase, a list of requirements was put together as a basis for the design of a new concept. The purpose of the design was to verify the conclusions of the exploration as a whole and explore new possibilities to learn. Moreover, in this design-inclusive research project, the design activities were also used as reﬂective practice to reason upon the conclusions of the exploration. 2 3 Evaluation 2.1.3. Qualitative Observation and In-Depth Interview Before usage, questions were asked regarding the problem of microﬁber pollution and its urgency as well as about the test subjects’ ﬁrst impression of the eﬀectivity of the product and the manner of 6 of 14 Sustainability 2020, 12, 2244 cleaning. During product use, participants were asked to ﬁll in a task sheet to allow post evaluation. Questions included the clearness of the manual, the amount and type of clothes that were used for the experiment, whether the clothes ﬁtted within the Guppyfriend bag, and what type of washing detergent was used (liquid, powder, etc.). After usage, questions were asked about the cleanness and smell of the clothes, the actual cleaning of the product, and the overall evaluation of the complete user journey. 3.1.2. Evaluation of Existing Products Based on the participants’ evaluation of the existing products, we found that the most relevant indicated property is eﬀectiveness (1). Durability (2) comes in second place. Usability (3) and price (4) are considered more important than extra time (5) spent. Based on this list, we could conclude that time is considered less relevant compared to the other aspects. Yet, it is important to note that 75% of respondents also indicated that they do not want to spend more than ﬁve minutes extra on the product per washing cycle. Appearance (6) is the least important for the majority (78%). Besides, it is interesting to see that, in another question price (48%), usability (18%) and convenience (13%) 7 of 14 Sustainability 2020, 12, 2244 are considered the most important factors to decide on buying or not buying a microﬁber ﬁltering product. This suggests price is more important to respondents during the purchase decision, whereas eﬀectiveness, durability, and usability are more important during usage. Detailing the durability of the product, almost unanimously, 91% of the respondents want a reusable solution. Awareness creation and supporting people’s sustainable behavior is considered as an important (side)function of these types of products. This was conﬁrmed in the study, as on average, people consider it as important (4/5) to see or know the impact they have on the environment by using the solution. On the other hand, there seemed to be less need to share the environmental impact of the ﬁlter with other people (only 1/5 score by most people). In contrast to our expectations, there is no signiﬁcant diﬀerence between the diﬀerent age categories. Regarding the physical installation of their washing machine, the drain of the washing machine is easily accessible for 77% of the respondents, which means that 23% (almost a quarter) cannot reach the drain on a frequent basis or after complete installation. Consequently, for those people, the solution oﬀered by removable cartridges mounted at the drain, such as the Filtrol 160, is not applicable. Considering the economic motivator or demotivator of cost, 42% of the respondents are willing to pay 20 to 50 euros for a solution that lasts 3–5 years. 23% does not want to spend more than 20 euros and only 12% would pay more than 100 euros. A ﬁnancial advantage when purchasing the product is mostly chosen by people that are willing to pay 20–50 euros and less than 20 euros. 3.1.2. Evaluation of Existing Products Since they do not want to pay too much for the solution, they welcome a ﬁnancial advantage. To conclude, 68% of the respondents that do not want to pay more than 20 euro on a solution chose the Cora Ball, because of the low price and its convenience. 23% chose the Filtrol 160, which is a lot more expensive than the Cora ball but gives a more eﬀective and durable impression, conﬁrming the importance of eﬀectivity as stated earlier. Of the respondents that are willing to pay more than 100 euros, 69% chose the Filtrol 160, which is the most expensive solution, although 26% would choose the Cora ball and only 5% the Guppyfriend. Overall, the Guppyfriend is the least favorite solution (16% in total), and the Cora Ball the most popular (49%). From these results, it can be stated that the Filtrol 160 is considered the most eﬀective and durable, but a lot of people are held back by the high price and lack of user-friendliness. The Cora Ball is the most user-friendly and convenient, but people are skeptical about the eﬀectiveness. Next to the functionality and requirements regarding the product itself, external measurements that could persuade people to adopt the product are examined. From an economic perspective, we found that when asked for a desirable reward for their ecological behavior, 49% of the respondents choose a ﬁnancial advantage when purchasing the product (for example a reduction in price). After that follows a donation to an environmental NGO (23%) and receiving a free product when purchasing the solution (16%). A ﬁnancial advantage when collecting microﬁbers (9%) and a voucher (3%) are less popular options. 3.2. Explorative Qualitative Study Based on the qualitative research, the following results came out. When asked on what would be the best way to make people use the product, seven out of eight responded said it is most important to make people aware of the problem, thus focusing on expanding knowledge and literacy. During use, most people use ﬂuid detergent (6/8), 3/8 use powder, and 2/8 use capsules. Only one person reported her clothes were less clean in the Guppyfriend, and they all stated their clothes smelled the same as the clothes washed without the Guppyfriend. For half of the respondents, all their synthetic clothes from the washing load ﬁt into the Guppyfriend. The other half had to leave some clothes out of the bag. Four people reported the Guppyfriend was too small to ﬁt all their synthetic clothes. Some suggested making a ﬁlter(bag) that includes all of the clothes, for example around the drum. Someone else said the Guppyfriend should be a lot bigger because otherwise they would have to wash their synthetic clothes in a half-empty machine. Most people were positive about the manual (average of 8/10 considered the manual clear). However, some reported ambiguity with the number of clothes that can ﬁt in the Guppyfriend. The 8 of 14 Sustainability 2020, 12, 2244 manual says at one point “3 medium size clothes” and at another point “maximum half full,” which does not always mean the same. Furthermore, it was not always clear what materials should be put inside the Guppyfriend, as it was mentioned that only synthetics should be included. However, not everyone knows which materials are synthetic. Although everyone knew cotton is a natural material, there was some confusion about viscose and cellulose, which are artiﬁcial but not synthetic materials. Two people reported they cut out the labels after buying the pieces, so they could not know which clothes to put inside the Guppyfriend. Evaluation of the manual is important to get an idea of how intuitive the product is and how much information should be provided. Furthermore, respondents were asked how they would intuitively clean the Guppyfriend. Several people (3) answered ‘rinsing under the tap’, which would cause a counterproductive eﬀect. However, after a ﬁrst thought, they quickly reconsidered and answered they would turn the Guppyfriend inside out to take out the ﬁbers and put them in the bin (residual waste). 3.2. Explorative Qualitative Study Two people had questions about the destination of the ﬁbers: “Are they burned? Where do I put them? Recyclable waste, residual waste, or perhaps hazardous waste?” One person asked whether the ﬁbers are recycled and how that could be possible. p When cleaning the Guppyfriend, two people could not ﬁnd any ﬁbers at all. Five people reported the ﬁbers were too small and not visible enough. Mostly the older interviewees could not see the ﬁbers well. One of them used a magnifying glass. Because of the size of the ﬁbers, the results did not look very spectacular. This demotivated several people. They stated one really has to believe in the impact. Not everyone reacted the same way: one person stated he would not use the Guppyfriend since it does not seem to capture any ﬁbers. However, another one said although they could not see many ﬁbers in the bag, they believed in the impact and would continue using it. Six out of eight respondents found the cleaning of the Guppyfriend diﬃcult and annoying. The two remaining respondents did not ﬁnd any ﬁbers. The main reasons for complaints were the small ﬁber size, the diﬀusion of the ﬁbers over the whole bag and the disposal of the ﬁbers. A lot of ﬁbers were thrown on the ground or fell on the ground by accident as it was not clear where the ﬁbers were after washing. Some people collected the ﬁbers on a piece of paper, after which they threw the whole package in residual waste. Someone suggested using disposable ﬁlters. Seven out of eight respondents stated they would “sometimes” use the Guppyfriend in the future. Two said they might forget to use it after a while. The overall score given to the usability of the Guppyfriend was 5/10. Seven out of eight respondents thought informing people and making them aware of the negative eﬀects of microplastic ﬁber pollution was the most important aspect to motivate people. Six respondents wanted to see their impact on the environment. The order of relevant properties was almost the same as at the survey. Only usability and durability were interchanged. Providing proof of the eﬀectiveness of the solution remains a key factor in convincing people to buy our product. It was mentioned by all eight respondents. 3.3. Preliminary Conclusion Based on the Exploration 3.2. Explorative Qualitative Study At last, all respondents stated they would prefer a diﬀerent solution: either a product that ﬁlters all the wastewater (2/8), such as the Filtrol 160, or a device built into the machine (6/8). One person suggested shifting from consumer to industrial laundry, but, as previously mentioned, in the literature, it is found that the impact of all household laundry is bigger than that of industrial laundry. 3.3. Preliminary Conclusion Based on the Exploration Based on the explorative research actions, it can be concluded that • Besides providing a product, also information should be given. People need to be made aware of the problem and expand their knowledge. • Besides providing a product, also information should be given. People need to be made aware of the problem and expand their knowledge. • The most important property is visible eﬀectiveness and proof that the solution works. Therefore, the visibility of the ﬁbers and/or the contact of the users with the ﬁbers seemed to be given as crucial arguments. • The most important property is visible eﬀectiveness and proof that the solution works. Therefore, the visibility of the ﬁbers and/or the contact of the users with the ﬁbers seemed to be given as crucial arguments. • Usability is the second most important factor, with a speciﬁc focus on convenience—(i) people should be able to wash all their clothes with the product, so they do not need to think about the • Usability is the second most important factor, with a speciﬁc focus on convenience—(i) people should be able to wash all their clothes with the product, so they do not need to think about the 9 of 14 Sustainability 2020, 12, 2244 textile material; (ii) people do not easily change their washing behavior; as such, if the eﬀort would be too large, they would probably abandon the product and revert to their old washing routine. textile material; (ii) people do not easily change their washing behavior; as such, if the eﬀort would be too large, they would probably abandon the product and revert to their old washing routine. • As the cleaning process is a new step in their washing habits, this phase should be easy and clear. People should not be able to do it the wrong way - as their ﬁrst reaction is often to rinse it under the tap, which will allow the ﬁbers to enter the wastewater again. 3.4.1. User-Related Design Requirements 3.4.1. User-Related Design Requirements The following user-related requirements are extrapolated from the results of the explorative research: • The operating and cleaning processes should be short and unambiguous; • The operating and cleaning processes should be short and unambiguous; • The product or marketing of the product should raise awareness of the problem; • The product should be accessible from the front of the machine; • The impact should be visible for the user(s), to keep them motivated; • The scientiﬁcally proven eﬀectiveness should be communicated objectively; • The product should be durable and last for several years; • The cleaning process should not rely on the visibility of the ﬁbers; • The location of the ﬁbers should be clear to support optimal cleaning; • Synthetic materials should not have to be selected or separated ﬁrst; • The product should be perceived as sustainable by itself (energy consumption, . . . ); • The product should allow a full load to be washed. • The operating and cleaning processes should be short and unambiguous; • The product or marketing of the product should raise awareness of the problem; • The product should be accessible from the front of the machine; • The impact should be visible for the user(s), to keep them motivated; • The scientiﬁcally proven eﬀectiveness should be communicated objectively; • The product should be durable and last for several years; • The cleaning process should not rely on the visibility of the ﬁbers; • The location of the ﬁbers should be clear to support optimal cleaning; • Synthetic materials should not have to be selected or separated ﬁrst; • The product should be perceived as sustainable by itself (energy consumption, . . . ); • The product should allow a full load to be washed. 3.2. Explorative Qualitative Study Next, the process of cleaning should not require too much time or should not be necessary every time. • Many respondents also want to know where the ﬁbers will go after disposal, showing that the impact on the environment is very valuable, as can be concluded from the survey. • Many respondents also want to know where the ﬁbers will go after disposal, showing that the impact on the environment is very valuable, as can be concluded from the survey. 3.4. Synthesis and Design of the Product Concept 3.4. Synthesis and Design of the Product Concept 3.4.1. User-Related Design Requirements 3.5. Evaluation of the Concept 3.5. Evaluation of the Concept When asked, almost all participants were interested in a product that tackles microplastic fiber pollution from domestic washing machines: in fact, 96% of respondents answered 'yes'. The purpose and working principles were both clear to 98% of respondents. 95% would use the product if they got it for free. The two participants who were willing to use a microfiber filter, but do not want to use this one, indicated that their washing machine is currently completely built-in, so they do not have any place to put the filter on top of their machine. When asked, almost all participants were interested in a product that tackles microplastic ﬁber pollution from domestic washing machines: in fact, 96% of respondents answered ’yes’. The purpose and working principles were both clear to 98% of respondents. 95% would use the product if they got it for free. The two participants who were willing to use a microﬁber ﬁlter, but do not want to use this one, indicated that their washing machine is currently completely built-in, so they do not have any place to put the ﬁlter on top of their machine. On average people estimate the product costs 157 euro, whereas the estimated selling prices was calculated to be at least 185 euros. However, 47% of respondents stated they would buy the product for 185 euro. It should be noticed that the largest group of people who would buy the product are people with a master's degree (55%). This is not unexpected since often people with a higher degree have a higher-paid job and thus more financial resources. When looking at the working situation, the majority of self-employed people would buy the product (68%). The explanation for this is similar to the one previously stated. However, this is still a very positive result, since it was predicted the price could pose an issue. No less than 90% would use the product for 20 euro a year. From this, it can be deduced that a rental system, where people rent the product for a certain amount per year, could be a good alternative to just selling the product to customers. On average people estimate the product costs 157 euro, whereas the estimated selling prices was calculated to be at least 185 euros. However, 47% of respondents stated they would buy the product for 185 euro. 3.4.2. Concept Design The following product Fibio is an external ﬁltering device that captures ﬁbers that are shed by the domestic washing of synthetic clothes. It is placed on top of a front loader washing machine and connected directly to the drain of the washing machine (Figure 2). It ﬁts the top surface of the washing machine (60 x 60 cm) and is 15 cm high. It needs no electricity and works purely mechanically. Other products can still be placed on top and it also serves as a stacking kit to put a dryer on top. The product consists of three ﬁlters, that can be removed as drawers. During simultaneous research, the principle of the concept employing three ﬁlters (of diﬀerent mesh sizes) showed a 97% eﬃciency in preventing the microﬁbers reaching the wastewater [42]. When the dot (right of the front panel, see Figure 3) is red, the ﬁlter is clogged. By pushing the drawer, it opens, and the ﬁlter can be cleaned with a vacuum cleaner or a scraper. Since the ﬁbers need to be dry, the user has to wait a couple of hours (approximately half a day). 10 of 14 cleaned ouple of Sustainability 2020, 12, 2244 g ) , with a vacuum cleaner Figure 2. Fibio product integrated in the washing environment. Figure 2. Fibio product integrated in the washing environment. FOR PEER REVIEW Figure 2. Fibio product integrated in the washing environment. Figure 2. Fibio product integrated in the washing environment. FOR PEER REVIEW Figure 2. Fibio product integrated in the washing environment. Figure 2. Fibio product integrated in the washing environment. OR PEER REVIEW Figure 2. Fibio product integrated in the washing environment. Figure 2. Fibio product integrated in the washing environment. OR PEER REVIEW Figure 3. Fibio filtering product, showing a clogged filter. Figure 3. Fibio ﬁltering product, showing a clogged ﬁlter. Figure 3. Fibio filtering product, showing a clogged filter. Figure 3. Fibio ﬁltering product, showing a clogged ﬁlter. 4. Discussion More demographic questions should have been asked to get more data out of the study, especially the ﬁrst explorative questionnaire. Furthermore, the participants were selected by snowball sampling, which allows little control on the distribution of the participants. Regarding the topic of the questionnaire (ecological knowledge and behavior), the answers could be inﬂuenced by the participants tending to give the desired answers instead of the reality. Their knowledge is expanded while doing the questionnaire, which inﬂuences the answers later. The study lets participants report their intention to buy or use certain products, which does not say anything about actual behavior. According to the results, more women wash than men, but studies reveal the share of men is increasing [23]. This is due to the higher rate of divorces resulting in a higher amount of men living alone and having to do their laundry [23]. Although most people wash at 40 ◦C, the number of people that wash at 30 ◦C is increasing compared to some years ago [23]. There is not enough correlation to state that people who wash at 30 ◦C are more ecologically minded than people that do not. There are many other potential reasons for this behavior. Here, 57% of respondents always wash a full machine. This number is also increasing due to rising awareness of the importance of sustainability [43]). This means a transition is happening towards more environmentally friendly behavior. Respondents gave contradictory answers regarding the extra time they are willing to spend on using a product that captures microﬁbers. They agree that using a solution is important, and one should be willing to sacriﬁce time, but when they are concretely confronted with the actual time, they will have to spend each day, they are less enthusiastic. It is important to take into account that there is a diﬀerence in what people say and what they actually do, which is called the attitude-behavior gap [44]. In this research, only people’s intention to take action is examined. Only three products were included in the research, although more solutions exist. The alternatives were not on the market while doing this research (November 2017), but should be included in a larger comparison in the future. The survey results were very descriptive. In further studies, it would be interesting to ﬁnd stronger correlations between diﬀerent survey results. 4. Discussion The respondents in the quantitative surveys could only give their opinion on the products through pictures, not while using it. 3.5. Evaluation of the Concept 3.5. Evaluation of the Concept It should be noticed that the largest group of people who would buy the product are people with a master’s degree (55%). This is not unexpected since often people with a higher degree have a higher-paid job and thus more ﬁnancial resources. When looking at the working situation, the majority of self-employed people would buy the product (68%). The explanation for this is similar to the one previously stated. However, this is still a very positive result, since it was predicted the price could pose an issue. No less than 90% would use the product for 20 euro a year. From this, it can be deduced that a rental system, where people rent the product for a certain amount per year, could be a good alternative to just selling the product to customers. When looking at the people that already live and behave sustainably (have a green energy supplier, use reusable bottles, buy expensive but ecological clothes, etc.), it can be seen that, as expected, mainly, people who already act toward ecological living and sustainability are interested in the product. They would be the early adopters. This intention is most significant for people who pay attention to the origin and material of clothes; 61% of this group would buy the product. When looking at the people that already live and behave sustainably (have a green energy supplier, use reusable bottles, buy expensive but ecological clothes, etc.), it can be seen that, as expected, mainly, people who already act toward ecological living and sustainability are interested in the product. They would be the early adopters. This intention is most signiﬁcant for people who pay attention to the origin and material of clothes; 61% of this group would buy the product. 11 of 14 Sustainability 2020, 12, 2244 No correlation is found between the number of people in one household or the presence of children, and the willingness to clean the ﬁlters. The ﬁlter needs to be cleaned every 15–17 washing cycles and takes 10 minutes. The absolute majority of respondents would not mind cleaning the ﬁlters with this frequency (95%). 5. Conclusions Microscopic plastic particles released by synthetic clothes (microﬁbers) cause considerable environmental problems to the oceans and inland waters. In this research, consumer laundry behavior and knowledge of the problem, supplemented by the possibilities and needs for a ﬁltering device for domestic washing machines that aims to prevent microﬁbers released by synthetic clothes from reaching the environment, were examined. Based on a survey with 411 respondents, user observation with eight test subjects, and evaluation of a new design concept by means of a survey with 227 respondents, the following propositions could be constructed: Proposition 1: In contrast to the fact that the problem of the plastic soup and microplastics is relatively well known, the problem of microﬁbers is not. Hence, the impact of people’s washing behavior is also underestimated/not recognized. Proposition 2: Eﬀectiveness is seen as the most important characteristic for products that support consumers in their washing habits to catch microﬁbers. This eﬀectiveness should be not only be scientiﬁcally proven, but also visual/experienced by the users to see and recognize their reduced eﬀect Sustainability 2020, 12, 2244 12 of 14 on the environment. Respondents argued that this feedback probably oﬀers the largest opportunity for long term usage. on the environment. Respondents argued that this feedback probably oﬀers the largest opportunity for long term usage. Proposition 3: Next to eﬀectiveness, durability is considered a very important factor that determines the willingness to adopt a product that tackles the microﬁber problem. This is a logical addition to the eﬀectiveness characteristic. Both factors ensure long term usage. Proposition 4: In order to spend the least amount of time on operating the product, thus encouraging consumers to keep on using the product for a long time, the usage should be straightforward and user friendly. Next to the technical side, the human-centered side is important to consider. Proposition 5: Cost is seen as a less important factor, but nonetheless something to carefully consider. To distribute the product widely, which is necessary to tackle the plastic ﬁber issue, people from all socio-economic classes should be able to purchase the product. Proposition 6: As cleaning is an important and new action in the washing habit, it should be fast, easy and foolproof (i.e., avoid possibility to clean by rinsing for example). The suggested amount of cleaning every 15–17 washing cycles, duration of cleaning: 10 minutes, was considered as achievable. 5. Conclusions Current research is limited by the fact that none of the products are used over a longer period of time, whereas this would be essential for microﬁber catching. Future research is needed to eﬀectively experience long term behavior change and usage, and to investigate the possibilities to recycle the collected ﬁbers. Many respondents also want to know where the ﬁbers will go after disposing and how they should be disposed optimally. This circular thinking could encourage people better to optimally use a washing machine ﬁlter. Additional research can be done to investigate the possibilities to close the loop for the collected ﬁbers. Author Contributions: Conceptualization, L.H. and E.D.B.; methodology, L.S.; validation, R.W., C.C.C., and L.S.; formal analysis, L.H..; investigation, L.H. and C.C.C.; data curation, L.H.; writing—original draft preparation, L.H. and E.D.B.; supervision, R.W., E.D.B., and L.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Ethical Statement: All subjects agreed upfront to give consent for inclusion before they participated in the study. Author Contributions: Conceptualization, L.H. and E.D.B.; methodology, L.S.; validation, R.W., C.C.C., and L.S.; formal analysis, L.H..; investigation, L.H. and C.C.C.; data curation, L.H.; writing—original draft preparation, L.H. and E.D.B.; supervision, R.W., E.D.B., and L.S. All authors have read and agreed to the published version of the manuscript. References Synthetic microﬁbers in the marine environment: A rev on their occurrence in seawater and sediments. Mar. Pollut. Bull. 2018, 127, 365–376. [CrossRef] 10. Cesa, F.S.; Turra, A.; Checon, H.H.; Leonardi, B.; Baruque-Ramos, J. 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Synthetic ﬁbers as microplastics in the marine environment: A review from textile perspective with a focus on domestic washings. Sci. Total Environ. 2017, 598, 1116–1129. [CrossRef] [PubMed] comte, M.; Janssen, C. De Verwijdering Van Microplastics in Rioolwaterzuiveringsinstallaties: Een Case-Study or Vlaanderen; Ghent University: Ghent, Belgium, 2015. 7. Leslie, H.; Moester, M.; de Kreuk, M.; Vethaak, D. Verkennende Studie Naar Lozing Van Microplastics Door Rwzi’s. Available online: https://pure.tudelft.nl/portal/en/publications/verkennende-studie-naar- lozing-van-microplastics-door-rwzis(d980ca07-b54b-409a-a17d-bfa72b7655f8)/export.html (accessed on 28 January 2020). 13 of 14 13 of 14 Sustainability 2020, 12, 2244 8. De Falco, F.; Gullo, M.P.; Gentile, G.; Di Pace, E.; Cocca, M.; Gelabert, L.; Brouta-Agnésa, M.; Rovira, A.; Escudero, R.; Villalba, R.; et al. Evaluation of microplastic release caused by textile washing processes of synthetic fabrics. Environ. 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https://openalex.org/W4236898557	https://www.qeios.com/read/CF6VXA/pdf	English	null	Congenital Spondylolysis, Lumbosacral Region	Definitions	2,020	cc-by	71	Open Peer Review on Qeios Open Peer Review on Qeios Congenital Spondylolysis, Lumbosacral R i National Cancer Institute National Cancer Institute Qeios ID: CF6VXA · https://doi.org/10.32388/CF6VXA Qeios · Definition, February 7, 2020 Source National Cancer Institute. Congenital Spondylolysis, Lumbosacral Region. NCI Thesaurus. Code C34502. Code C34502. A defect in the pars interarticularis of the vertebral arch of a lumbar or sacral vertebra that is present at the time of birth. 1/1
https://openalex.org/W3107582253	https://www.nature.com/articles/s41467-020-19771-0.pdf	English	null	High-resolution combinatorial patterning of functional nanoparticles	Nature communications	2,020	cc-by	8,347	ARTICLE Results Electric ﬁeld-assisted patterning of NPs. The EFASP process is schematically illustrated in Fig. 1a. A charged nanopattern is ﬁrst written on a ﬂuorine polymer substrate (electret) by applying a localized high voltage (-40 to -90 V) using a conductive atomic force microscope (AFM) tip. Here, the localized high voltage decomposes the ﬂuorine polymer surface with nanoscale preci- sion and creates de-ﬂuorinated nanopatterns, which can be used for effectively attracting colloidal nanoparticles (Fig. 1b, c). Then, an oil dispersion of NPs is applied to the substrate. NPs in the nonpolar solvent is enriched at the areas of electrostatic nano- patterns due to the electrostatic attraction and adsorbed on the patterned areas because of their high surface energy. In the unpatterned area, no adsorption occurs since the surface energy of the ﬂuoride substrate is low and all NPs are carried away by the solution. With this method, we can accurately assemble perovskite quantum dots (QDs) in an area of 100 × 800 μm2, as shown in the photoluminescence (PL) image of Nanjing University Logo, composed of 9481 pixels, approximately 4.7 × 105 10-nm CsPbBr3 NPs (Fig. 1d). No nonspeciﬁc adsorption was found in the uncharged area, as shown in the enlarged PL, SEM, AFM images in Fig. 1e. In other words, the error ratio (i.e., the ratio of the number between nonspeciﬁc adsorbed and correctly printed NPs) is lower than 2 × 10-6. This extremely low error ratio ensures the EFASP a reliable nanofabrication technique. Moreover, EFASP enjoys a very high resolution and accuracy. The pitch size can reach 200 nm (125,000 DPI), or even a smaller value, as supported by the Kelvin probe force microscopy (KPFM) potential maps (Fig. 1f, g) and the corresponding AFM height maps of the printed structure (Fig. 1h, i), with the positioning accuracy better than 10 nm (Supplementary Fig. 1). The nanoprinting method is robust and fast, which can be completed by not only spin coating but also brushing or dip coating of the NP dispersion in just seconds. (Supplementary Movie 1) y The difﬁculty of achieving robust nanopatterning of func- tional NPs is rooted in the complexity of the NPs behaviors at the solution/substrate interface25. To align NPs at deﬁned locations with nanometer precision, it requires a series of interactions between NPs and substrate. High-resolution combinatorial patterning of functional nanoparticles Xing Xing1,5, Zaiqin Man1,5, Jie Bian1,5, Yadong Yin 2✉, Weihua Zhang 1,3✉& Zhenda Lu 1,4✉ Xing Xing1,5, Zaiqin Man1,5, Jie Bian1,5, Yadong Yin 2✉, Weihua Zhang 1,3✉& Zh Fast, low-cost, reliable, and multi-component nanopatterning techniques for functional col- loidal nanoparticles have been dreamed about by scientists and engineers for decades. Although countless efforts have been made, it is still a daunting challenge to organize dif- ferent nanocomponents into a predeﬁned structure with nanometer precision over the milli- meter and even larger scale. To meet the challenge, we report a nanoprinting technique that can print various functional colloidal nanoparticles into arbitrarily deﬁned patterns with a 200 nm (or smaller) pitch (>125,000 DPI), 30 nm (or larger) pixel size/linewidth, 10 nm position accuracy and 50 nm overlay precision. The nanopatterning technique combines dielectrophoretic enrichment and deep surface-energy modulation and therefore features high efﬁciency and robustness. It can form nanostructures over the millimeter-scale by simply spinning, brushing or dip coating colloidal nanoink onto a substrate with minimum error (error ratio < 2 × 10−6). This technique provides a powerful yet simple construction tool for large-scale positioning and integration of multiple functional nanoparticles toward next- generation optoelectronic and biomedical devices. 1 College of Engineering and Applied Sciences, State Key Laboratory of Analytical Chemistry for Life Science, and Jiangsu Key Laboratory of Artiﬁcial Functional Materials, Nanjing University, Nanjing 210093, PR China. 2 Department of Chemistry, University of California, Riverside, CA 92521, USA. 3 MOE Key laboratory of Intelligent Optical Sensing and Manipulation, Nanjing University, Nanjing 210023, PR China. 4 Research Center for Environmental Nanotechnology (ReCENT), Nanjing University, Nanjing 210023, PR China. 5These authors contributed equally: Xing Xing, Zaiqin Man, Jie Bian. ✉email: yadong.yin@ucr.edu; zwh@nju.edu.cn; luzhenda@nju.edu.cn 1 NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 N N anofabrication, the process of making miniature struc- tures with nanometer precision, is the foundation of integrated electronics that has profound impacts on our modern society. With advanced photolithographic tools, people can now produce transistors with feature sizes below 10 nm over 12 inch Si wafers at high yields. However, when it comes to the emerging areas, such as photonic, optoelectronic, and bio- medical nanodevices, where materials are often incompatible with the conventional CMOS processes, more nanofabrication tools are highly desirable. Results It needs long-range particle-substrate attraction to enrich the NPs on the substrate in order to realize efﬁcient and fast assembly, strong short- range attraction to ﬁx the NPs at the designed locations, and counter-interactions to avoid nonspeciﬁc deposition of NPs at undesired areas. Moreover, all these interactions are sensitive to the subtle changes of assembly parameters, such as the surface property of the substrate, solvent polarity, and particle mor- phology, and they are often coupled and affecting each other. A robust nanoprinting technique will need to carefully optimize these interactions to enable effective NP assembly. Combinatorial patterning. The EFASP technique can be directly extended to color patterning by repeating the writing/deposition cycles. Here, we used a dual-color pattern with two different NPs, green CsPbBr3 and red CsPbI3 QDs as an example to illustrate the process (Fig. 2a). First, CsPbBr3 QDs were printed, forming a hollow NJU pattern. Then, we scanned the as-formed pattern with AFM, aligned the probe using the topography image as the refer- ence, and wrote a new pattern for the second NP deposition cycle (Fig. 2b, c). This process guarantees the overlay accuracy between different printing cycles, and in our work, it reaches 58 nm (Sup- plementary Fig. 3). After that, as shown in Fig. 2d we deposited CsPbI3 QDs by spin coating, and complete the second color part for the binary pattern. Figure 2e shows the PL images with green and red bandpass ﬁlters, and no spatial overlap was found between the green and red areas, indicating that there is no undesired cross- contamination between the two separate printing cycles. This can be further conﬁrmed with micro-area PL spectra (Fig. 2f), in which every pixel only exhibits a single PL peak. y In this work, we report an electric ﬁeld-assisted surface- sorption nano-printing (EFASP) method for patterning multi- ple functional nanoparticles with high resolution, precision, and scalability. As an additive nanofabrication method, it uti- lizes a tip-based high-voltage writing process to generate nanoscale de-ﬂuorinated patterns in a ﬂuorinated surface, which not only enables electrostatic trapping but also creates a high local surface potential in contrast to the ﬂuorinated area, both favoring highly efﬁcient site-speciﬁc NP assembly. By combining long-range electrostatic attractions in the solvent, short-range surface adsorption, and low surface energy of the ﬂuoride substrate, EFASP allows fast, nanometer precision printing of various functional NPs over large areas with high reproducibility. High-resolution combinatorial patterning of functional nanoparticles One possible solution is the additive fabrication through wet chemical self-assembly, which can organize pre-synthesized colloidal nanoparticles (NPs) as functional building blocks at the desired positions to build devices from bottom up1–3. With the recent signiﬁcant advances in colloidal synthesis, NPs of metal, semiconductor, and dielectrics with unique electrical, magnetic, and optical properties can now be produced routinely in large quantity and high quality4,5. However, until now it still remains a great challenge in ﬁnding robust assembly approaches to effectively introduce NPs of different compositions and functions to the pre-designed locations6,7, although several methods have been proposed in the past two decades to tackle this issue, including colloidal self-assembly8,9, microcontact printing10,11, dip-pen nanolithography12, inkjet printing13, optical tweezers14, biomolecule-induced assembly15, patterning within predeﬁned features16 or chemically modiﬁed surface17, nanoxerography18–23, and so on. These previous methods are not yet ready for broad practical applications as they suffer from various limitations such as small scale, slowness, low resolution, and inﬂexibility with the choice of NPs and substrates. Techniques for robust, fast, multi- component NP patterning with true nanometer precision and high scalability are still highly demanded, which, if made available, are believed to revolutionize the manufacturing processes toward many advanced nanodevices7,24. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 a C F F C O O V C F F C F F C F F C F F C F F - - - r (nm) Electrical potential b 0 50 100 Fele 1 - - - + + - - r b d (nm) Potential Uele 0 -Uspin c 0 100 –100 - - - C F F C O O C F F 2 d Udefluo-Ufluo Ufluo c Potential f 0 V –2.8 V d 0 V g –1.5 V f d g e 23 nm i 32 nm h 20 nm i i h h e Fig. 1 Nanoprinting of perovskite QDs by EFASP. a Schematic illustration of the EFASP process, which involves high-voltage electrostatic nanopattern generation, surface modiﬁcation, and site-speciﬁc NP assembly by electrical trapping. b Electrical trapping potential in a nonpolar solvent as a function of the distance from the center of the written charge (dash circle 1 in a). The electric ﬁelds can extend for hundreds of nanometers, leading to the high efﬁciency of NP collection. c Surface potential modulation along the nanopattern surface (dash circle 2 in a) De-ﬂuorination through high voltage induces signiﬁcant surface energy modulation, providing strong short-range interactions to ﬁx the NPs. d A large-scale photoluminescence (PL) image obtained by assembling CsPbBr3 NPs using the EFASP process, with a total area of 100 × 800 μm2 with 9481 pixels, a pitch of 800 nm, and approximately 4.7 × 105 10- nm CsPbBr3 NPs. Scale bar, 50 μm. e High-magniﬁcation PL, SEM, and AFM height images of the marked area in d. Scale bar, 2 μm. f–i Extremely high- resolution EFASP can be produced by modulating patterns, reaching a pitch of 200 nm (125,000 DPI). The high-magniﬁcation KPFM potential map and corresponding AFM height map after NP assembly (f and h, scale bar: 2 μm) and their details (g and i, scale bar: 500 nm). The corresponding large area PL image is shown in Supplementary Fig. 2. and then mapped its electrostatic potential distribution using the KPFM. As shown in Fig. 3a, the ﬁelds were strongly localized in the vicinity of the charge line, causing electrostatic forces, Fele on the surrounding NPs. each printing cycle are shown in Supplementary Fig. 4. Again, no cross-contamination is observed in each pixel, further conﬁrming the robustness of EFASP for printing multiple functional NPs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 r (nm) d (nm) Electrical potential Potential Uele 0 -Uspin a b c d e 1 2 C F F C O O V C F F C F F C F F C F F C F F - - - f 0 V 0 V 32 nm 20 nm 23 nm g i 0 50 100 0 100 –100 - - - C F F C O O C F F 2 d Fele 1 - - - + + - - r h Udefluo-Ufluo Ufluo –2.8 V –1.5 V Fig. 1 Nanoprinting of perovskite QDs by EFASP. a Schematic illustration of the EFASP process, which involves high-voltage electrostatic nanopattern generation, surface modiﬁcation, and site-speciﬁc NP assembly by electrical trapping. b Electrical trapping potential in a nonpolar solvent as a function of the distance from the center of the written charge (dash circle 1 in a). The electric ﬁelds can extend for hundreds of nanometers, leading to the high efﬁciency of NP collection. c Surface potential modulation along the nanopattern surface (dash circle 2 in a) De-ﬂuorination through high voltage induces signiﬁcant surface energy modulation, providing strong short-range interactions to ﬁx the NPs. d A large-scale photoluminescence (PL) image obtained by assembling CsPbBr3 NPs using the EFASP process, with a total area of 100 × 800 μm2 with 9481 pixels, a pitch of 800 nm, and approximately 4.7 × 105 10- nm CsPbBr3 NPs. Scale bar, 50 μm. e High-magniﬁcation PL, SEM, and AFM height images of the marked area in d. Scale bar, 2 μm. f–i Extremely high- resolution EFASP can be produced by modulating patterns, reaching a pitch of 200 nm (125,000 DPI). The high-magniﬁcation KPFM potential map and corresponding AFM height map after NP assembly (f and h, scale bar: 2 μm) and their details (g and i, scale bar: 500 nm). The corresponding large area PL image is shown in Supplementary Fig. 2. Results The advantages of EFASP also include combi- natorial patterning of multiple functional nanoparticles for realizing multi-color printing by conveniently repeating the writing and assembly cycles. Combinatorial patterning is rare, and it has been only limited to the assembly of two types of NPs in conventional electric ﬁeld- assisted assembly processes26,27. More types of NPs may be assembled on the same substrate by successive assembly cycles, but it usually causes the damage or pollution of the previous assemblies. To further demonstrate the capability of combinator- ial patterning, we printed a pseudo-color image with four different NPs. Here15-nm Fe2O3 NPs were used to create the white color, and NaYF4:Yb,Er, CsPbBr3 and CdSe@ZnS NPs were used to produce the magenta, green and red colors in the image captured by the dark-ﬁeld microscopy (Fig. 2g). The images in NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 This work represents the large-scale deterministic assembly of NPs with multiple types of compositions and functionalities. Although persistent endeavors have been made to achieve high- quality multi-step NP assembly, the previous works failed majorly because the nonspeciﬁc adsorption accumulates the assembly errors, and the later cycles inevitably damage the previously assembled structures. In EFASP, however, the error accumulation problem is minimized because the error ratio is extremely low (<2 × 10-6) in a single step; and the strong surface adsorption ensures a high endurance of the printed structures to the successive assembly cycles. We can retrieve the charge density of the 1D pattern using the measured potential distribution, and further calculate the electro- static forces on the NPs in a given solution28 (see Supplementary Information Calculation). In our system, the charge density is typically at ~ 1 C/m2 level. The Fele is the sum of the Coulomb forces, FCoul, and gradient forces (dielectrophoretic forces), Fgra. Since nonpolar solvents are used and NPs are neutral, Coulomb forces are weak, and the trapping potential is mainly determined by gradient forces. For the case of perovskite QDs in a nonpolar solvent, the trapping potential is approximately 0.1 eV, and trapping forces is over 1 pico-Newton level at the surface (Fig. 3b). The usage of nonpolar solvent in this work brings several advantages comparing with the case of a commonly used polar solution, particularly water. First, it avoids the screening effects, and the electric ﬁelds can extend for hundreds of nanometers, leading to a high collection efﬁciency of NPs. On the contrary, the electric ﬁelds only extend for tens of nanometers in water (when ionic strength is 10-3 M) due to the screening effect29. Secondly, the dielectric constant of water (as well as other polar solution) is larger than that of NPs. This makes gradient forces repulsive and Electrostatic potential and forces. The high performance of EFASP is the result of joint forces of long-range electrostatic interactions and short-range adsorption interactions between NPs and substrate. To have a quantitative understanding of the process, we measured the interaction strengths and calculated how they inﬂuence the detailed performance of EFASP. To determine the strength of electrostatic interactions, we ﬁrst wrote a charged line of 10 μm by applying -90 V on the AFM tip, 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 V a b c d g e f 400 600 800 Wavelength/nm Intensity/a.u. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 The white color is created by 15-nm γ-Fe2O3 NPs, while the magenta, green and red colors are produced by NaYF4:Yb,Er, CsPbBr3 and CdSe@ZnS NPs, respectively. Scale bar, 10 μm. d h d fﬁ f f h h d h f d ﬁ d h d Distance (nm) 0 100 200 300 -0.16 -0.12 -0.08 -0.04 0 Trapping potential (eV) 0 1.0 2.0 3.0 Gradient force (pN) Trapping force Trapping potential Potential (V) 0.0 -2.0 -4.0 0 2.0 X (m) 1.0 3.0 4.0 0 2.0 Z (m) - - - Fele Binding energy (eV) XPS signal (a.u.) 286 290 294 298 a b c 76° 112° Fluoride area De-fluoride area x y z Fig. 3 Quantitative analysis of NP–substrate interactions. a Electrostatic potential distribution along the x-z plane induced by a charged line with 10 μm length (y axis) and 50 nm width (x axis), which is written by applying a voltage of -90 V on the substrate. The electric ﬁeld is highly localized in the vicinity of the charge line, inducing strong attraction to the surrounding NPs. b The calculated electrostatic force and potential of the 1D charged pattern to NPs in a nonpolar solvent, based on the measured potential distribution (a). Inset is the schematic illustration of the electrostatic attraction force between the charged pattern and dispersed NPs. c XPS spectral analysis of carbon 1 s on charged (de-ﬂuorinated) and uncharged (ﬂuoride) area. The original spectra are shown by the black dotted curve, which is well ﬁtted by the sum (red solid curves) of four Gaussian lines (solid black curves). The high voltage leads to a decrease in the peak at 290.5 and 291.8 eV, which can be assigned as diﬂuoride carbons, and an increase in the peak at 289.3 and 293.0 eV, which can be assigned as the carboxylation carbons. Insets are the corresponding water contact angle of ﬂuoride and de-ﬂuoride area, decreasing from 112o to 76o. Potential (V) 0.0 -2.0 -4.0 0 2.0 X (m) 1.0 3.0 4.0 0 2.0 Z (m) a x y z Binding energy (eV) XPS signal (a.u.) 286 290 294 298 c 76° 112° Fluoride area De-fluoride area c a Distance (nm) 0 100 200 300 -0.16 -0.12 -0.08 -0.04 0 Trapping potential (eV) 0 1.0 2.0 3.0 Gradient force (pN) Trapping force Trapping potential - - - Fele b b Fig. 3 Quantitative analysis of NP–substrate interactions. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 CsPbBr3 CsPbI3 15 nm 27 nm 0 V –1.2 V Fig. 2 Color printing of multiple functional NPs. a Schematic illustration of the assembly of different NPs by repeating the EFASP process. b AFM height image after printing the ﬁrst cycle of 12-nm CsPbBr3 QDs as a hollow N pattern. Scale bar, 1 μm. c Electrical potential image after writing the second charge pattern N. Scale bar, 1 μm. d AFM height mapping of the binary pattern after depositing the second cycle of CsPbI3 QDs. Scale bar, 1 μm. e PL image of the dual-color pattern and its corresponding images with green and red bandpass ﬁlters (marked by the white solid rectangle). No spatial overlap was found between the green and red areas, indicating no cross-contamination occurs. Scale bar, 5 μm. f PL spectra of two kinds of QDs on one pixel corresponding to (e). g pseudo-color image captured by the dark-ﬁeld microscopy of a color pattern, which is composed of four types of NPs with 606 pixels. The white color is created by 15-nm γ-Fe2O3 NPs, while the magenta, green and red colors are produced by NaYF4:Yb,Er, CsPbBr3 and CdSe@ZnS NPs, respectively. Scale bar, 10 μm. V a b c d g e f 400 600 800 Intensity/a.u. CsPbBr3 CsPbI3 15 nm 27 nm 0 V –1.2 V b g d c f 400 600 800 Wavelength/nm Intensity/a.u. CsPbBr3 CsPbI3 f f u. e Fig. 2 Color printing of multiple functional NPs. a Schematic illustration of the assembly of different NPs by repeating the EFASP process. b AFM height image after printing the ﬁrst cycle of 12-nm CsPbBr3 QDs as a hollow N pattern. Scale bar, 1 μm. c Electrical potential image after writing the second charge pattern N. Scale bar, 1 μm. d AFM height mapping of the binary pattern after depositing the second cycle of CsPbI3 QDs. Scale bar, 1 μm. e PL image of the dual-color pattern and its corresponding images with green and red bandpass ﬁlters (marked by the white solid rectangle). No spatial overlap was found between the green and red areas, indicating no cross-contamination occurs. Scale bar, 5 μm. f PL spectra of two kinds of QDs on one pixel corresponding to (e). g pseudo-color image captured by the dark-ﬁeld microscopy of a color pattern, which is composed of four types of NPs with 606 pixels. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 a Electrostatic potential distribution along the x-z plane induced by a charged line with 10 μm length (y axis) and 50 nm width (x axis), which is written by applying a voltage of -90 V on the substrate. The electric ﬁeld is highly localized in the vicinity of the charge line, inducing strong attraction to the surrounding NPs. b The calculated electrostatic force and potential of the 1D charged pattern to NPs in a nonpolar solvent, based on the measured potential distribution (a). Inset is the schematic illustration of the electrostatic attraction force between the charged pattern and dispersed NPs. c XPS spectral analysis of carbon 1 s on charged (de-ﬂuorinated) and uncharged (ﬂuoride) area. The original spectra are shown by the black dotted curve, which is well ﬁtted by the sum (red solid curves) of four Gaussian lines (solid black curves). The high voltage leads to a decrease in the peak at 290.5 and 291.8 eV, which can be assigned as diﬂuoride carbons, and an increase in the peak at 289.3 and 293.0 eV, which can be assigned as the carboxylation carbons. Insets are the corresponding water contact angle of ﬂuoride and de-ﬂuoride area, decreasing from 112o to 76o. decreases the adsorption efﬁciency of NPs further. Third, this process enables access to many high-quality nanoparticles, such as semiconductor quantum dots as most of them are synthesized in nonpolar solvents at elevated temperatures via hot injection or solvothermal processes. decreases the adsorption efﬁciency of NPs further. Third, this process enables access to many high-quality nanoparticles, such as semiconductor quantum dots as most of them are synthesized in nonpolar solvents at elevated temperatures via hot injection or solvothermal processes. Surface modiﬁcation and short-range interactions. Compared with the electrostatic interactions, the short-range interaction induced by surface modiﬁcation is more complicated. To conﬁrm the chemical modiﬁcation and consequent surface energy increases of the patterned area, several characterizations before NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 it is possible for NPs to be adsorbed in the nearby area through electrostatic interaction, deteriorating the position accuracy of the EFASP method. Similar to the case of error ratio estimation, we can estimate this uncertainty and after charge writing were performed, including X-ray pho- toelectron spectroscopy (XPS), lateral force measurements, and contact angle measurements. The carbon 1 s XPS spectra of the substrate surface were shown in Fig. 3c. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 Four peaks at 290.5, 291.9 289.3, and 293.0 eV were analyzed by Gaussian ﬁtting, which can be assigned to diﬂuoride carbons and de-ﬂuorinated chemical species, respectively. The intensity decrease of the peaks at 290.5 and 291.8 eV, together with the increase at 289.3 and 293.0 eV, indicate the occurrence of de-ﬂuorination induced by the high-voltage writing process (Supplementary Fig. 5). The de-ﬂuorination process can intro- duce strong modulation of local surface energy, which conse- quently alters the local frictions. This change in the local friction force was indeed observed in our experiment with lateral force measurement (Supplementary Fig. 6). δ ¼ Pele=Pdefluo / e UdefluoUfluo ð Þ=kBT ð6Þ ð6Þ which is decided by the surface energy modulation depth, Udefluo Ufluo, caused by the de-ﬂuorination. For a 10 nm spherical NPs, ΔU is around 0.24 eV, and this ratio is 7 × 10−5. This leads to a clean-cut between the modiﬁed and unmodiﬁed area, which is also demonstrated in the printed interdigitated nanoelectrode by Au NPs, where variation less than 10 nm can be achieved for patterns with a line width less than 150 nm (Fig. 4c, d). Tunable pixel size. The pixel size in EFASP can be tuned pre- cisely by controlling the number of NPs adsorbed in the patterned area, The surface energy modulation induced by de-ﬂuorination is signiﬁcant. As shown in the insets of Fig. 3c, the contact angle with water is changed considerably (from 112 degrees to 76 degrees) by the de-ﬂuorination process. With the help of the combining relation29, we can estimate the adhesion energy difference of a 10 nm spherical particle with the substrate before and after it is de-ﬂuorinated, ΔW = 0.24 eV (see Supplementary Information Calculation). In the case of a cubic NP, this value can be even larger. Overall, the adhesion resulting from the change in surface energy is much larger than the electrostatic interaction and provides a strong binding force for the NPs. N / e UdefluoþUeleUspin ð Þ=kBT ð7Þ ð7Þ To demonstrate this, we ﬁxed the parameters of spin-coating and adjusting Uele (0.15 to 1.5 V) by changing the writing voltage applied on the AFM from -40 to -90 V (Supplementary Fig. 7). It is evident that high surface potential leads to a large pixel size (Fig. 4a, b). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 For a quantitative understanding, the particle volume in the AFM topographic graphs and ﬂuorescence intensity in the PL images were statistically analyzed. All the 100 printed pixels in the 10 × 10 charged spots separated by 1.0 μm were counted, as shown in Supplementary Fig. 8. Both the two plots (Supplemen- tary Fig. 8e, f) demonstrate that there is a near-linear relationship between the average particle number and the surface potential. Spatial distribution of NPs. We can further calculate adsorption possibilities of NPs at different areas using Boltzmann-Gibbs distribution P rð Þ / eU rð Þ=kBT ð1Þ ð1Þ g p p We can push the pixel size down to few-NP level by lowering the pattern writing potential to -0.15 V. Only several NPs are adsorbed in one pixel, while still maintaining 100% yield based on the charged spots. Taking 15-nm Fe2O3 NPs as an example, only 2–5 NPs are assembled at each spot when the surface potential is low at -0.25 V (Supplementary Fig. 10c), corresponding to a pixel size less than 30 nm. This few-NP level control capability is particularly useful for making QD nanopatterns since single QDs can be used for generating single photons, which have many applications in the ﬁeld of quantum information. For illustrating this, a 10 × 10 array of CsPbBr3 QDs was assembled at the surface potential of -0.15 V, and low-temperature PL spectra (at 4 K) show that there are only a few well-separated ﬂuorescence peaks at each pixel (Supplementary Fig. 9), indicating that QDs can be precisely located at the desired position at the few-NP level since one CsPbBr3 QD normally only has one emission peak at low temperature. Here, P is the possibility of a nanoparticle being at location r, and U is the adsorption energy. From Fig. 1c, we learn that the adsorption energy at the patterned area (de-ﬂuorinated area), electrostatic interaction area, and ﬂuoride surface is Udeﬂuo + Uele −Uspin, Uele + Uﬂuo −Uspin, and Uﬂuo −Uspin, respectively. Consequently, the possibility for a nanoparticle to adsorb on the surface is Pdefluo / e UdefluoþUeleUspin ð Þ=kBT ð2Þ Pele / e UeleþUfluoUspin ð Þ=kBT ð3Þ Pfluo / e UfluoUspin ð Þ=kBT ð4Þ ð2Þ ð3Þ ð4Þ Based on this, some of the key parameters of EFASP can be evaluated in a quantitative fashion. NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications Discussion h In this work, we have developed a nanofabrication technique, EFASP, which is efﬁcient, fast, robust, accurate, and widely applicable. By regulating the long-range and short-range forces between the oil-dispersed NPs and the substrate, as well as the surface energy of the substrate, which can be achieved by high- voltage tip-based writing, we can print the NPs into nanopatterns with feature size less than 30 nm, without any nonspeciﬁc adsorption. The EFASP is versatile for NPs dispersed in nonpolar solvents, such as QDs, magnetic NPs, upconversion ﬂuorescent nanocrystals and metal NPs. Moreover, we have demonstrated that multiple NPs could be assembled accurately on the same substrate with an arbitrary arrangement, just by repeating suc- cessive charge-writing/spin-coating cycles. The printing process is intrinsically scalable, and samples over millimeter sizes can be produced if the high-voltage writing is carried out using tip arrays or large-area molds. This work provides a platform for con- venient, ﬂexible, and rapid manufacturing of non-silicon based Au NPs were synthesized as follows34. For the synthesis of 5 nm Au nanoparticles, 0.0925 g dodecyldimethylammonium bromide (DDAB) and 0.046 g HAuCl4·4H2O were added into 10 mL of toluene with ultrasonication until the complete dissolution of metal salts. In total 40 μL of freshly prepared aqueous NaBH4 (9.4 M) solution were added dropwise under vigorously stirring to initiate the gold reduction. After 20 min, 0.8 mL 1-dodecanethiol was added into the as- prepared solution, and the stirring was continued for 5 minutes. After adding ethanol, Au NPs were precipitated and separated through centrifugation. Finally, the resulting Au NPs were re-dispersed into 10 ml cyclohexane. Upconversion ﬂuorescent NaYF4:18%Yb, 2%Er NPs were synthesized as follows35. YCl3·6H2O (242.7 mg), YbCl3·6H2O (70.0 mg), and ErCl3·6H2O (7.6 mg) were dissolved in 100 μL water ﬁrst and then mixed with oleic acid (6 mL) and 1- octadecene (15 mL) in a three-neck ﬂask. The solution was degassed at 150 °C under Ar for 30 min and then cooled down to room temperature. A 10-mL methanol solution containing NaOH (0.1 g) and NH4F (0.1481 g) was added and stirred for 30 min. After that, the solution was slowly heated to 110 °C and kept at 110 °C for 0.5 h to remove methanol and a small amount of water. Then, the solution was quickly heated to 320 °C and aged for 1 h under Ar protection. Methods Synthesis. All the perovskite QDs were synthesized by the hot injection method32. Take the typical perovskite CsPbBr3 QDs as an example, Cs2CO3 (0.407 g) was loaded into a 50 ml three-neck ﬂask with octadecene (ODE, 20 ml) and oleic acid (1.5 ml). The mixture was degassed for 0.5 h at 120 °C, and then heated to 150 °C under Ar to dissolve the solid completely, as the Cs-oleate stock solution. In another three-neck ﬂask, PbBr2 (0.14 g) was added into a mixed solvent of ODE (10 ml), oleylamine (2 ml), and oleic acid (1 ml). The mixture was then heated to 120 °C under vacuum, followed by Ar gas ﬂow. After complete solubilization of the PbBr2 salt, the temperature was raised to 150 °C, and followed by a quick injection of above Cs-oleate solution (1 ml). The reaction was stopped after 5 s by an ice- water bath. After the dispersion of perovskite NCs was cooled down to 30 °C, antisolvent such as acetone with volume ratio 1:1 to original dispersion was added to precipitate NCs. The resulting CsPbBr3 NCs were collected by centrifugation, and then re-dispersed into 3 ml cyclohexane. With the same steps, CsPbI3 NCs can be synthesized by replacing the PbBr2 salts with the same mole amount of PbI2 salts. Finally, it is worth noting that there is no limit on the printing size of the EFASP technique. Currently, the size is only limited by the nanopattern writing tool (i.e., the AFM), and this potential problem can be solved by many existing techniques, e.g., tip arrays or a large area mold30,31. In addition, we can set the scan AFM head on the motorized stage to address the concerns about the slow and small scale about AFM based technology. As demonstrated in Supplementary Fig. 11, by combining the tip scanning and stage moving, we can easily make millimeter-scale charge patterns in 15 min, requiring no manual tip- repositioning step. Superparamagnetic γ-Fe2O3 NPs were synthesized using a thermolysis process33. Fe(CO)5 (0.2 mL, 1.52 mmol) was added to a mixture containing 10 mL of ODE and 1.28 g of oleic acid at 100 °C. The solution was then heated to 290 °C under Ar atmosphere and maintained for 1 h. Then the solution was cooled down to 200 °C and bubbled with air for 2 h. After cooling down to room temperature, acetone was added to precipitate γ-Fe2O3 nanoparticles. nanodevices, such as integrated photonic chips, biochips, and wearable devices. nanodevices, such as integrated photonic chips, biochips, and wearable devices. properties of the ink materials, and therefore, all synthesized functional NPs dispersed in a nonpolar solvent can be used as the ink for effective nanoscale printing. We tested several different NPs including magnetic, metal, semiconductor, dielectric nano- material (e.g., γ-Fe2O3, Au, CdSe, and NaYF4, respectively) with the size from a few nanometers to tens of nanometer, and high quality printed structure can be obtained for all of these materials without exception (Supplementary Fig. 10). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 One of the key parameters for the assembly-based nanofabri- cation technique is the error ratio, which determines whether the technique is useful in industrial settings. In the EFASP process, it can be deﬁned as the ratio between the adsorption possibility in the unpatterned ﬂuoride area and patterned de-ﬂuorinated area, Printing continuous nanostructured assemblies. In addition to the few-NP level pixel control, the EFASP technique is suitable for fabricating continuous nanostructured assemblies, which are particularly useful in electronics, e.g., conductive nanowires, antennas, as well as nano-optoelectronic devices. As an example, we prepared interdigitated nanoelectrodes printed using Au NP (10 nm, Fig. 4c, d) with 1 μm electrode separation and <150 nm ﬁnger width. Note that nonpolar solvents are used in this work and NPs are neutral, and therefore, the NPs are densely packed when they are assembled on the patterns. This is different from the water-dispersed NPs which are repulsive to each other due to the existence of surface charges. ER ¼ Pfluo=Pdefluo / e UdefluoþUeleUfluo ð Þ=kBT ð5Þ ð5Þ We can see that the error ratio is solely decided by the attraction potential, ΔU ¼ Udefluo þ Uele Ufluo. Estimation shows that ΔU ~0.35 eV for a 10 nm QD (see Supplementary Information Calculation), and the corresponding error ratio is about 9 × 10−7. This explains the extremely clean background over a large area of printed nanostructures demonstrated in Fig. 1d. Versatility of the patterning technique. The EFASP technique is versatile for various nanoparticle inks, insensitive to printing parameters, and easy to scale up. As a physical adsorption pro- cess, the EFASP technique is not sensitive to the chemical High spatial accuracy. Another important parameter is the accuracy of the EFASP method. Ideally, all NPs should be limited in the de-ﬂuorinated area. However, from Fig. 1b, we can see that TURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications 5 ARTICLE Methods Finally, the resulting black powder was collected by centrifugation and re-dispersed into 10 mL cyclohexane. A NP th i d f ll 34 F th th i f 5 A NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 –0.25V –0.60V –1.00V 0 V 11 nm 0 V 12 nm 0V 17 nm Charge potential / V –1.5 –1.2 –0.9 –0.6 –0.3 Number of particles 0 50 100 150 8 20 53 70 80 89 125 a c 0 V b 33 nm Dx ~10 nm d –0.3 V –0.7 V –1.2 V –7 V Fig. 4 Tunable pixel size and printed interdigitated nanoelectrodes. a Tuning the pixel size (the number of NPs on each spot) by adjusting the charge potential. From left to right, they are KPFM potential mapping, corresponding AFM height mapping, SEM, and PL images (inset) of CsPbBr3 QD patterns. Scale bar, 500 nm. b The near-linear relationship between the average number of NPs on each spot and the charge potential. Error bars represent the standard deviation of the NPs numbers on 100 spots of each potential. c KPFM potential image and AFM height mapping of interdigitated nanoelectrode printed with 10-nm Au NPs. Scale bar, 1 μm. d Corresponding SEM of the interdigitated nanoelectrode and an enlarged part, showing a ﬁnger width of 150 nm with variation less than 10 nm. Scale bar, 1 μm (left), and 200 nm (right). –0.25V –0.60V –1.00V 0 V 11 nm 0 V 12 nm 0V 17 nm a –0.3 V –0.7 V –1.2 V Charge potential / V –1.5 –1.2 –0.9 –0.6 –0.3 Number of particles 0 50 100 150 8 20 53 70 80 89 125 b b a Charge potential / V c 0 V 3 n –7 V Dx ~10 nm d d c Fig. 4 Tunable pixel size and printed interdigitated nanoelectrodes. a Tuning the pixel size (the number of NPs on each spot) by adjusting the charge potential. From left to right, they are KPFM potential mapping, corresponding AFM height mapping, SEM, and PL images (inset) of CsPbBr3 QD patterns. Scale bar, 500 nm. b The near-linear relationship between the average number of NPs on each spot and the charge potential. Error bars represent the standard deviation of the NPs numbers on 100 spots of each potential. c KPFM potential image and AFM height mapping of interdigitated nanoelectrode printed with 10-nm Au NPs. Scale bar, 1 μm. d Corresponding SEM of the interdigitated nanoelectrode and an enlarged part, showing a ﬁnger width of 150 nm with variation less than 10 nm. Scale bar, 1 μm (left), and 200 nm (right). NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 5. Talapin, D. V., Lee, J.-S., Kovalenko, M. V. & Shevchenko, E. V. Prospects of colloidal nanocrystals for electronic and optoelectronic applications. Chem. Rev. 110, 389–458 (2010). The ﬁnal NaYF4:Yb,Er NPs was re-dispersed in 5 mL of cyclohexane after washing with cyclohexane/acetone two times. y The CdSe/ZnS core/shell NP dispersions were purchased from Suzhou Xingshuo Nanotech Co., Ltd. 6. Begley, M. R., Gianola, D. S. & Ray, T. R. Bridging functional nanocomposites to robust macroscale devices. Science 364, eaav4299 (2019). g All the TEM images of the functional NPs are shown in Supplementary Fig. 12. All the TEM images of the functional NPs are shown in Supplementary Fig. 12. 7. von Bojnicic-Kninski, C. et al. Combinatorial particle patterning. Adv. Funct. Mater. 27, 1703511 (2017). Charge writing and NP assembly. Charge writing was performed using a cus- tomized AFM-based direct electrostatic charge writing system, which contains an atomic force microscope from NT-MDT NTEGRA PROBE NANOLABORATORY mounted with TipsNano HA_NC/Pt tips (235/140 kHz, 12/3.5 N/m, 20-30 nm Pt coating) and an external high voltage ampliﬁer manufactured by THORLABS (model: HVA200). A commercial ﬂuorine polymer CYTOP (perﬂuoro (1-butenyl vinyl ether)) and its special dilution solvent (perﬂuorocarbons, PFC) were used as a substrate (purchased from ASAHI GLASS COMPANY, Japan). Typically, CYTOP powder was diluted to 1–3 wt% with solvent and spin-coated onto silicon wafers, with spin speed, spin time, and acceleration set as 2500 rpm, 1 min, and 1200 r/ min2, respectively. Then the substrate was baked on a hot plate at 100 oC for 10 min to remove residual solvent. For writing charged patterns on the polymer substrate, the tips were loaded at high voltage and driven using the Litho mode of Nova software. The z-feedback was adjusted to control the tip–substrate distance during charge writing. In order to import a preset pattern, Vector mode was selected to write the simple dot charge matrix, and Raster mode was used to write the complex charge patterns. The time on a single point was ﬁxed at 100 ms and 18 ms for Vector and Raster mode, respectively. 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The typical spin speed, spin time, and acceleration were set as 6000 rpm, 25 s, and 1200 r/min2, respectively. The typical dispersion concentration was 1.5 × 1013 NPs/mL. As long as the dispersion covered the charge pattern, NPs could be successfully assembled onto the designated area. For Supplementary Movie 1, we just put one drop of the NP dispersion (10 μL) next to the pattern, and then use a needle to swipe the drop through the patterns (around 0.5 cm/s). The NPs can be assembled on the patterns quickly and efﬁciently. All the operations can be performed in the open system, generally in the fume hood at RT, including spin-coating, dip coating, or brushing. 18. Jacobs, H. O., Campbell, S. A. & Steward, M. G. Approaching nanoxerography: the use of electrostatic forces to position nanoparticles with 100 nm scale resolution. Adv. Mater. 14, 1553–1557 (2002). 100 nm scale resolution. Adv. Mater. 14, 1553–1557 (2002 19. Jacobs, H. O. & Whitesides, G. M. Submicrometer patterning of charge in thin-ﬁlm electrets. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 Science 291, 1763 (2001). 20. Mesquida, P. & Stemmer, A. Attaching silica nanoparticles from suspension onto surface charge patterns generated by a conductive atomic force microscope tip. Adv. Mater. 13, 1395–1398 (2001). 21. Palleau, E., Sangeetha, N. M., Viau, G., Marty, J.-D. & Ressier, L. Coulomb force directed single and binary assembly of nanoparticles from aqueous dispersions by AFM nanoxerography. ACS Nano 5, 4228–4235 (2011). Measurement and characterization. All the AFM-based experiments were car- ried on an INTEGRA SPM Controller of NT-MDT. After AFM charge writing, the surface potential of the charge patterns (relative to the surrounding uncharged polymer substrate) was measured by Kelvin Probe Force Microscopy (KPFM) in semi-contact mode. In the KPFM experiments, double path measurement was used, and the typical lift height was 10 nm. After NP assembling, the height images were obtained by tapping mode scanning (carrying an NSG10 tip of TipsNano). Th ﬂ f d b d 22. Seemann, L., Stemmer, A. & Naujoks, N. Selective deposition of functionalized nano-objects by nanoxerography. Microelectron. Eng. 84, 1423–1426 (2007). 23. Seemann, L., Stemmer, A. & Naujoks, N. Local surface charges direct the deposition of carbon nanotubes and fullerenes into nanoscale patterns. Nano Lett. 7, 3007–3012 (2007). 24. Palleau, E. & Ressier, L. Combinatorial particle patterning by nanoxerography. Adv. Funct. Mater. 28, 1801075 (2018). The ﬂuorescence images of quantum dot patterns were obtained using a ﬂuorescence microscope (Newton EMCCD, ANDOR) with a 405-nm laser. The PL spectrum was measured using an SR-303i-B spectrograph of Andor (EU). The single point FL spectrums of low temperature were measured by Andor spectrograph at 4 K. 25. Min, Y., Akbulut, M., Kristiansen, K., Golan, Y. & Israelachvili, J. The role of interparticle and external forces in nanoparticle assembly. Nat. Mater. 7, 527 (2008). 26. Morales, D., Teulon, L., Palleau, E., Alnasser, T. & Ressier, L. Single-step binary electrostatic directed assembly of active nanogels for smart concentration-dependent encryption. Langmuir 34, 1557–1563 (2018). g The SEM images were captured using a Zeiss Ultra55 ﬁeld-emission SEM operated at 15 kV. X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Scientiﬁc, UK) were in situ measured after writing dense charge lattice with 200 × 200 μm area. The TEM images were captured by FEI Tecnai F20 G2 TEM. 27. Sangeetha, N. M. et al. 3D assembly of upconverting NaYF4 nanocrystals by AFM nanoxerography: creation of anti-counterfeiting microtags. Nanoscale 5, 9587–9592 (2013). Received: 15 February 2020; Accepted: 22 October 2020; 30. Barry, C. R., Gu, J. & Jacobs, H. O. Charging process and Coulomb-force- directed printing of nanoparticles with sub-100-nm lateral resolution. Nano Lett. 5, 2078–2084 (2005). 31. Barry, C. R., Lwin, N. Z., Zheng, W. & Jacobs, H. O. Printing nanoparticle building blocks from the gas phase using nanoxerography. Appl. Phys. Lett. 83, 5527–5529 (2003). Discussion h After the solution was cooled down, acetone was added to precipitate the nanoparticles. 6 ARTICLE ARTICLE NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunicati Data availability 28. Palleau, E., Sangeetha, N. M. & Ressier, L. Quantiﬁcation of the electrostatic forces involved in the directed assembly of colloidal nanoparticles by AFM nanoxerography. Nanotechnology 22, 325603 (2011). The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. nanoxerography. Nanotechnology 22, 325603 (2011). 29. Israelachvili, J. N. Intermolecular and Surface Forces. 3 edn, (Academic Press, 2011). Received: 15 February 2020; Accepted: 22 October 2020; Received: 15 February 2020; Accepted: 22 October 2020; NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19771-0 Author contributions 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/. X.X., Z.M., and J.B. contributed equally to this study. W.Z. and Z.L. conceived the project and designed the experiments. X.X. and Z.L. synthesized the nanoparticles dispersed in the oil-phase. W.Z. Z.M. and J.B. developed the nanoscale charging and deﬂuorination tech- nique for ﬂuorinated substrates. Z.M. and X.X. characterized the samples and collected all the data. W.Z. carried out the theoretical calculations. Z.L., Z.W., and Y.Y. wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing interests p g The authors declare no competing interests. Acknowledgements This study was supported by grants from the National Key Technologies R&D Program of China (No. 2016YFA0201104), the National Basic Research Program of China (No.2015CB659400), the National Natural Science Foundation of China (No. 11574142, 11621091, and 22075128). This study was also supported by the Fundamental Research Funds for the Central Universities (14380166), Jiangsu Innovative and Entrepreneurial Talent Award, and State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS2017). We thank Prof. Xiaoyong Wang for the measurement of emission spectra of perovskite nanocrystals on the single-pixel at 4 K. We also thank the assistance from the Technical Center of Nano Fabrication and Characterization, Nanjing University for the TEM characterization. Peer review information Nature Communications thanks Yong Lei, Etienne Palleau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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Additional information © The Author(s) 2020 Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-19771-0. © The Author(s) 2020 Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-19771-0. NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS \| (2020) 11:6002 \| https://doi.org/10.1038/s41467-020-19771-0 \| www.nature.com/naturecommunications
https://openalex.org/W2864095105	https://europepmc.org/articles/pmc6334715?pdf=render	English	null	A Web-Based Interactive Tool to Reduce Childhood Obesity Risk in Urban Minority Youth: Usability Testing Study	JMIR formative research	2,018	cc-by	15,438	A Web-Based Interactive Tool to Reduce Childhood Obesity Risk in Urban Minority Youth: Usability Testing Study Sandra Verdaguer1,2, MPH, MSc; Katrina F Mateo1,2, MPH; Katarzyna Wyka2, PhD; Tracy A Dennis-Tiwary3,4, PhD; May May Leung1, PhD, RDN 1School of Urban Public Health, Hunter College, The City University of New York, New York, NY, United States 2Graduate School of Public Health and Health Policy, The City University of New York, New York, NY, United States 3School of Arts and Sciences, Hunter College, The City University of New York, New York, NY, United States 4The Graduate Center, The City University of New York, New York, NY, United States Corresponding Author: May May Leung, PhD, RDN School of Urban Public Health Hunter College The City University of New York 2180 Third Avenue New York, NY, 10035 United States Phone: 1 212 396 7774 Email: maymay.leung@hunter.cuny.edu Original Paper A Web-Based Interactive Tool to Reduce Childhood Obesity Risk in Urban Minority Youth: Usability Testing Study Sandra Verdaguer1,2, MPH, MSc; Katrina F Mateo1,2, MPH; Katarzyna Wyka2, PhD; Tracy A Dennis-Tiwary3,4, PhD; May May Leung1, PhD, RDN 1School of Urban Public Health, Hunter College, The City University of New York, New York, NY, United States 2Graduate School of Public Health and Health Policy, The City University of New York, New York, NY, United States 3School of Arts and Sciences, Hunter College, The City University of New York, New York, NY, United States 4The Graduate Center, The City University of New York, New York, NY, United States Corresponding Author: Verdaguer et al JMIR FORMATIVE RESEARCH JMIR FORMATIVE RESEARCH Verdaguer et al Original Paper Abstract Background: Childhood obesity is a serious public health issue among minority youth in the United States. Technology-enhanced approaches can be effective for promoting healthy behavior change. Objective: The purpose of this study was to test the usability of prototypes of a Web-based interactive tool promoting healthy dietary behaviors to reduce childhood obesity risk in urban minority youth. The Web-based tool comprised a manga-style comic with interactive features (eg, sound effects, clickable pop-ups), tailored messaging, and goal setting, and was optimized for use on tablet devices. Methods: Latino and black/African American children ages 9 to 13 years were recruited to participate in two rounds of usability testing. A modified think-aloud method was utilized. Self-reported surveys and field notes were collected. Audio recordings and field notes from usability testing sessions were systematically reviewed by extracting and coding user feedback as either positive comments or usability or negative issues. The quantitative data from self-reported questionnaires were analyzed using descriptive statistics. Results: Twelve children (four female; eight black/African American) with a mean age of 10.92 (SD 1.16) years participated. Testing highlighted overall positive experiences with the Web-based interactive tool, especially related to storyline, sound effects, and color schemes. Specific usability issues were classified into six themes: appearance, content, special effects, storyline, terminology, and navigation. Changes to the Web-based tool after round 1 included adding a navigation guide, making clickable icons more visible, improving graphic designs, and fixing programming errors. In round 2 of testing (after modifications to the Web-based tool were incorporated), many of the usability issues that were identified in round 1 did not emerge. Conclusions: Results of testing will inform further development and finalization of the tool, which will be tested using a two-group pilot randomized study, with the goal of reducing childhood obesity risk in minority, low-income youth. (JMIR Formativ Res 2018;2(2):e21) doi:10.2196/formative.9747 JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.1 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ 1School of Urban Public Health, Hunter College, The City University of New York, New York, NY, United States 2Graduate School of Public Health and Health Policy, The City University of New York, New York, NY, United States 3School of Arts and Sciences, Hunter College, The City University of New York, New York, NY, United States 4The Graduate Center, The City University of New York, New York, NY, United States Background Childhood obesity continues to be a serious public health challenge [1]. In the United States, the prevalence of obesity among youth is 18.5% [2]. The challenge remains pronounced particularly in low-income, minority populations. Latino and black/African American children have the highest rates at 25.8% and 22.0%, respectively [2]. Furthermore, adolescents (12-19 years) have the highest prevalence (20.6%) compared to school-aged (6-11 years; 18.4%) and preschool-aged children (2-5 years; 13.9%) [2]. Childhood obesity leads to negative health outcomes, such as type 2 diabetes, cardiovascular disease, and hypertension, which can continue through to adulthood [3-5]. This complex epidemic has been attributed to, among other behaviors, the increased consumption of energy-dense and low-fiber foods [6,7] as well as the reduced consumption of nutrient-dense fruits and vegetables [8,9]. The comic component of the Web-based tool was guided by the narrative transportation theory. The narrative transportation theory explains how narrative communication, such as manga comics, could contribute to changes in health-related beliefs and behaviors by transporting the reader into the narrative world [29]. According to the narrative transportation theory, transportation into a narrative world is believed to lead to acceptance of persuasive messages within a story through multiple mechanisms, which include positive relationships with story characters, lowered resistance to story messages, and similarities to real-world experiences [30-34]. If a reader likes or identifies with a specific character, the events experienced by the character or statements made by the character may have a greater effect in shifting the reader’s beliefs [33,34]. As a result, narrative messages may be more effective than fact-based evidence, particularly when the messages are not similar to one’s own beliefs [29]. Additionally, readers tend to be more engaged with stories that are similar to their personal experiences and cultural values [29]. Thus, embedding health messages into storylines with realistic and relatable scenarios could further engage readers, and thus potentially impact health-related attitudes and beliefs. The narrative transportation theory also suggests that images are most impactful when they are embedded in a story rather than provided in isolation as it could enhance the narrative influence [35]. Therefore, visual images relevant to the story’s message, such as those incorporated in manga comics, may further impact attitudes and beliefs. Effective, yet innovative interventions are needed to capture the attention of children living in a multimedia environment. KEYWORDS ity testing; interactive technology; mHealth, childhood obesity; minority; health nutrition; health educatio JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.1 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ JMIR FORMATIVE RESEARCH Verdaguer et al form of entertainment in many countries, including the United States, irrespective of gender, nationality, or age [25-28]. Although such popularity increases the opportunity for reach of manga comics, components such as story plot and character details (eg, physical features, language use, personal preferences) can be developed to tailor these comics for specific minority populations. Background The pervasiveness of technology and new media use in youth, particularly within the Latino and black/African American population [10-12], highlights opportunities and potential new avenues to engage with this priority population [13]. A systematic review indicated that Web-based programs, as part of a multicomponent intervention, could reduce obesity and overweight in school-aged children [14]. Web-based and technology-assisted interventions, particularly if developed using human-centered approaches and informed by theory [15], have the potential to increase access, improve convenience, decrease cost, and increase participant engagement with dietary behavior change strategies, especially among culturally diverse and hard-to-reach communities [16-19]. At the same time, these types of interventions that allow for flexible engagement with health-related material may require more intrinsic motivation to initiate and maintain engagement over time. Thus, innovative dietary-focused interventions targeting youth should not only incorporate technology, but also integrate engaging features and components to sustain interest and use. Social cognitive theory is a frequently used framework in effective dietary behavior change interventions [36,37], and it also lends explanation to ways in which a manga comic may influence health behavior in youth [22,23]. Exposure to characters in the storylines may facilitate observational learning and influence health behaviors, particularly when readers relate to the comic characters and consider them role models [38]. With input from members of the priority population throughout development, character personalities, interests, and appearances can be designed to increase the likelihood that readers may see them as relatable, and thus role models. The development of similar entertainment-education narratives draws greatly on social cognitive theory by using role models to perform new behaviors [39-41]. Further, the use of relatable characters to illustrate the positive effects of healthy eating and the negative effects of unhealthy eating operationalizes the construct of outcome expectations for comic readers. Thus, an engaging manga comic informed by the narrative transportation theory, which includes health messages and content guided by social cognitive theory, may be an effective vehicle to promote healthy eating behaviors. http://formative.jmir.org/2018/2/e21/ JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.2 (page number not for citation purposes) Theoretical Basis and Content of Intervention INC Theoretical Basis and Content of Intervention INC Intervention INC is a theory-informed, Web-based interactive tool promoting healthy dietary behaviors, specifically increased fruit and vegetable or water intake, with the goal to reduce childhood obesity risk in Latino and black/African American youth. The main component of the Web-based tool is a novel interactive manga comic, optimized for use on tablet devices. Although research is limited, Japanese comic art, commonly known as manga, has previously been used as part of cognitive behavioral therapy to improve depressive symptoms in Japanese adults [20], as a mental health campaign for youth in England [21], and as an obesity prevention tool for minority children in the United States [22,23]. Unlike Western-style comic books, manga are a unique form of multimodal narrative media that stimulate a reader’s attention by combining detailed visual images and text to create more of a subjective viewpoint of a story [24]. Another distinct feature of manga comics is their wider range of genres. Manga comics are an increasingly popular Another key component of the Web-based tool is goal setting. Goal setting is discussed in several behavior change theories, http://formative.jmir.org/2018/2/e21/ JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.2 (page number not for citation purposes) XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al including social cognitive theory and goal-setting theory, and involves a commitment to change through small steps [42-44]. These theories similarly relate goals to outcome expectancy and self-efficacy, both of which are needed for goal commitment and attainment. Further, goal setting and self-monitoring are approaches through which self-regulation is operationalized. In the Web-based tool, goal setting, weekly assessment of goals, and tailored messages and feedback (based on initial screening questions and goal assessments) are integrated as theory-guided approaches to support healthy behavior change. including social cognitive theory and goal-setting theory, and involves a commitment to change through small steps [42-44]. These theories similarly relate goals to outcome expectancy and self-efficacy, both of which are needed for goal commitment and attainment. Further, goal setting and self-monitoring are approaches through which self-regulation is operationalized. In the Web-based tool, goal setting, weekly assessment of goals, and tailored messages and feedback (based on initial screening questions and goal assessments) are integrated as theory-guided approaches to support healthy behavior change. controlled trial (RCT). Study Overview This study is part of a larger study that aims to design, develop, and evaluate the Intervention INC tool. Textbox 1 outlines the multiple phases and research activities of the overall study; research activities specific to this study are marked. The methods described focus on the two rounds of usability testing conducted during the development phase with children using prototypes of the Intervention INC tool. Theoretical Basis and Content of Intervention INC This study also aims to add to the limited literature related to usability testing of Web-based tools with youth by describing usability testing methods used to evaluate a Web-based tool with urban minority preadolescents. The Concept of Usability Testing According to the US Department of Health and Human Services, usability testing refers to evaluating a product or service by testing it with representative users [45]. Usability testing is a crucial step in the development of online health tools and mobile health (mHealth) apps and technologies to ensure that they are accessible, understandable, and useful to end users, and are delivered in an efficient, effective, satisfying, and culturally competent manner [46,47]. Although several studies have emphasized how usability testing can improve technology-based tools [43-45], there is limited research detailing usability testing methods for mHealth tools with youth users, especially younger than 13 years of age [48-53]. A challenge often cited is that traditional usability testing approaches, whether via survey or qualitative methods, are designed for adults and may require different practical, methodological, and ethical considerations with children. The literature also highlights the importance of taking into account individual characteristics that may make it easier or more difficult to participate in these verbal reporting methods, such as level of “extraversion” and “friendliness” [51,53-55]. For example, usability testing done with very young children (younger than 7 years) have highlighted issues related to impatience during testing, unpredictable reactions (especially if the child is uncertain about what to do), and minimal remarks made by users while using a typical think-aloud protocol [55,56]. At the same time, authors have emphasized how behavioral observation (especially during “free play”) often provides the most useful information and insight into usability [55,56]. Although simplifying usability survey questions or think-aloud verbal probes may address issues of literacy and understandability in children, this may also diminish the depth of relevant feedback provided by youth users. Thus, more research is needed to demonstrate successful approaches to usability testing among youth, and particularly among the understudied preadolescent population (9 to 12 years). The lack of published studies in this area suggests that Web-based health promotion tools are being developed without formal involvement or evaluation by potential users, which can impact their potential usefulness, relevance, and effectiveness. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.3 (page number not for citation purposes) Web-Based Tool Description The Web-based tool tested in round 1 and round 2 consisted of a three-chapter interactive nutrition comic with a character profiles section and embedded interactive features (eg, sound effects, character voice-overs, clickable pop-up windows) to engage users. At the end of each chapter, a tailored message from a character was provided to the user promoting either fruit and vegetable or water intake, as well as a prompt to select a goal related to either increasing fruit and vegetable or water intake (tailored content based on initial screening questions and goal assessments). Figure 1 shows a flowchart of the Web-based tool components that were tested within the current study. Sample Participants Formative phase Focus groups or interviews with children and parents Development phase • Internal development of initial Web-based tool concepts • Codesigning of Web-based tool content and design with children and parents • Usability testing of Web-based tool prototypes with children* and parents Evaluation phase Two-group pilot randomized controlled trial to evaluate feasibility and acceptability of Web-based tool with parent-child dyads Textbox 1. Phases and activities of Intervention INC tool design and evaluation. Research activity specific to this study. Formative phase Focus groups or interviews with children and parents Development phase • Internal development of initial Web-based tool concepts • Codesigning of Web-based tool content and design with children and parents • Usability testing of Web-based tool prototypes with children and parents Evaluation phase Two-group pilot randomized controlled trial to evaluate feasibility and acceptability of Web-based tool with parent-child dyads Phases and activities of Intervention INC tool design and evaluation. Research activity specific to this study. Figure 1. Flowchart of Web-based tool components. F/V: fruit/vegetable. Figure 1. Flowchart of Web-based tool components. F/V: fruit/vegetable. The final version of the Web-based tool will include an additional three chapters (six chapters in total), post-chapter trivia questions, and rewards for correct answers, as well as expanded health information and fun-fact pop-ups throughout each chapter to reinforce health messages. Screening questions will also be incorporated and asked first to determine whether the child receives messages, goals, and comic content focused on either fruit and vegetable or water intake. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.4 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ Sample Participants English-speaking Latino and black/African American children ages 9 to 13 years were recruited to participate in two rounds of usability testing to provide feedback and identify problems to help inform final development of the Web-based tool. Participants were recruited from a contact list of 36 children, who had participated in previous formative phase focus groups ot interviews (manuscript under review) and early development phase study sessions (manuscript in preparation). These youth were originally recruited via a community-based organization primarily serving children in high-need New York City neighborhoods and local outreach near businesses within the East Harlem, New York, neighborhood. Eligibility criteria for this prior study sample consisted of the child being between the ages of 9 and 12 years; the child self-identifying as Latino and/or black/African American; the child being English-speaking; the child having internet access, as well as access to a mobile phone or tablet; and the child having an interest in talking about food and technology. We did not screen for reading or digital literacy level as content in the Web-based tool was delivered via multiple mediums, including text, audio, and images. Literature suggests that pictures and audio-assisted reading improves reading comprehension and lowers literacy level of the text [57,58]. Children meeting eligibility criteria were scheduled for a one-on-one usability testing session with a study staff member. Round 1 sessions were conducted in June 2017 and round 2 sessions in July 2017 after certain modifications were made to the Web-based tool. The goal was to recruit five to eight children in each round of usability testing as it has been reported that usability testing with five users will reveal 85% of usability issues [59,60]. Child assent was obtained prior to study participation, in addition to parental permission and a photo release form. Participants received a US $10 gift card and a round trip Metrocard on completion of the session. All study activities were approved by the Institutional Review Board at Hunter College in New York, NY. The purpose of this study was to conduct usability testing with Latino and black/African American preadolescents to evaluate prototypes of Intervention INC. Study results will be used to finalize the tool, which will be evaluated in a pilot randomized http://formative.jmir.org/2018/2/e21/ http://formative.jmir.org/2018/2/e21/ XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al Textbox 1. Phases and activities of Intervention INC tool design and evaluation. Research activity specific to this study. Think-Aloud Training A note-taking guide was also developed for use by the note taker to record observations of participant’s responses (especially nonverbal) during usability testing of the comic section. The note-taking guide included screenshots of each panel of the comic, along with multiple checkboxes (eg, to check which automatic animations displayed automatically), yes or no options (eg, to indicate if user selected a goal), and reminders for the note taker to record start and end times and note general comments. Using this guide, data related to time taken to complete each comic chapter, number of usability issues, frequency of interaction with clickable features within the comic, and specific comments made in each panel of the comic could be collected. When using think-aloud with adults, examples from the literature suggest starting with a practice session where a moderator or evaluator asks the participant to do an example task similar to the target tasks to orient participants to the practice of talking out loud (as opposed to explaining) before actually engaging with the developed tool or system [62,63]. Once the participant starts interacting with the tool or system, evaluators should only intervene when a participant stops verbalizing their thoughts, and only use simple, short, and nondirective prompts such as “keep on talking” to minimize biasing the user to change their behavior. Prior to the usability testing with our Web-based tool, the moderator explained to participants the purpose of the session (eg, to test an early version of a website using a tablet to get feedback and suggestions on how to make it better) and provided instructions on how to “think aloud” while testing different sections of the website. Our protocol included the moderator explaining the concept (eg, “I want you to say out loud what you are thinking as you use the tablet to go to the website”) and providing an example to practice (eg, “I want you to raise the volume of this tablet while thinking out loud”). This example was practiced until the child demonstrated an understanding of how to “think aloud” (eg, explained out loud that he or she is looking for the volume button on the side of the tablet and pressing the “up” button to raise the volume). Data Collection Testing sessions for round 1 and round 2 followed similar procedures. They were conducted in private rooms in a college campus building with two trained researchers (a moderator and a note taker). Demographic information (eg, age, gender, race or ethnicity) and technology use and preferences (eg, “What devices do you use to access the internet or download apps?”) of scheduled child participants were previously assessed during formative phase study sessions via a questionnaire. However, these data were collected from any unscheduled child participants, who attended usability sessions (additional details in Participant Characteristics section). During the usability testing sessions, a combination of qualitative and quantitative methods were implemented. Both methods are essential in the iterative design cycle [61]. Each session consisted of brief think-aloud training, usability testing of the Web-based tool with a modified think-aloud protocol and moderator guide (with The Web-based tool was optimized for use on tablet devices as formative research with a similar population highlighted that most parents reported owning mobile phones and tablet devices, and a majority of children reported preferring tablets over laptops (manuscript in preparation). For the purposes of comic development, tablets were preferred over mobile phones because of the larger display size and also to maintain the touchscreen capabilities. Throughout development, the tool was tested on devices with iOS, Android, or Windows operating systems and across Safari, Chrome, Firefox, and Internet Explorer Web browsers. XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al reading the comic, the participants were specifically encouraged to read aloud, verbalize reactions, and share initial thoughts with the moderator. Examples of think-aloud prompts included, “What is the first thing you notice on this page?” “Can you tell me what you’re doing?” and “Is there anything you would change?” Prompts were also provided to encourage specific feedback once a child experienced any special effects in the comic or interacted with clickable icons (eg, “What did you think about that animation?” “Why did you click that?” “What do you think of that pop-up message?”). examples of prompts to encourage verbalized feedback from participants throughout testing), and a questionnaire to assess usability and acceptability. Each participant accessed the Web-based tool using a touchscreen laptop (Microsoft Surface Pro) as it provided flexibility for the participant to use the device as a computer or tablet. Usability testing sessions were audio-recorded and field notes were taken to document participant’s comments, performance, behaviors, and nonverbal body language. Perceived Usability and Acceptability The perceived usability and acceptability of the Web-based tool was assessed using a questionnaire provided to each participant after the usability testing session. The questionnaire was administered via pen and paper, and the moderator was available to answer any questions about the survey or clarify words that the participant did not understand. The moderator additionally highlighted that this questionnaire was not meant to test the child but was a way for the child to express how easy or hard it was to use the Web-based tool so that the developers could improve it for future users. The questionnaire combined and modified items from the System Usability Scale (SUS) [64], the Usefulness, Satisfaction, and Ease of use (USE) questionnaire [65], and an acceptability/usability measure questionnaire [66] in order to assess five usability domains: usability, usefulness, ease of use, ease of learning, and satisfaction. The combined questionnaire consisted of 37 items scored on a five-point Likert scale from strongly disagree to strongly agree. The usability domain comprised the 10 items from the SUS questionnaire. Two items comprised the usefulness domain (one from the USE and the other from the acceptability/usability measure). Ease of use domain was assessed using 13 items (10 from the USE and three from the acceptability/usability measure). Ease of learning was assessed by the same four items found in the USE questionnaire. The satisfaction domain comprised eight items (all from the USE, except for one that was added from the acceptability/usability measure). http://formative.jmir.org/2018/2/e21/ Think-Aloud Training In addition, as there is limited literature on using the think-aloud method with youth, moderators were prepared to use more directed prompts and questions in the case that child participants forgot to verbalize their thoughts while using the tool. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.5 (page number not for citation purposes) Technology Use Prior to accessing the Web-based tool, participants were asked whether they would prefer to receive messages about the Web-based tool through text message or email. Participants’ preference was text message (7/12, 58%) over email (5/12, 42%). The most common devices used to access the internet or download apps were tablets (11/12, 92%) and mobile phones (10/12, 83%), followed by desktop computer or laptop (8/12, 67%) and xBox (5/12, 42%). Other devices participants reported to use to access the internet or download apps were iPod, Wii, and Kindle. Mobile phones and tablets were the top two devices used most often. However, mobile phones were the preferred devices among participants (8/12, 67%). Although the majority of participants used mobile phones, two of 12 indicated they did not use mobile phones. Among the participants who used mobile phones, four of 10 shared their mobile phones with someone else in the family, normally with their mom and siblings. Table 1 summarizes the participants’ demographic and technology characteristics. To ensure the reliability of the content analysis, the coding and themes were continually validated by two other researchers throughout the analysis process. Specifically, the primary analyst coded the data and then presented the analysis to two other researchers, who reviewed code application to comments or verbalizations. If any inquiries or disagreements arose regarding codes and themes, the three researchers discussed and resolved any discrepancies. Coding revision and theme refinement continued until data analysis was complete. Field notes were reviewed to help inform analysis. Data Analysis Analysis of think-aloud data, including coding categories and themes were guided by approaches used in previous literature [46,67,68]. Audio recordings from usability testing sessions were not transcribed verbatim as the context of user interactions with the tool (eg, audio of character dialog prompted by touching interactive icons) would be more evident from listening to and directly analyzing audio recordings [69,70]. Microsoft Excel version 15.33 was used to assist with data organization and analysis. For both round 1 and round 2, the audio recordings and field notes were systematically reviewed. First, child utterances during usability testing were extracted and coded as either positive comments or usability issues (which also included negative comments verbalized by participants). Similar or related comments were then grouped into themes and subthemes. Each code was counted in coding units. Coding units consisted of sentences or reactions from the participants and programming glitches counted during usability testing. The major coding rules were as follows: (1) multiple sentences or reactions that referred to the same matter were coded as one unit (eg, if a participant made multiple comments about a picture being too small, they were all counted as one unit); (2) agreements between participants on the same matter in dyad sessions were counted as two units (eg, if a participant made a comment and his or her pair agreed, the two comments were counted separately); and (3) programming glitches that occurred during dyad sessions were counted as one unit. Usability Testing of Web-Based Tool Participants were first asked if they would prefer to receive a message with a link to the website by text or email. They were then provided with a printed sample text or email message that included the website URL. The moderator asked the participant if he or she knew what to do next (ie, click on website URL or open a browser to type in the URL). Once the website URL was entered into a browser on the tablet, a log-in page was displayed with a form to enter a username and password. The moderator provided the username and password for participants, and observed if the participant was able to enter in the information to log in. Once logged in, participants were allowed to navigate freely through the different sections of the Web-based tool, but were guided to cover all the sections, which included comic chapters, goals, the message board, and character profiles (see Figure 1). The combined questionnaire was pilot tested in earlier development phase study sessions with a similar population of children ages 9 to 12 years (manuscript in preparation). Based on this previous testing, some modifications were made to tailor the questionnaire according to children’s literacy levels for this study. For example, the item “I found the system very Throughout usability testing of the Web-based tool, participants were encouraged to think aloud to explain what they were thinking as they were navigating through the sections. While XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al cumbersome to use” was replaced with “I found the website very awkward to use,” and “I would imagine most people would learn to use this system very quickly” was changed to “I think most people my age would learn to use this website very quickly.” Additionally, changes were made to make the questions more appropriate for our Web-based tool. For example, the word “system” or “tool” was replaced with the term “website.” Participant Characteristics A total of 12 children (n=6 per round) were recruited. Round 1 consisted of two dyad sessions and two individual sessions and round 2 consisted of six individual sessions. Although dyad sessions were not a part of the initial study design, they were conducted in round 1 as two scheduled children brought their relatives. The overall age of participants was mean 10.92 (SD 1.16) years (range 9 to 13 years). The mean age of participants in round 1 was slightly higher than in round 2 (mean 11.17, SD 1.33 years and mean 10.67, SD 1.03 years, respectively). The majority of participants were male (n=8) and black/African American (n=8). Among the 12 participants, eight (three in round 1; five in round 2) were involved in the codesigning process of the Web-based tool and participated in a previous usability session of the first prototype. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.6 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ Type of smartphone, n iPhone Participants who share mobile phone with other family members, n Participants who have been involved in the codesigning process of the Web-based tool, n Participants who have been involved in a previous usability session, n although they thought the guide was useful, it was not necessary to include it at the start of each chapter. Appearance referred to the impressions of how the Web-based tool looked and included the design, layout, illustrations, font, and colors. Participants approved of the comic illustrations and the overall design of the Web-based tool. One participant mentioned this referring to the illustrations of the comic: “I like it’s [the comic] anime.” However, they had complaints on the colors as the comic was in black and white with only some instances of color. One participant expressed “I would like it [the comic illustrations] better in color, we are in 2017!” Special effects were comic features, including sound effects, voice-overs of some selected character dialog, clickable pop-up windows with additional information, and animation, meant to increase immersion into and engagement with the comic. Participants commented positively on them and asked for more special effects. Suggestions were even provided as to specific scenes in the comic where additional special effects could be incorporated. Some of the quotes were: “It is funny that he’s [the chameleon] blinking his eyes” and “It would be cool if they [the characters] were moving. Kind of funny too.” Content included information that was delivered through the Web-based tool. Participants found the information provided in the character profiles section most interesting. Participants expressed particular interest in the character’s favorite recipes, hobbies, and favorite links (ie, external online games and apps). One participant mentioned wanting to know the character’s favorite color. Participants also reported liking the fun facts. For example, one participant said “Interesting, I didn’t know that [basketball fun fact].” However, three round 2 participants felt that some of the pop-ups and post-chapter messages were “off topic” or not relevant to the story. Participants in round 1 suggested that a guide could be added to learn how to use the interactive features within the comic. However, after the guide was included, some round 2 participants commented that Storyline comprised any comment related to the plot of the nutrition comic. Overall, the storyline was positively received, especially the flashbacks (ie, of character memories) and the “love triangle” between characters. Usability Testing Themes Overall mean testing time was 65 (SD 12) minutes with mean time in round 1 slightly higher than in round 2 (mean 67, SD 8 minutes vs mean 63, SD 15 minutes, respectively). Testing revealed a total of 586 comments or reactions. A greater number of comments and reactions were collected in round 1, especially during dyad usability sessions (329 collected in round 1, 257 collected in round 2). Multimedia Appendix 1 provides a summary of participants’ comments and reactions identified from the content analysis, which have been classified under six themes: appearance, content, special effects, storyline, terminology, and navigation. Additional comments were labeled under general feedback. Participants’ comments and reactions were further categorized as either positive comments or usability issues. Overall, there were more positive comments (70.8%, 233/329 in round 1; 65.8%, 169/257 in round 2) compared to usability issues (29.2%, 96/329 in round 1; 34.2%, 88/257 in round 2) in both rounds. The quantitative data from self-reported questionnaires about participant’s usability and acceptability of the Web-based tool across the five domains were analyzed using SPSS version 22 and Microsoft Excel version 15.33 to calculate the means, standard deviation, and ranges (minimum-maximum) for the overall score as well as subscales. For usability domain questions (10 items), separate means, standard deviation, and ranges were also calculated based on the SUS scoring protocol [64]. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.6 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Table 1. Demographic characteristics and technology use of participants. http://formative.jmir.org/2018/2/e21/ JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.7 (page number not for citation purposes) Usability Testing Themes Total (N=12) Round 2 (n=6) Round 1 (n=6) Characteristics 10.92 (1.16) 10.67 (1.03) 11.17 (1.33) Age (years), mean (SD) Gender, n 8 4 4 Male 4 2 2 Female Race, n 8 4 4 Black/African American 4 2 2 Latino Preferred notification platform, n 7 3 4 Text message 5 3 2 Email Devices used to access internet or download apps, n 11 5 6 Tablet 10 5 5 Mobile phone 8 5 3 Desktop computer or laptop 5 2 3 Xbox 3 1 2 Other (eg, iPod, Wii, Kindle) Type of smartphone, n 6 2 4 Android (eg, Samsung) 4 3 1 iPhone 2 1 1 Do not use a phone 4 1 3 Participants who share mobile phone with other family members, n 5 3 2 Participants who have been involved in the codesigning process of the Web-based tool, n 3 2 1 Participants who have been involved in a previous usability session, n Verdaguer et al JMIR FORMATIVE RESEARCH JMIR FORMATIVE RESEARCH Verdaguer et al Modifications to the Web-Based Tool Between Rounds 1 and 2 Terminology referred to the words, abbreviations, and onomatopoeia used in the Web-based tool. There were a few words that participants had trouble reading, such as “high-fructose corn syrup” and “hypertonic solution.” Participants stated they did not know the meaning of some words and abbreviations (“What does NPS mean?” “What is an athlete?”). No problems were encountered with the onomatopoeia as children correctly identified the intended sounds. Although round 1 participants provided multiple suggestions and different usability issues were detected, modifications to the Web-based tool between round 1 and round 2 had to be prioritized. Prioritization adjustments were based on what the researchers believed would have the largest positive effect on usability. Additionally, adjustments were chosen based on the time, resources, and skills available on the development team. The issues and problems highlighted by round 1 users that we sought to address with modifications between round 1 and round 2 included the following: (1) clickable icons for information pop-ups, sound effects, or character dialog were not obvious; (2) the touch feature to “swipe” pages was not intuitive; (3) the siblings in the comic story did not look related; and (4) multiple programming errors were identified (eg, tips not displaying after goals being selected, sound effect of “swiping” page not playing). Modifications to the Web-based tool to address these issues after round 1 included (1) making clickable icons more obvious and visible (changing shape, color, and pop-out effect), and improving graphic design, such as a making a more unified and vibrant color scheme, forms; (2) adding background and pop-up images; (3) adding a navigation guide to highlight how to identify and use touch features, including clickable icons and “swiping” the comic pages; (4) altering or improving comic illustrations; and (5) fixing programming errors. Figures 2-4 are screenshots of some of the additions and modifications to the Web-based tool. Navigation reflected the way a user navigated the Web-based tool to complete tasks. For one participant, the steps that should have been followed to access the Web-based tool (ie, open a browser and typing in URL) were unclear. Three participants also pointed out that they did not know what to do after completing a section or a task. Their suggestions included adding some guidance texts such as “type this link into your browser” and “check back next week for a new chapter.” On the other hand, participants also provided positive comments related to navigation. Type of smartphone, n Participants were generally very engaged while reading the comic, often using vocal inflections to express reactions or, at times, reading character dialog out loud and mimicking the character voice. In general, participants thought the comic was humorous and chapters had interesting endings, which made them eager to read subsequent chapters. Participants also mentioned liking the characters and relating to at least one of them. However, there were parts of XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al it [the Web-based tool], I want it on my phone!” and “I would give it [the Web-based tool] a 9.9!!!” it [the Web-based tool], I want it on my phone!” and “I would give it [the Web-based tool] a 9.9!!!” the storyline where the older participants had other expectations. One participant mentioned, “That’s it? The worm thing...Oh, I thought it would be something different.” JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.9 (page number not for citation purposes) Modifications to the Web-Based Tool Between Rounds 1 and 2 Turning pages was often an issue for participants as the touch area to “swipe” was narrow and not as obvious to users. Four participants were confused on how to go back to the main page, commenting that “you should make Home link bigger and more obvious.” One participant said, “I love being able to swipe and zoom in.” General feedback included any other broad comments related to the Web-based tool. Overall, participants’ general feedback was very positive. For example, two participants said, “I liked Figure 2. Screenshot of the navigation guide added as a modification to the Web-based tool after round 1 of usability testing. Figure 2. Screenshot of the navigation guide added as a modification to the Web-based tool after round 1 of usability testing. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.8 (page number not for citation purposes) JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.8 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ http://formative.jmir.org/2018/2/e21/ JMIR FORMATIVE RESEARCH Verdaguer et al 3. Screenshots of the clickable icon modification added to the Web-based tool after round 1 of usability testing premodification (top) and odification (bottom). modifications may have impacted users’ usability in round though none of the six round 1 participants clicked the al effects icons initially without being prompted by the rator, all six round 2 participants selected these clickable without any prompts. Also, the proportion of participants navigated the comic pages by swiping was higher in round m 1/6 in round 1 to 3/6 in round 2). Additionally, it may been clearer to round 2 users that they needed to select a goal at the end of each comic chapter (see Figure 4). In round 1, only 2 of 6 participants understood that they had to select a goal after viewing the goal-setting page for the first time at the end of the chapter. However, all six round 2 participants selected a goal without prompting by the moderator. Lastly, there was an 80% reduction in unique programing glitches and errors in round 2 after modifications to the Web-based tool were made after round 1 (20 reported in round 1, 4 reported in round 2). the clickable icon modification added to the Web-based tool after round 1 of usability testing premodification (top) and 3. Modifications to the Web-Based Tool Between Rounds 1 and 2 Screenshots of the clickable icon modification added to the Web-based tool after round 1 of usability testing premod dification (bottom). Figure 3. Screenshots of the clickable icon modification added to the Web-based tool after round 1 of usability testing premodification (top) and postmodification (bottom) Figure 3. Screenshots of the clickable icon modification added to the Web-based tool after round 1 of usability testing premodification (top) and postmodification (bottom). The modifications may have impacted users’ usability in round 2. Although none of the six round 1 participants clicked the special effects icons initially without being prompted by the moderator, all six round 2 participants selected these clickable icons without any prompts. Also, the proportion of participants who navigated the comic pages by swiping was higher in round 1 (from 1/6 in round 1 to 3/6 in round 2). Additionally, it may have been clearer to round 2 users that they needed to select a goal at the end of each comic chapter (see Figure 4). In round 1, only 2 of 6 participants understood that they had to select a goal after viewing the goal-setting page for the first time at the end of the chapter. However, all six round 2 participants selected a goal without prompting by the moderator. Lastly, there was an 80% reduction in unique programing glitches and errors in round 2 after modifications to the Web-based tool were made after round 1 (20 reported in round 1, 4 reported in round 2). JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.9 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al Figure 4. Screenshots of the goal setting modification added to the Web-based tool after round 1 of usability testing premodification (top) postmodification (bottom). Figure 4. Screenshots of the goal setting modification added to the Web-based tool after round 1 of usability testing premodification (top) and t difi ti (b tt ) igure 4. Screenshots of the goal setting modification added to the Web-based tool after round 1 of usability testing premodification (top) and ostmodification (bottom). JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.10 (page number not for citation purposes) Participant’s Usability and Acceptability Questionnaire p y p y Q Table 2 highlights the mean scores of the five usability domains (usability, usefulness, ease of use, ease of learning, and satisfaction) for round 1, round 2, and combined (rounds 1 and 2). The combined total score of perceived usability and acceptability of the Web-based tool was high (total mean 4.29, SD 0.99, range 3.19-4.81). Additionally, all five usability domains had combined scores of over 4.00. Specifically, the ease of learning and satisfaction domains had the highest combined scores (mean 4.60, SD 0.71, range 3.25-5.00 and mean 4.48, SD 0.83, range 2.88-5.00, respectively). In round 1 specifically, all domains had a mean score higher than 4.00, ranging from mean 4.40 (SD 1.04, range 3.80-4.80) for usability to mean 4.96 (SD 0.20, range 4.75-5.00) for ease of learning. In round 2, three out of five domains had a mean score of 4.00 or greater. The usefulness and usability domains scored lowest with scores of mean 3.67 (SD 1.23, range 3.00-5.00) to mean 3.80 (SD 1.16, range 3.10-4.60), respectively. Only two individual questionnaire items of the 37 had a mean score lower than 3.00. In round 1, the item “I can use it without written instructions” (item under ease of use domain) had a mean score of 2.67 (SD 1.03, range 1.00-4.00). However, the same item in round 2 had a mean score of 4.00 (SD 1.10, range 2.00-5.00). In round 2, the item “I felt very confident using the website” (item under usability domain) had a mean of 2.33 (SD 1.51, range 1.00-5.00). This same item in round 1 had a mean score of 4.67 (SD 0.52, range 4.00-5.00). In separate scoring of the usability domain questions (10 items) according to the SUS protocol [64], the overall usability was relatively high (total mean 77.08, SD 13.97), with round 1 participants rating the usability of the Web-based tool higher than round 2 participants (mean 85.00, SD 8.94 and mean 69.17, SD 14.11, respectively). Usability testing also reaffirmed the feasibility and acceptability of embedding health information into narratives, as well as the importance of using interactive features to enhance engagement and assist with accessibility. For example, interactive features such as pop-ups with specific health information and accompanying images may increase engagement with the content. In addition, the use of embedded (clickable) audio recordings for long character dialog can help with the literacy of the comic [72]. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.10 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al Table 2. Participant’s perceived usability and acceptability of the Web-based tool.a Combined (n=12) Round 2 (n=6) Round 1 (n=6) Domain (37 items) Range Mean (SD) Range Mean (SD) Range Mean (SD) 3.10-4.80 4.10 (1.14) 3.10-4.60 3.80 (1.16) 3.80-4.80 4.40 (1.04) Usability (10 items) 3.00-5.00 4.17 (1.09) 3.00-5.00 3.67 (1.23) 3.50-5.00 4.67 (0.65) Usefulness (2 items) 2.85-4.92 4.25 (0.98) 2.85-4.92 4.00 (1.04) 4.31-4.69 4.50 (0.85) Ease of use (13 items) 3.25-5.00 4.60 (0.71) 3.25-5.00 4.25 (0.85) 4.75-5.00 4.96 (0.20) Ease of learning (4 items) 2.88-5.00 4.48 (0.83) 2.88-5.00 4.23 (1.02) 4.38-5.00 4.73 (0.49) Satisfaction (8 items) 3.19-4.81 4.29 (0.99) 3.19-4.81 4.00 (1.07) 4.41-4.76 4.58 (0.81) Total aAssessment questionnaire was developed by using a combination of items from the System Usability Scale [64], Usefulness, Satisfaction, and Ease of use questionnaire [65], and acceptability/usability measure [66]. Response options ranged from 1 (strongly disagree) to 5 (strongly agree). Table 2. Participant’s perceived usability and acceptability of the Web-based tool.a aAssessment questionnaire was developed by using a combination of items from the System Usability Scale [64], Usefulness, Satisfaction, and Ease of use questionnaire [65], and acceptability/usability measure [66]. Response options ranged from 1 (strongly disagree) to 5 (strongly agree). We observed that round 1 participants did not engage with interactive clickable icons. Further probing revealed that, in most cases, users overlooked these icons despite moderators noting that there were interactive features in the comic. Adding a “Guide to Interactive Features” at the beginning of each chapter may have addressed this usability issue, evident by the fact that all round 2 participants clicked on the icons without prompting by the moderator. Except for incorporating a guide, no other content was added to the Web-based tool. However, three round 2 participants mentioned that certain pop-ups and post-chapter messages were “off topic” or not relevant. As each comment made by these participants was counted as a usability issue, this may have contributed to a higher number of content issues noted in round 2. Also, some participants were not familiar with browsers and one participant experienced difficulties when asked to type in URL links. Usability testing revealed the importance of providing training or including user guides for technology-based tools. Participant’s Usability and Acceptability Questionnaire Overall, the use of a comic-style narrative to communicate health information is an approach to delivering content to low-literate readers [73,74]. In our study, all participants demonstrated great interest in the comic storyline and interactive features (ie, special effects, interactive pop-up, and swiping pages), and in some cases even provided suggestions on how to increase interactivity with the tool. This finding supports other usability studies conducted by the Nielsen Norman Group (leading user interface and user experience consulting firm), which concluded youth younger than age 12 This is consistent with a previous study in which youth participants needed a short training session prior to engaging with a Web-based program which was focused on increasing physical activity [71]. Although youth are familiar with technology and tablet-optimized tools such as apps, they may need some training at the beginning of Web-based interventions to learn how to access online tools. http://formative.jmir.org/2018/2/e21/ JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.11 (page number not for citation purposes) Principal Findings This study describes the methods and results of usability testing of Intervention INC, a Web-based tool to promote healthy dietary behaviors in Latino and black/African American youth. Overall evaluation of the prototypes tested over two rounds revealed positive experiences with the Web-based interactive tool and opportunities to incorporate additions to increase engagement and improve usability. XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al years prefer animation and sound effects and enjoy “hunting for things to click” [75]. participants were not asked to read aloud, most of the kids preferred to. This allowed us to successfully identify reading and comprehension issues, which were addressed in the final Web-based tool. From the usability issues identified during round 1, modifications were made, such as incorporating an interactive feature guide and improving the comic’s graphic designs (eg, improved clickable icons and character features). These modifications appeared to enhance round 2 usability based on observation and qualitative feedback. In addition, the improved score for the questionnaire item “I can use it without written instructions” from round 1 to round 2 may be a positive indicator of the impact of an incorporated user guide. However, in general, scores from the usability and acceptability questionnaire (both from the overall questionnaire and just the SUS usability questionnaire items) were slightly higher in round 1 than in round 2. Although the sample was not large enough to make powered comparisons, the scores may have dropped because round 2 had a higher number of participants who were involved in the codesigning process or a previous usability session. Those who participated in previous Web-based tool development activities may have had higher expectations of the Web-based tool than the participants who did not have prior exposure. Age could be another explanation as children become substantially more Web-savvy as they get older [76,77]. Round 1 participants were, on average, half a year older than round 2 participants. Half a year may be a significant amount of time in relation to cognitive or literacy development, particularly with school-aged youth [78]. This age difference (and possible differences in reading or computer and digital literacy associated with age) could also explain why round 1 participants scored the item “I felt very confident using the website” with a much higher mark than round 2 participants. Implications for Future Research Our study highlights the need for further research to be conducted to refine the approaches utilized and to further elaborate on our initial findings related to usability testing with youth, particularly with minority, urban preadolescents. However, multiple insights were gained during this study. First, the modified think-aloud approach used with preadolescents, especially in dyad sessions, were successful in collecting meaningful feedback. In future usability studies, we would continue to engage dyads, in combination with individuals, to evaluate Web-based tools with youth. Secondly, although we encouraged the participants to read the comic aloud, this was not mandatory. During future testing, we would request that all participants read aloud as this would allow for the proper assessment of literacy levels and identification of any reading and comprehension issues across all participants. From an evaluation perspective, we were unable to make direct comparisons between round 1 and round 2 as the participants differed between these two rounds of testing. Thus, future research may consider using the same participants across usability testing rounds, such that direct assessments and comparisons could be conducted. This approach may also have the added benefit of the ability to assess the impact of added or changed components (such as a navigation guide) on usability. Lastly, including a third round of usability testing to study how participants interact with the final product in a real-world setting (without guidance of a moderator) would have been informative. During this third round of usability testing, uninterrupted observational approaches such as screen recordings to capture interactions, along with participants’ voice (audio) as they are completing key tasks, would provide insight into any usability issues that may be encountered outside of a testing setting. Modified Think-Aloud Approach Used With Our Participants Previous studies recommend conducting usability testing with potential users prior to outcome assessment in studies involving larger samples [48,79,80]. The think-aloud method is commonly used as a usability testing approach among adults [81-83]. However, there are limited references in the literature describing the think-aloud approach in youth usability testing, and most have been conducted with older youth [46,84]. For this study, we modified this method by helping youth to express what they were thinking with directed questions and probes. We found that using a modified think-aloud approach was successful in eliciting important feedback to improve user experience. Usability guidelines recommend limiting testing sessions with youth to less than 25 minutes or using multiple stations to break up and vary the modes of engagement [85]. However, we were able to successfully keep youth engaged in usability testing for more than 60 minutes. Our approach provided structured and continuous opportunities for participants to verbalize their thoughts and encourage feedback. In addition, although Principal Findings In addition, since some round 1 participants interacted with the tool as a dyad, they may have perceived the tool as having higher usability because they were able to navigate through the tool with a partner. Even if a child may have encountered a usability issue, these may not have been captured or explicitly experienced if the other child was not experiencing the same issue or helped the other child either consciously or unconsciously. It should be noted that our protocol aimed to conduct individual sessions. However, two dyad sessions were conducted in round 1, and we observed a greater number of comments in round 1 compared to round 2. One explanation for this is that having two participants in round 1 sessions provided many more comments than the individual sessions. Future usability testing of Web-based tools with youth using a modified think-aloud approach should consider dyad assessments (rather than individual) to facilitate more meaningful feedback in a peer-to-peer environment. Indeed, some of the limited evidence of usability testing with youth have discussed the benefits of a similar approach, referred to as “constructive interaction,” and the impact of different factors (eg, nonacquainted vs acquainted dyads, same gender dyads) on the identification of usability problems [86-89]. http://formative.jmir.org/2018/2/e21/ JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.12 (page number not for citation purposes) Conclusions Usability testing is critical during the developmental process of Web-based tools because it can enhance a tool’s usefulness, engagement, and potential effectiveness for end users. This study adds to the limited literature related to usability testing of Web-based tools with youth by describing modified usability testing methods used to evaluate the Intervention INC tool with urban minority preadolescents. The authors engaged youth during usability testing sessions using a combination of a modified think-aloud approach with directed questions and prompts, behavioral observation of users interacting with the tool, and a usability questionnaire. Usability findings suggest that this Web-based tool was acceptable to youth and could be an engaging approach to communicate and promote healthy dietary behaviors among urban minority youth. In addition, although the usability questionnaire used in this study was informed by several usability questionnaires commonly used in the literature [64-66], the final combined version is not a validated tool and was only pilot tested in previous development phase study sessions. The general high usability ratings among users and the lack of difference between round 1 and round 2 scores on the self-reported questionnaires for participant’s usability and acceptability of the Web-based tool are also suggestive of response bias, which has been observed in other studies using usability questionnaires with youth [91]. Furthermore, although there is always a risk of social acceptability bias while administering surveys with a moderator present, which may be higher with youth [91,92], it was important to ensure that a study staff member was available to clarify terminology or address questions, especially as children have varying levels of literacy. Lastly, one of the usability sessions in round 2 was not recorded due to technical issues. Although field notes were taken during this session, some comments and reactions may not have been documented. Results from this study will inform further development and finalization of the Web-based tool, which will be tested using a two-group pilot RCT targeting fruit and vegetable or water intake to reduce childhood obesity risk in black/African American and Latino youth. The final tool will be a six-chapter comic with one chapter being released each week. Conclusions If such a tool is found to be effective in larger scale studies, it could be disseminated as a publicly available online health promotion tool that could be implemented in various settings, such as health care clinics, after school-based programs, and public schools, which highlights its potential for high reach. Conflicts of Interest None declared. Limitations It is acknowledged that this study is not without its limitations. First, the data analysis was performed by one researcher. However, the coding process was continually validated by two other researchers. Secondly, some participants had previously participated in the initial development process of the Web-based http://formative.jmir.org/2018/2/e21/ JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.12 (page number not for citation purposes) XSL•FO RenderX JMIR FORMATIVE RESEARCH Verdaguer et al tool or a previous usability testing session. This may have contributed to biases regarding certain preconceived ideas for how the Web-based tool would look like or how the storyline was actualized in the comic. However, engaging the same participants throughout tool and intervention development builds on prior knowledge and exposure to the tool, which may contribute to more relevant and informed feedback regarding needed improvements and criticisms [90]. Acknowledgments This project was supported by grant number R21H5024117 from the Agency for Healthcare Research and Quality (AHRQ). 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PLoS One 2015;10(9):e0135612 [FREE Full text] [doi: 10.1371/journal.pone.0135612] [Medline: 26352436] JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.16 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ Abbreviations Edited by G Eysenbach; submitted 29.12.17; peer-reviewed by K Riiser, S Taki, YP Bruton, J Wright; comments to author 30.03.18; revised version received 25.05.18; accepted 06.07.18; published 01.11.18 Please cite as: Verdaguer S, Mateo KF, Wyka K, Dennis-Tiwary TA, Leung MM A Web-Based Interactive Tool to Reduce Childhood Obesity Risk in Urban Minority Youth: Usability Testing Study JMIR Formativ Res 2018;2(2):e21 URL: http://formative.jmir.org/2018/2/e21/ doi:10.2196/formative.9747 PMID: JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.17 (page number not for citation purposes) http://formative.jmir.org/2018/2/e21/ ©Sandra Verdaguer, Katrina F Mateo, Katarzyna Wyka, Tracy A Dennis-Tiwary, May May Leung. Originally published in JMIR Formative Research (http://formative.jmir.org), 01.11.2018. 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 Formative Research, is properly cited. The complete bibliographic information, a link to the original publication on http://formative.jmir.org, as well as this copyright and license information must be included. JMIR Formativ Res 2018 \| vol. 2 \| iss. 2 \| e21 \| p.18 (page number not for citation purposes) JMIR FORMATIVE RESEARCH Verdaguer et al ©Sandra Verdaguer, Katrina F Mateo, Katarzyna Wyka, Tracy A Dennis-Tiwary, May May Leung. Originally published in JMIR Formative Research (http://formative.jmir.org), 01.11.2018. 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W3020504136.txt	https://publications.muet.edu.pk/index.php/muetrj/article/download/1601/425	en		Estimating Sectoral Water Demand for Sindh Province of Pakistan	Mehran University research journal of engineering and technology	2,020	cc-by	5,063	Mehran University Research Journal of Engineering and Technology Vol. 39, No. 2, 398 - 406, April 2020 p-ISSN: 0254-7821, e-ISSN: 2413-7219 DOI: 10.22581/muet1982.2002.16 Estimating Sectoral Water Demand for Sindh Province of Pakistan Heman Das Lohano1 and Fateh Muhammad Marri2 RECEIVED ON 24.02.2019, ACCEPTED ON 30.01.2020 ABSTRACT Water resources in Sindh province of Pakistan are under significant pressure due to increasing and conflicting water demand from municipalities for domestic users, agriculture and industries, and requirements of environmental flows. Population growth and climate change are likely to pose serious challenges to households and economic sectors that depend on water. This study estimates the present water demand from municipalities, agriculture and industries, and its future projections by the year 2050 in Sindh. The study also evaluates the impact of climate change on sectoral water demand and assesses the water requirements for the environmental flows. The results show that presently the total water demand for these sectors in Sindh is 44.06 Million Acre Feet (MAF). Agriculture is the largest consumer of water, accounting for 95.24 percent of the total water demand. Municipal water demand accounts for 2.61 percent while industrial water demand accounts for 1.88 percent. The demand for water in these sectors is expected to rise by 10 percent from 2018 to 2050. Moreover, depending on climate change scenario, the total water demand in these three sectors is likely to rise by 16 to 25 percent from 2018 to 2050. In additions, water requirements for the environmental flows have been indicated as 10 MAF in the National Water Accord of 1991. The findings of this study call for policy measures and strategies for management of water resources in Sindh. Keywords: Water, Demand Projections, Climate Change, Sindh, Pakistan [1]. Water resources in the province are distributed through the Indus Basin Irrigation System (IBIS), which comprises three main barrages on the Indus River in Sindh. These barrages divert water into fourteen main canals that serve a total cultivable command area of 6.8 million hectares [2]. 1. INTRODUCTION W ith a population of nearly 50 million, Sindh is the second most populous province in Pakistan. Its economy accounts for more than one third of economy of Pakistan. Sindh is the most downstream province in the Indus River Basin and depends almost exclusively on the water resources of the Indus River. Users of water resources include municipalities for domestic users, agriculture, industries and environment. Agriculture, which accounts for almost 20 percent of GDP and 70 percent of employment in Sindh, has traditionally been the largest user of water resources 1 2 Undoubtedly, the Indus River system has shaped the economic development of Sindh, and has delivered critical water services. However, water resources in Sindh – as in the larger Indus Basin – are under significant pressure, a situation which is likely to be exacerbated by climate change. This has also been underscored at the country level in National Water Policy of Pakistan [3]. Under the framework of the national water policy, each province including Sindh Professor of Economics, IBA, Karachi, Sindh, Pakistan. Email: hlohano@iba.edu.pk (Corresponding Author) Member, Higher Education Commission, Islamabad, Pakistan. Email: fateh.marri@hec.gov.pk Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 398 Estimating Sectoral Water Demand for Sindh Province of Pakistan is required to develop its own water resources management plan keeping in view the challenges facing the province. Water use sectors are generally categorized into four sectors, namely municipal (domestic users), agriculture, industry, and environmental flows. Three users of water including municipal, agriculture and industry place their demand for water, while the environmental flow is the water required for ecosystem, which provides various ecosystem services. separately for each sector including municipalities, agriculture and industries. The details about data, methods and assumptions for estimating and forecasting water demand for each sector are given in the following section along with the results. The requirements for environmental flow are assessed by reviewing the literature including National Water Accord 1991. 3. RESULTS AND DISCUSSION 3.1 Water Demand Projections and Climate Change Impacts Due to diverse geography of Sindh and conflicting water demands, climate change and weather variability are likely to create a great deal of uncertainty about water sector and pose serious challenges to economic sectors that depend on water. Sindh is very vulnerable to climate shocks due to its geographic location. It is already a hot region with mid-summer temperatures shooting as high as 50oC in some regions, so further increases in temperature can be very detrimental to crops and lives in form of heat waves. Sindh being the lowest riparian province is at high risk of droughts and floods in case of relevant water activity upstream. Thus, there is a need to estimate water demand to input for formulating policies and developing strategies that would facilitate managing water resources and climate change adaptation in Sindh to mitigate the negative impacts of climate change on the water sector. In this section, we analyze and forecast the sectoral and total demand for water and potential climate change impacts on water demand. We also assess the water requirements for environmental flows. 3.1.1 Municipal Water Demand Water demand from municipalities for domestic users depends on several factors including population, proportion of urban and rural population, and climatic conditions. Table 1 presents the urban, rural and total population in Pakistan and Sindh. Population data for 1981, 1998 and 2017 come from census conducted in the respective years. Future projections for Pakistan are provided by the United Nations [4]. Future projections for Sindh have been estimated. Data show that Sindh’s share in Pakistan’s population has remained 23 percent in the years 1981, 1998 and 2017, and thus the same share has been assumed for future projections. Projections of rural and urban population of Sindh have been estimated based on the growth pattern of urban population relative to total population between 1998 and 2017. The main objective of this study is to estimate the present water demand from municipalities, agriculture and industries, and its future projections by the year 2050 in Sindh. The study also evaluates the impact of climate change on sectoral water demand and assesses the water requirements for the environmental flows. Table 2 presents the municipal water demand for domestic users. According to the National Drinking Water Policy [5], the daily per capita water requirement is 45 and 120 liters for rural and urban dwellers, respectively. Using this water requirement and population estimates given in Table 1, we estimate the baseline municipal water demand on annual basis for the years 2018, 2030 and 2050. Table 2 reports the baseline (without climate change) municipal water demand for 2018, 2030 and 2050. Table 2 also reports 2. METHODOLOGY To estimate the sectoral water demand, this study uses secondary data and various analytical methods including descriptive analysis, regression analysis and past trend analysis. Furthermore, this study uses the parameters estimated in the literature and also estimates some other parameters using secondary data. Based on the nature of the sector, appropriate data and methods are used for estimating the water demand Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 399 Estimating Sectoral Water Demand for Sindh Province of Pakistan Table 1: Population in Pakistan and Sindh: Urban, Rural and Overall Year Pakistan (millions) Sindh (millions) Urban Rural Total Urban Rural Total 1981 23.84 60.41 84.25 8.24 10.79 19.03 1998 43.04 89.32 132.35 14.84 15.60 30.44 2017 75.58 132.22 207.80 24.91 22.97 47.90 2018 77.42 135.39 212.82 25.51 23.54 49.05 2030 99.39 144.81 244.20 29.96 26.33 56.29 2050 160.20 146.70 306.90 38.91 31.84 70.70 Source: Pakistan Census Reports [8], Pakistan Economic Survey [7], UN (2018) and author’s calculations Table 2: Municipal Water Demand in Pakistan and Sindh Annual Water demand (MAF) under each scenario Pakistan Sindh 2018 2030 2050 2017 2030 2050 Without climate change 4.55 5.46 7.64 1.22 1.41 1.80 Climate scenario with 1 °C - 5.51 7.77 - 1.42 1.83 Climate scenario with 3 °C - 5.76 8.25 - 1.49 1.94 this scenario, the municipal water demand is expected to rise to 8.25 MAF in Pakistan and 1.94 MAF in Sindh in 2050. the future projections of municipal water demand under two climate change scenarios with 1 °C and 3°C increase in the average temperature. The projections of increase in municipal water demand under each climate scenario are based on the percent increase estimates provided by Amir and Habib [6]. 3.1.2. Agricultural Water Demand Agriculture sector includes not just crops but also livestock, fisheries, agro-forestry and rangelands [7]. We estimate the agricultural water demand using data of both surface water and groundwater. Total agriculture water is calculated by adding the water availability at farm gate and the groundwater used by agriculture sector. To estimate the demand for agriculture water in Pakistan, we estimated a trend line of total agriculture water by regressing the natural log of agriculture water on time using data for past 21 years. The upward trend was statistically significant at 10 percent level. The regression results show that agricultural water use has increased at the rate of 0.16 percent per annum in the past 21 years, which we use to compute future projections for 2030 and 2050. The results show that the municipal water demand for Pakistan will increase from 4.55 MAF in 2018 to 7.64 MAF in 2050 without accounting for the climate change. Similarly, the municipal water demand for Sindh will increase from 1.22 MAF in 2018 to 1.80 MAF in 2050 if there is no climate change. The estimated increase in municipal water demand in Pakistan in 2050 attributable to climate change show that the municipal water demand will be higher by 1.7 percent under the climate scenario of 1 °C and by 7.8 percent under the climate scenario of 3 °C. This shows that the municipal water demand will be even higher under the climate change regime. We find that if the temperature increases by 1 °C, then the municipal water demand in Pakistan will rise from the existing demand of 4.55 MAF (in 2018) to 7.77 MAF in 2050. Along the same lines, if the average temperature increases by 3 °C in 2050, it will put immense pressure on the current water resources of the country. Under Table 3 reports the average (per year) availability of surface water at farm gate and use of groundwater in Pakistan and Sindh in the last ten years, computed using data from 2007-08 to 2016-17. Data on amount Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 400 Estimating Sectoral Water Demand for Sindh Province of Pakistan Table 3: Availability of Surface Water at Farm Gate and Groundwater per Year: Average during 2007-08 to 2016-17 Share of Sindh Pakistan Sindh (percent) Surface water at farm gate (MAF) 85.21 34.54 40.54 Groundwater (MAF) 49.87 8.10 Total (MAF) 135.08 42.64 Source: Author’s computation based data from Pakistan Statistical Yearbook (GOP, 2017) 16.24 31.57 of water at farm gate and groundwater use for Pakistan are available in Statistical Year Book [9]. As data on canal water withdrawal was available for both Pakistan and Sindh, we estimated water at farm gate for Sindh assuming that the ratio of farm gate to canal water withdrawal is the same in Pakistan and Sindh. Table 3 shows that, during last ten years, Sindh’s share was 40.54 percent in surface water at farm gate and 16.24 percent in groundwater. Overall, Sindh used 31.57 percent of total agricultural water in Pakistan. We compute the future projections of total agricultural water demand for Sindh assuming that the growth in water demand in Sindh will mimic the pattern in Pakistan and Sindh will require 31.57 percent of total agricultural water demand of Pakistan. under the climate change regime, it is expected that the agricultural water demand will be even higher. We find that if the temperature increases by 1 °C, then the agricultural water demand in Pakistan will rise from the existing demand of 132.91 MAF (in 2018) to 148.24 MAF in 2050. Similarly, the agricultural water demand in Sindh will rise from the existing demand of 41.96 MAF (in 2018) to 46.80 MAF in 2050. If the average temperature increases by 3 °C in 2050, it will put immense pressure on the current water resources of the country. Under this scenario, the agricultural water demand is expected to rise to 159.70 MAF in Pakistan and 50.41 MAF in Sindh in 2050. Table 4 presents the agricultural water demand for Pakistan and Sindh. The table reports the baseline (without climate change) water demand for 2018, 2030 and 2050, and future projections of water demand under two climate change scenarios with 1 °C and 3 °C increase in the average temperature. The projections of increase in agricultural water demand under each climate scenario are based on the percent increase estimates provided by Amir and Habib [6]. The results show that the agriculture water demand for Pakistan will increase from 132.91 MAF in 2018 to 139.78 MAF in 2050 without accounting for the climate change. Similarly, the agriculture water demand for Sindh will increase from 41.96 MAF in 2018 to 44.13 MAF in 2050 if there is no climate change. For estimating the industrial water demand in Pakistan, we assumed water requirement per rupee of industrial value addition in GDP in 2008. Data on industrial water withdrawal in 2008 were collected from AQUASTAT [10] while data on industrial value addition in GDP were collected from Pakistan Economic Survey reports [7; 11]. For future projections of industrial value addition in GDP, we used its average growth rate (3.74 percent) during 2007-08 to 2017-18. For estimating the industrial water demand in Sindh, we estimated the water share based on Sindh’s share in industrial value addition in GDP of Pakistan, which was 42.2 percent in 2012-13 according to Pasha [12]. For future projections, we assume that Sindh’s share in industrial value addition in GDP of Pakistan will remain the same. The agricultural water demand in 2050 in Pakistan attributable to climate change shows that the agricultural water demand will increase by 6 percent under the climate scenario of 1°C and by 14.2 percent under the climate scenario of 3°C. This shows that Table 5 presents the industrial water demand for Pakistan and Sindh. Table 5 reports the baseline (without climate change) water demand for 2018, 2030 and 2050, and future projections of water demand under two climate change scenarios with 1°C 3.1.3 Industrial Water Demand Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 401 Estimating Sectoral Water Demand for Sindh Province of Pakistan Table 4: Agricultural Water Demand in Pakistan and Sindh Annual Water demand (MAF) under each scenario Pakistan Without climate change Sindh 2018 2030 2050 2018 2030 2050 132.91 135.45 139.78 41.96 42.76 44.13 Climate scenario with 1 °C - 140.14 148.24 - 44.24 46.80 Climate scenario with 3 °C - 145.62 159.70 - 45.97 50.41 Table 5: Industrial Water Demand in Pakistan and Sindh Annual Water demand (MAF) under each scenario Pakistan Sindh 2018 2030 2050 Without climate change 2.08 3.00 Climate scenario with 1 °C - Climate scenario with 3 °C - and 3°C increase in the average temperature. The projections of increase in industrial water demand under each climate scenario are based on the percent increase estimates provided by Amir and Habib [6]. 5.52 2018 0.88 2030 1.27 2050 2.33 3.21 6.17 - 1.35 2.60 3.44 6.82 - 1.45 2.88 3.1.4 Analysis of Total Water Demand Table 6 summarizes the total and sectoral water demand from municipalities, agriculture and industries in Sindh. Agriculture is the largest consumer of water in Sindh. In 2018, out of total water of 44.06 MAF, agriculture used 41.96 MAF, accounting for 95.24 percent of total water used by these three sectors. Municipal (domestic) water demand was 1.22 MAF (2.77 percent) while industrial water demand was 0.88 MAF (1.99 percent). The results show that the industrial water demand for Pakistan will increase from 2.08 MAF in 2018 to 5.52 MAF in 2050 without accounting for the climate change. Similarly, the agriculture water demand for Sindh will increase from 0.88 MAF in 2018 to 2.33 MAF in 2050 if there is no climate change. The total water demand from these three sectors in Sindh will increase from 44.06 MAF in 2018 to 48.26 MAF in 2050, which is 10 percent increase, if there is no climate change. The industrial water demand in Pakistan in 2050 attributable to climate change shows that the industrial water demand will increase by 11.8 percent under the climate scenario of 1°C and by 23.5 percent under the climate scenario of 3°C. This shows that under the climate change regime, it is expected that the industrial water demand will be even higher. We find that if the temperature increases by 1°C, then the industrial water demand in Pakistan will rise from the existing demand of 2.08 MAF (in 2018) to 6.82 MAF in 2050. Similarly, the industrial water demand in Sindh will rise from the existing demand of 0.88 MAF (in 2018) to 2.60 MAF in 2050. If the average temperature increases by 3 °C in 2050, it will put more pressure on the current water resources of the country. Under this scenario, the industrial water demand is expected to rise to 6.82 MAF in Pakistan and 2.88 MAF in Sindh in 2050. Under the climate change scenario of just 1°C increase in temperature by 2050, the total water demand in these three sectors from 2018 to 2050 will rise by 7.18 MAF (16.29 percent). The total water demand is expected to increase by 11.18 MAF (25.38 percent) under the climate change scenario of 3 °C increase in temperature. We find that if the temperature increases by 1°C, then the total water demand in Sindh will rise from the existing demand of 44.06 MAF (in 2018) to 51.23 MAF in 2050, which 16 percent increase. If the average temperature increases by 3 °C in 2050, it will put more pressure on the current water resources. Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 402 Estimating Sectoral Water Demand for Sindh Province of Pakistan Table 6: Summary of Sectoral Water Demand in Sindh Annual Sectoral Water demand 2018 2030 2050 Increase from 2018 to 2050 (MAF) in Sindh under each scenario MAF MAF MAF MAF Percent Municipalities (domestic users) 48 1.22 1.41 1.80 Without climate change 0.58 Climate scenario with 1 °C - 1.42 1.83 0.61 50 Climate scenario with 3 °C - 1.49 1.94 0.72 59 42.76 44.13 2.17 5 Agriculture Without climate change 41.96 Climate scenario with 1 °C - 44.24 46.80 4.84 12 Climate scenario with 3 °C - 45.97 50.41 8.46 20 Industries Without climate change 0.88 1.27 2.33 1.45 165 Climate scenario with 1 °C - 1.35 2.60 1.73 197 Climate scenario with 3 °C - 1.45 2.88 2.00 227 45.44 48.26 4.20 10 Total Without climate change 44.06 Climate scenario with 1 °C - 47.02 51.23 7.18 16 Climate scenario with 3 °C - 48.91 55.24 11.18 25 meet the water demand not only from the municipal, agricultural and industrial sectors but also for the environmental flows for sustaining the ecosystem in the Indus delta. Under this scenario, the total water demand in Sindh is expected to rise from 44.06 MAF in 2018 to 55.24 MAF in 2050, which is 25 percent increase. 3.2 Water Requirements for Environment 3.3 Challenges of Water Resources Management Environmental flows for escapage to sea are the most important for sustaining ecosystem and minimizing the losses due to sea intrusion. Low environmental flow has threatened the mangroves, disturbed the ecological balance and created extreme hardship for the fisher folks and other communities residing in the Indus delta and near the coastline. Furthermore, maintaining a continuous flow throughout the year in downstream of Kotri Barrage is critical for sustaining the ecosystems of the Indus Delta [13] The sectoral water demand analysis given above shows that due to population growth, urbanization and climate change, the demand for water will increase in future. The ecosystem of Indus Delta has already been degraded as discussed above and the situation can further be aggravated with continuous increase in water demand from other sectors and due to climate change. In order to ensure adequate water for the three economic sectors and for the environment and ecosystems including wetlands, mangrove forests and irrigated forestry, substantial quantity of water will be required in Sindh. The National Water Accord of 1991 [14] recognized the need to ensure a minimum environmental flow below Kotri for escapage to sea. The proposal of a minimum 10 MAF for environmental flow was discussed at length. However, as the other studies have suggested more/less than 10 MAF as a minimum environmental flow each year, it was decided in the accord that further studies would be conducted to determine the minimum environmental flow below Kotri for escapage to sea [14]. Thus, Sindh needs to Historically, groundwater use has been increasing to meet part of the increasing demand. However, there are limitations of groundwater due waterlogging and salinity issues in Sindh and declining water table across the country. Thus, there will be immense Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 403 Estimating Sectoral Water Demand for Sindh Province of Pakistan pressure on the Indus river water from different sectors. 4. CONCLUSION The sectoral water demand analysis of municipalities, agriculture and industries shows that due to population growth, urbanization and climate change, the demand for water in Sindh is expected to increase substantially in future. Depending on climate change scenario, the total water demand in these three sectors is likely to rise by 16 to 25 percent from 2018 to 2050. Furthermore, the environment and ecosystems require minimum annual water flow as well as a continuous flow throughout the year in downstream of Kotri Barrage for escapage to sea. This shows that substantial quantity of water will be required in Sindh for the three economic sectors, environment and ecosystems. To ensure food and water security and protection of environment and ecosystems, Sindh needs to develop integrated water resource management (IWRM) plan that not only has the sectoral water demand management plan but also the plan for increasing water supply by reducing the water losses in the Indus river system and exploring rain harvesting. The overall water efficiency in the Indus River system in Sindh is only 35 percent, indicating huge water losses [15]. Furthermore, total water productivity in Pakistan is very low ‒ USD 1.38 per cubic meter of water. Pakistan ranks eighth lowest in the world in terms of total water productivity [13]. Agriculture sector is using water inefficiently. Despite using 95.24 percent of total water, there is a lot of farmland uncultivated mainly due to unavailability of water. Total cultivated area in Sindh is 5.198 million hectares while culturable waste is 1.607 million hectares, most of which is not cultivated due to water shortage [2]. Similarly, there is a limited access to safe and reliable water for household and industries. For food and water security and protection of ecosystem, Sindh needs to develop integrated water resource management (IWRM) plan that not only has the sectoral water demand management plan but also the plan for increasing water supply by reducing the water losses in the Indus river system and exploring rain harvesting. In the following sections, we present the way forward. 3.4 Impact of Adaptation Investment 5. RECOMMENDATIONS A study by the World Bank [15] evaluated the impacts of adaptation investments in Pakistan. The results are presented in Table 7. They evaluated three adaptation investment options (i) investments in agricultural technologies to increase crop yield (such as developing new seed varieties), (ii) investment for canal and watercourse efficiency improvements, and (iii) construction of new reservoirs to introduce an additional 13 MAF. Table 7 presents the main findings of the World Bank study, showing the impacts on GDP, agricultural value-added to GDP, and household income. The findings show that the climate change is likely to reduce agricultural value-added to GDP by around 5 percent if there is no investment made for climate change adaptation. The investment in agricultural technologies to increase crop yield is likely to increase the agricultural value-added to GDP by around 17 percent and the investment for canal and watercourse efficiency improvements would increase it by 9 percent. The findings of this study call for policy measures and strategies for management of water resources in Sindh, with focus on integrated water resources management (IWRM) plan, water demand management and conservation, investment in infrastructure and technology, and water information management. Integrated water resources management aims to achieve economic efficiency as well as equity among all users of water. Since all water users are competing for water share, it is very important to encourage a multi-sectoral approach rather than focusing on single sector only. Integrated waters resources management is a framework of interrelations between sources of water, its uses and better stakeholder engagement. It is a holistic and participatory approach to decision making spanning the entire watershed, management and development of resources such as land, water and related assets. This plan would require policy framework for institutional reforms, capacity building, Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 404 Estimating Sectoral Water Demand for Sindh Province of Pakistan Table 7: Impact of Adaptation Investments in Pakistan Change in GDP (percent) Average change without investments No investment Average change with investments Investments in agricultural to increase crop yield Investments for canal and watercourse efficiency improvements Construction of new reservoirs Source: Yu et al. [15] Change in Agricultural value-added to GDP (percent) Change in Household Income (percent) -1.10 -5.10 -2.00 3.66 16.70 5.42 2.09 9.32 3.21 0.29 1.50 0.64 Sindh can save a lot of water by making investments in improving canal and watercourses. More food and fiber products can be produced with development of agricultural technology and new seed varieties and breeds that give higher yields and are water efficient, drought tolerant and salinity tolerant. review of water pricing, spatial water distribution, improving water productivity and water efficiency, investment in infrastructure and technology adoption, raising awareness, and participation of all relevant stakeholders. Sindh needs to develop policies for water demand management and conservation to ensure food and water security and protection of ecosystem. Policy makers are increasingly recognizing the need for consideration and implementation of water demand management. For example, recently, Government of Sindh has imposed a ban on rice cultivation in some parts of Sindh along Ghotki feeder, Rohri canal and Nara canal, as rice crop not only uses high amount of water but may also cause waterlogging, salinity and land degradation in some type of soils. Water demand management is required for all sectors but most of the contribution for saving water is expected from the largest user of water, that is, agriculture sector. The investments in water informatics can assist in timely alerts for flood management, information of flow at barrages, optimum water escape to sea and timely weather forecasts through SMS and other channels to farmers, households, business community and other stakeholders. REFERENCES Investment in Infrastructure for Access to Safe Water by Households: Households place a high value for access to safe water and are willing to pay for better quality of water and reliable access to piped water [16]. There is a limited access to safe water for household. Around 33 percent of households in Sindh use hand pump as the main source of drinking water while majority of households in rural areas of Sindh (63 percent) use hand pump as the main source of drinking water [17]. Sindh needs to give priority to domestic use of water and charge water tariffs to ensure maintenance of the infrastructure. [1] Commonwealth Scientific and Industrial Research Organisation (CSIRO). Sindh Water and Agriculture Transformation (SWAT) Project. ACIAR, Australia. 2019. [2] Government of Sindh. Development Statistics of Sindh. Bureau of Statistics, Karachi. 2018. [3] Government of Pakistan. National Water Policy 2018. Ministry of Water Resources. 2018. [4] United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects: The 2017 Revision, Key Findings and Advance Tables. Working Paper No. ESA/P/WP/248. 2017 [5] Government of Pakistan. National Drinking Water Policy. Ministry of Environment. 2009 Mehran University Research Journal of Engineering and Technology, Vol. 39, No. 2, April 2020 [p-ISSN: 0254-7821, e-ISSN: 2413-7219] 405 Estimating Sectoral Water Demand for Sindh Province of Pakistan [6] Amir, P., and Z. Habib. Estimating the Impacts of Climate Change on Sectoral Water Demand in Pakistan. Islamabad, Pakistan: Action on Climate Today. https://cyphynets.lums.edu.pk/images /Readings_concluding.pdf. (accessed June 2019). [13] Young., William J., Arif Anwar, Tousif Bhatti, Edoardo Borgomeo, Stephen Davies, William R. Garthwaite III, E. Michael Gilmont, Christina Leb, Lucy Lytton, Ian Makin, and Basharat Saeed. Pakistan: Getting More from Water. Water Security Diagnostic. World Bank, Washington, DC. 2019. [7] Government of Pakistan. Pakistan Economic Survey 2018-19. Islamabad: Ministry of Finance. 2019 [14] [8] Government of Pakistan. Pakistan Census Report. Pakistan Bureau of Statistics. 2017 [9] Government of Pakistan. Pakistan Statistical Yearbook 2017. Pakistan Bureau of Statistics. 2017 [10] FAO. AQUASTAT Main Database - Food and Agriculture Organization of the United Nations (FAO). Website accessed on [29/06/2019 12:32]. 2019 [11] Government of Pakistan. Pakistan Economic Survey 2009-10. Islamabad: Ministry of Finance. 2010. [12] Pasha, H. A. Growth of the provincial economies. Institute for Policy Reforms (IPR). http://ipr. org. pk/wpcontent/uploads/2016/04/GROWTH-OFPROVINCIAL-ECONOMICS-. pdf. 2015. Government of Pakistan. Apportionment of Waters of Indus River System between the provinces of Pakistan. Indus River System Authority. 1991 [15] Yu, Winston, Yi-Chen Yang, Andre Savitsky, Donald Alford, Casey Brown, James Wescoat, Dario Debowicz, and Sherman Robinson. The Indus Basin of Pakistan: The Impacts of Climate Risks on Water and Agriculture. Washington, DC: World Bank. doi: 10.1596/978-0-8213-9874-6. License: Creative Commons Attribution CC BY 3.0. 2013. [16] Asim, S., and Lohano, H. D. Households’ Willingness to Pay for Improved Tap Water Services in Karachi, Pakistan. The Pakistan Development Review, 54(4): 507-526. [17] Government of Pakistan. Pakistan Social and Living Standards Measurement Survey 201415. Islamabad: Pakistan Bureau of Statistics. 2016. 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https://openalex.org/W1976416008	https://bmcneurosci.biomedcentral.com/counter/pdf/10.1186/1471-2202-10-105	English	null	Preliminary evidence that both blue and red light can induce alertness at night	BMC neuroscience	2,009	cc-by	9,300	BioMed Central BioMed Central Open Research article Preliminary evidence that both blue and red light can induce alertness at night Mariana G Figueiro, Andrew Bierman, Barbara Plitnick and Mark S Rea Corresponding author Received: 30 December 2008 Accepted: 27 August 2009 © 2009 Figueiro 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. BMC Neuroscience Open Access Background Cajochen and colleagues showed that low levels of blue light (5 lx at the cornea of narrowband radiation peaking at 460 nm) for a duration of about 40 minutes at night increased heart rate and self- reported alertness as measured by the Karolinska Sleepi- ness Scale (KSS), as well as reduced melatonin levels [13]. More recently, Figueiro and colleagues demonstrated that, at night, self-reported alertness (Norris Scale) and the alpha attenuation coefficient (AAC; the ratio of the alpha power [812 Hz] when eyes are closed to the alpha power when eyes are open) in the EEG recordings were highly correlated and both increased monotonically with increasing levels of narrowband blue (peak at 470 nm) light (5, 10, 20 and 40 lx at the cornea) [14]. Moreover, they found these measures of alertness were highly corre- lated with predicted levels of light-induced nocturnal melatonin suppression for the same light stimuli. These results are consistent with the neurophysiological evi- dence that neural pathways from the SCN are important to sleep and to alertness, as recently elucidated by Saper and colleagues [15-17]. Together, these findings add weight to the inference that the SCN, through retinal stim- ulation by short-wavelength light, play an important role in nocturnal alertness in humans. g Alertness is a construct associated with high levels of envi- ronmental awareness. Alertness has been operationalized through many converging measurements, including sub- jective responses, behavior, and brain activity. Alertness is associated with self-reported high levels of wakefulness and low levels of fatigue, short response times, fast and more accurate tests of mental capacity, low power densi- ties in the alpha frequency range (812 Hz) and high power densities in the beta frequency range (1230 Hz) in electroencephalography (EEG) [1-9]. Since these outcome measures follow diurnal patterns, alertness also can be related to measures of endogenous hormones and core body temperature; alertness is expected to be low during the nighttime hours, when melatonin levels are high and core body temperature levels are low. In response to the natural light-dark solar patterns, humans have evolved endogenous circadian (circa = around; dies = day) rhythms (e.g., core body temperature variations, melatonin synthesis, sleep-wake behavior) that repeat approximately every 24 hours (average period of 24.2 hours). The internal mechanism that organizes these daily biological processes in mammals has been localized to small paired nuclei in the hypothalamus, the suprachiasmatic nuclei (SCN). Abstract Background: A variety of studies have demonstrated that retinal light exposure can increase alertness at night. It is now well accepted that the circadian system is maximally sensitive to short- wavelength (blue) light and is quite insensitive to long-wavelength (red) light. Retinal exposures to blue light at night have been recently shown to impact alertness, implicating participation by the circadian system. The present experiment was conducted to look at the impact of both blue and red light at two different levels on nocturnal alertness. Visually effective but moderate levels of red light are ineffective for stimulating the circadian system. If it were shown that a moderate level of red light impacts alertness, it would have had to occur via a pathway other than through the circadian system. Methods: Fourteen subjects participated in a within-subject two-night study, where each participant was exposed to four experimental lighting conditions. Each night each subject was presented a high (40 lx at the cornea) and a low (10 lx at the cornea) diffuse light exposure condition of the same spectrum (blue, λmax = 470 nm, or red, λmax = 630 nm). The presentation order of the light levels was counterbalanced across sessions for a given subject; light spectra were counterbalanced across subjects within sessions. Prior to each lighting condition, subjects remained in the dark (< 1 lx at the cornea) for 60 minutes. Electroencephalogram (EEG) measurements, electrocardiogram (ECG), psychomotor vigilance tests (PVT), self-reports of sleepiness, and saliva samples for melatonin assays were collected at the end of each dark and light periods. Results: Exposures to red and to blue light resulted in increased beta and reduced alpha power relative to preceding dark conditions. Exposures to high, but not low, levels of red and of blue light significantly increased heart rate relative to the dark condition. Performance and sleepiness ratings were not strongly affected by the lighting conditions. Only the higher level of blue light resulted in a reduction in melatonin levels relative to the other lighting conditions. Conclusion: These results support previous findings that alertness may be mediated by the circadian system, but it does not seem to be the only light-sensitive pathway that can affect alertness at night. Page 1 of 11 (page number not for citation purposes) BMC Neuroscience 2009, 10:105 http://www.biomedcentral.com/1471-2202/10/105 Background Circadian rhythms are syn- chronized to the 24-hour solar day by photic and non- photic cues, the light-dark cycle being the strongest. Light and dark patterns are conveyed from the retina to the SCN via the retino-hypothalamic tract (RHT). Alertness and performance are strongly influenced by the timing of the circadian clock; therefore, the impact of light on alertness has gained recent attention. Bright white light (2500 to 10,000 lx at the cornea) has been shown to increase alertness at night but not during the day, suggest- ing a role for the circadian system in evoking alertness [1- 9]. An early study by Badia and colleagues [1], for exam- ple, tested the effects of bright white light (5000 to 10,000 lx at the cornea) on core body temperature, alertness, and performance during the day and at night. In their study, subjects who normally would be awake during the day and asleep at night were exposed to 90-minute blocks of alternating bright and dim (50 lx at the cornea) light dur- ing daytime and nighttime hours. Core body temperature, beta power (1821 Hz) from the EEG recordings, and per- formance were higher after exposure to bright light than after exposure to dim light during the nighttime hours, but not during daytime hours. These results suggest a role for the circadian system in modulating alertness, but the mechanisms associated with the alerting effects of light through the circadian system cannot be unambiguously established by simply introducing "bright" light into the experimental protocol. It is not completely clear, however, whether light-induced alertness can arise from other neural pathways. For exam- ple, some evidence suggests that red light, which is ineffec- tive for stimulating the circadian system at moderate light levels, can be more stimulating than blue light [18,19]. Studies have also suggested that perception of red color prior to executing an important task impairs performance relative to the perception of green or achromatic color [19,20]. The present experiment was conducted to look at the impact that both blue and red light at two different levels might have on human subjective and objective measures of alertness, on performance and on melatonin levels during the night. Background It is well established now that the human circadian system is maximally sensitive to short-wavelength radiation (blue light) [10-12]. Not only does this mean that the circadian system is quite insensitive to low levels of long-wave- length, red light, it also means on a quantitative basis that the efficacy of "dim" blue light can be computed to be equivalent to that of "bright" white light for stimulating the circadian system [12]. Cajochen and colleagues showed that low levels of blue light (5 lx at the cornea of narrowband radiation peaking at 460 nm) for a duration of about 40 minutes at night increased heart rate and self- reported alertness as measured by the Karolinska Sleepi- ness Scale (KSS), as well as reduced melatonin levels [13]. More recently, Figueiro and colleagues demonstrated that, at night, self-reported alertness (Norris Scale) and the alpha attenuation coefficient (AAC; the ratio of the alpha power [812 Hz] when eyes are closed to the alpha power when eyes are open) in the EEG recordings were highly correlated and both increased monotonically with increasing levels of narrowband blue (peak at 470 nm) light (5, 10, 20 and 40 lx at the cornea) [14]. Moreover, they found these measures of alertness were highly corre- lated with predicted levels of light-induced nocturnal melatonin suppression for the same light stimuli. These results are consistent with the neurophysiological evi- dence that neural pathways from the SCN are important to sleep and to alertness, as recently elucidated by Saper and colleagues [15-17]. Together, these findings add weight to the inference that the SCN, through retinal stim- ulation by short-wavelength light, play an important role in nocturnal alertness in humans. It is well established now that the human circadian system is maximally sensitive to short-wavelength radiation (blue light) [10-12]. Not only does this mean that the circadian system is quite insensitive to low levels of long-wave- length, red light, it also means on a quantitative basis that the efficacy of "dim" blue light can be computed to be equivalent to that of "bright" white light for stimulating the circadian system [12]. http://www.biomedcentral.com/1471-2202/10/105 http://www.biomedcentral.com/1471-2202/10/105 http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 experiment at 03:45. During every session, each subject was presented a high (40 lx) and a low (10 lx) light expo- sure condition of the same spectrum (blue or red). The presentation order of the light levels was counterbalanced across sessions for a given subject; light spectra were coun- terbalanced across subjects within sessions. Every 45- minute light exposure condition was preceded by a 45- minute period of inactivity in a dark anteroom (< 1 lx of red light at the cornea). During the inactive dark periods, subjects remained quiet and were not allowed to perform any task (e.g., talk, read, or computer work) except for the prescribed data sampling specified in the experimental protocol. Each data collection period (after each dark and after each light exposure condition), which included EEG measurements, saliva collection and PVT tests, lasted about 15 minutes. In sum then, in each nighttime session there was a total of four, 45-minute light-and-dark condi- tions (a dark condition always preceded one of the four light conditions), and each condition was continued for 15 minutes for data collection. Figures 1 and 2 show the experimental design and the data collection activity peri- ods, respectively. completed a Munich Chronotype Questionnaire (MCTQ) prior to the study [21]; those who were late or extremely late chronotypes were excluded from the experiment. All subjects provided an informed consent approved by Rens- selaer's Institute Review Board. Subjects were asked to refrain from alcohol and caffeine on the days of the exper- iment and were asked not to sleep after awakening for the day. Of the sixteen subjects, nine males and five females completed the entire experiment, and the results of their data are reported here. Four experimental lighting conditions, two spectra (blue and red) at two levels (10 and 40 lx), were delivered to individual subjects from 0.6 × 0.6 × 0.6 m light boxes, each fitted with arrays of light-emitting diodes (LEDs). The arrays (ICove, Color Kinetics) were located behind the front box apertures to be outside the subject's direct view, thereby creating a uniform, non-glaring distribution of light within the box. During light exposures, subjects placed their chin on a rest mounted near the front of the box, ensuring delivery of the prescribed light exposure. http://www.biomedcentral.com/1471-2202/10/105 The spectral emissions of the blue LEDs peaked at 470 nm with a full width at half maximum (FWHM) of 25 nm. Light from the red LEDs peaked at 630 nm with a FWHM of 25 nm. Before the experiment, each of the light boxes was calibrated using a Gigahertz illuminance photometer to provide the prescribed corneal illuminance levels when subjects were positioned in the chinrest. The spectral radi- ances of the red and blue conditions were measured prior to the experiment with a calibrated spectroradiometer (Photoresearch, model PR705a) and diffuse white reflect- ance standard (Labsphere, model SR 099) and were used to calibrate the Gigahertz illuminance readings. Two boxes provided blue light (40 μW/cm2 at 40 lx and 10 μW/ cm2 at 10 lx) and two emitted red light (19 μW/cm2 at 40 lx and 4.7 μW/cm2 at 10 lx); light levels could be adjusted with an electronic dimmer to reach the prescribed light levels without significantly affecting the relative spectral distributions of the LED emissions. Measurements of pupil area completed after the experiment with a different group of subjects (n = 5) were: red at 10 lx, 34 mm2; red at 40 lx, 22 mm2; blue at 10 lx, 10 mm2; blue at 40 lx, 6.5 mm2. Procedures and apparatus Sixteen subjects (21 to 46 years of age) were recruited to participate in the study from an electronic posting at Rens- selaer Polytechnic Institute in Troy, N.Y. All subjects were screened for major health problems and except for women taking birth control pills, subjects reported not taking any pharmaceuticals or medications. Every subject Page 2 of 11 (page number not for citation purposes) Page 2 of 11 (page number not for citation purposes) PVT and KSS lf l i Self-luminous personal digital assistants (PDA; Tungsten E2 models from Palm Inc.) were used to record subjects' performance levels and sleepiness self-reports [22,23]. A battery of three psychomotor vigilance tests (PVT; Brain Checkers 2.75 from Behavioral Neuroscience Systems LLC) were used to measure performance: (a) a Simple Reaction Times (RT) test where the subject had to tap a symbol displayed on the PDA screen with a stylus as soon it appeared; (b) a Two-choice Reaction Time (TCRT) test where the subject had to choose, by tapping the screen with a stylus, whether a previously, briefly displayed numeral on the screen was either a "2" or a "3" or a "4" or a "5"; and (c) a Matching-to-Sample (MTS) test where the subject had to indicate with the stylus which of two spe- cific patterns of squares had previously been displayed on the screen. The KSS was used to probe self-assessments of sleepiness [24]. Subjects performed tests every 15 minutes (three times for every light-and-dark condition) through- out a four-hour session. Tests were performed while sub- jects were sitting comfortably in a chair, either adjacent to the light box or in the dark anteroom. The study was conducted over the course of several weeks in April/May 2008 and in March 2009. Groups of four subjects participated in two sessions separated by at least one week. Subjects were asked to arrive at the laboratory at 22:00 to receive instructions, become familiar with the performance tests, and be fitted with scalp electrodes for EEG recordings. Because only one EEG machine was avail- able, data collection was staggered. The first subject in a session started at 23:00, the second at 23:10, the third at 23:20, and the last at 23:30; the last subject completed the PVT and KSS data were downloaded to an Excel spread- sheet for subsequent analyses using the BCDataMan 2.0.10 software from Behavioral Neuroscience Systems LLC. Both a response time and a score were determined for the TCRT and MTS tasks. Both scores were calculated by dividing the percentage of correct responses by the mean response times for each test during a given record- ing period. Page 3 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 Experimental design Figure 1 Experimental design. Each subject participated in the experiment over the course of two nights. Melatonin l Melatonin, a hormone produced at night and under con- ditions of darkness, is used as a marker of circadian system timing. In darkness, melatonin levels rise in the evening, reach a peak between 02:00 and 04:00 and decline to day- time levels between 07:00 and 09:00. Light can attenuate the rate of melatonin production during the nighttime; depending upon the characteristics of light exposure (intensity, spectrum, duration), melatonin synthesis will continue to rise, but at a slower rate, in the early part of the night and continue to fall, but at a faster rate, toward the end of the night. In the current study, 1 to 2 ml saliva sam- ples were collected at the end of each dark and each light exposure period using the Salivette system from Alpco Diagnostics (four samples per session per subject). The vessels containing the suspended saliva-impregnated cot- ton swabs were then spun in a centrifuge at 1000 × g for five minutes, causing the saliva to collect at the bottom of the centrifuge vessel. Saliva samples were frozen for trans- PVT and KSS lf l i On night 1, subjects saw two light levels (10 lux or 40 lux) of the same light spectra, either blue (B) or red (R) following a dark (D) condition; subjects experienced the other light spectra at both light levels on night 2. Note: Subjects 12 and 13 did not complete the experiment, so their data are not presented here. Experimental design Figure 1 Experimental design. Each subject participated in the experiment over the course of two nights. On night 1, subjects saw two light levels (10 lux or 40 lux) of the same light spectra, either blue (B) or red (R) following a dark (D) condition; subjects experienced the other light spectra at both light levels on night 2. Note: Subjects 12 and 13 did not complete the experiment, so their data are not presented here. Experimental design Figure 1 Experimental design. Each subject participated in the experiment over the course of two nights. On night 1, subjects saw two light levels (10 lux or 40 lux) of the same light spectra, either blue (B) or red (R) following a dark (D) condition; subjects experienced the other light spectra at both light levels on night 2. Note: Subjects 12 and 13 did not complete the experiment, so their data are not presented here. port to a laboratory for melatonin radioimmunoassay (Pharmasan, Osceola, WI). The sensitivity of the saliva assay was 0.7 pg/ml and the intra- and inter-assay coeffi- cients of variability (CVs) were 12.1% and 13.2%, respec- tively. EEG and ECG The Biosemi ActiveTwo system with active electrodes was used for EEG recordings. This system is battery powered, minimizing electrical interference from alternating cur- rent (ac) during recording sessions. Electrodes were placed on subjects' scalps according to the International 1020 system at Oz, Pz, Cz, and Fz [25]. Two additional electrodes serving as virtual reference electrodes for those attached to the scalp were attached to the right and to the left earlobes. Another electrode was placed approximately 5 cm below the left clavicle to measure an electrocardio- gram (ECG) signal. Data collection times for the first subject on an experimental night Figure 2 Data collection times for the first subject on an experimental night. During a 1-hr period, PVT and KSS data were collected twice during the first 45 minutes. In the last 15 minutes, EEG, saliva and a third PVT and KSS measures were col- lected. Four subjects were run each experimental night; start times for subsequent subjects were staggered by 15 minutes. Data collec Figure 2 j p g g Data collection times for the first subject on an experimental night. During a 1-hr period, PVT and KSS data were collected twice during the first 45 minutes. In the last 15 minutes, EEG, saliva and a third PVT and KSS measures were col- lected. Four subjects were run each experimental night; start times for subsequent subjects were staggered by 15 minutes. Page 4 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 dition. One AAC value was calculated for each of the four channels. Near the end of each dark and each light exposure period, just before collecting saliva samples, the scalp electrodes on each subject were attached to the EEG recording sys- tem. Six minutes of data were collected: three one-minute periods with the subject's eyes closed alternating with three one-minute periods with the eyes open. When the eyes were open and subjects were not sitting at the light box (dark condition), the subjects were asked to fixate on a specific marked point approximately one meter away. Similarly, when sitting at the light box, subjects fixated on specific point on the far wall of the box approximately 0.6 m away. Subjects were visually monitored by an experi- menter to ensure compliance with the protocol. The ECG data were digitally processed the same way as the EEG data up to the high-pass filtering. Results EEG Another program divided the filtered data into 5-second epochs, segregated by periods when the eyes were open and when they were closed during the six-minute record- ing period. Eye blink artifacts were eliminated by remov- ing epochs from all channels where voltage fluctuations of any epoch exceeded ± 100 μV. A Blackman window fol- lowed by a fast Fourier transform (FFT) was then applied to the data segments. This process yielded spectral power distributions from 150 Hz. The power spectra for each one-minute segment were then combined to give an aver- age spectral power distribution for each trial. The relative power levels for eyes open in the theta (57 Hz), alpha- theta (59 Hz), alpha (812 Hz), beta (1230 Hz), and gamma (3050 Hz) ranges were calculated as a percentage of overall power from 150 Hz [4]. These calculations were not performed for those intervals when the eyes were closed because these data were only used for the AAC (below). Since the a priori hypothesis was that the circadian system mediated light-induced nocturnal alertness, the experi- ment was designed so that the two levels of red light expo- sure would serve as controls for the two levels of blue light exposure. Initially then, a pair of repeated measures ANO- VAs (two [light spectra] by two [light levels] by four [chan- nels]) was performed, one using the percent power in the EEG recordings for the alpha frequency range (812 Hz) and the other using the percent power for the beta fre- quency range (1230 Hz), as recorded from four scalp elec- trode channels (Oz, Pz, Cz and Fz). There was no significant main effect of light spectra (F1,13 = 0.005, ns, and F1,13 = 1.09, ns, for alpha and beta power, respec- tively) or of light levels (F1,13 = 0.03, ns, for both meas- ures), nor was the interaction between these independent variables statistically significant for either dependent vari- able (F1,13= 3.27, ns, and F1,13 = 3.65, ns, for alpha and beta, respectively). There was a significant effect of chan- nels (F3,39 = 38.5, p < 0.0001, for alpha and F3,39 = 4.33, p < 0.01, for beta). EEG and ECG For the ECG analy- sis, the high-pass filtering -3dB cut-off was lowered to 0.2 Hz. Heart rates corresponding to the filtered ECG data were determined by two methods: 1) by taking the FFT of the ECG, whereby the frequency having the peak power within the range from 40 to 120 beats/minute is the heart rate, and 2) determining the mean elapsed time between successive QRS complexes of the ECG. The QRS complex represents ventricular depolarization. It is called a com- plex because there are three different waves in it (Q-wave, R-wave, and S-wave). The QRS complexes were located by the first derivative of the ECG falling below a negative threshold value (corresponding to the R-wave to S-wave transition) after individual normalization of the first derivative of the ECG. The values of the first derivative of the ECG at the QRS complexes were typically five to 10 times greater in magnitude than at any other part of the waveform, which enabled this simple algorithm to relia- bly find the QRS signatures. To further guard against spu- rious values and artifacts in the ECG data from affecting the mean heart cycle period and corresponding heart rate, only those periods between the 5% and 95% quantiles were included in the calculations of the reported means. The EEG signals were sampled at 16384 Hz and then low- pass filtered and downsampled to 2048 Hz for electronic storage by the Biosemi system. All subsequent EEG data processing and analyses were performed with Matlab, ver- sion R2008a, by The Mathworks™. The signals recorded from the two reference channels were averaged and these values were subtracted from those obtained from all of the other channels. The direct current (dc) offset of each chan- nel was eliminated by subtracting the mean value of each channel from itself. A low-pass finite impulse response (FIR) filter (f-3dB = 50 Hz) was applied and the data were downsampled to 512 Hz. Then a high-pass, 3rd order But- terworth filter (f-3dB = 4 Hz) was applied to the downsam- pled signals from each channel to eliminate slow trending in the data. Page 5 of 11 (page number not for citation purposes) Results EEG Importantly, the relative alpha power averages obtained under each of the four lighting condi- tions were always lower than those obtained in the pre- ceding dark conditions, and the relative beta power averages were always higher than those obtained in the previous dark conditions. The first t-test showed that the power in the alpha fre- quencies from all channels for combined dark conditions was higher than that for the combined light conditions (p < 0.0001); the power in the beta frequencies for the com- bined dark conditions was lower than the beta power for the combined light conditions (p < 0.0001). These two statistical comparisons simply mirror the results of the third ANOVA where the main effect of the variable dark versus light was significant. These effects are illustrated in the pair of panels labeled A in Figure 3; the comparison between light and dark for the alpha frequencies is shown in the left panel and for the beta frequencies in the right panel. Since there was no difference among the four lighting con- ditions in their effectiveness for inducing alertness at night compared to the previous darkness, the aggregated effects of light versus dark were examined using a third pair of repeated measures ANOVAs (two [dark versus light] by two [first and second sessions within the same night] by two [first and second nights] by four [channels]) was performed using the relative alpha power and the rel- ative beta power as dependent variables. There was a sig- nificant main effect of dark versus light for both the alpha and the beta frequencies (F1,13 = 6.34, p = 0.03 for alpha and F1,13 = 17.2, p = 0.001 for beta). No other term in the two ANOVAs interacted with this key variable. This analy- sis demonstrates, as inferred indirectly from the initial ANOVAs, that, simply, the four lighting conditions had an alerting effect on subjects. With regard to the combined red light conditions com- pared to the combined dark conditions preceding the red light exposures, the power in both alpha and beta fre- quencies were significantly different, in the expected direction (alpha, p < 0.0001; beta, p = 0.0002). These effects are illustrated in the pair of panels labeled B in Fig- ure 3. Results EEG As a check against potentially large indi- vidual differences masking the underlying treatment effects of interest, a second pair of ANOVAs was per- formed comparing the difference, for each subject, in alpha power and the difference, for each subject, in beta The AAC was computed from the ratio of the alpha power of eyes closed to the alpha power of eyes open for each of the three sessions in the six-minute recording period [26]. The three AAC values for a single measurement period were then averaged to arrive at an AAC value for that con- Page 5 of 11 (page number not for citation purposes) Page 5 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 power between measurements made in the dark and in the subsequent light conditions. Again, there were no sig- nificant main effects for light spectra (F1,13 = 0.16, ns, and F1,13 = 0.89, ns, for alpha and beta, respectively) or light levels (F1,13 = 0.61 and F1,13 = 0.19, ns, for alpha and beta, respectively) nor was the interaction statistically signifi- cant using the differences in EEG power for either the alpha or the beta frequencies (F1,13 = 2.78, ns, for both fre- quencies). Again, however, there was a significant effect of channels. could be compared to the mean alpha power for the asso- ciated dark condition preceding that light condition. Fig- ure 3 shows, in each pair of panels, how the aggregated mean values from all four channels for light and for dark were broken down. As this figure illustrates, eight pairwise comparisons were performed. To correct for multiple comparisons, the criterion alpha level (i.e., p < 0.05) was adjusted in accordance with the Bonferroni/Dunn method to p < 0.00625. Two-tail paired t-tests were per- formed for the alpha power and for the beta power using the combined data from all four channels. These findings show, in effect, that light of either spec- trum or either level is equally effective for inducing noc- turnal alertness because there was no statistically reliable difference between the blue light and the red light expo- sures at either light level. In other words, the EEG power averages in the alpha frequency range and in the beta fre- quency range were statistically the same for all four of the lighting conditions. Results EEG The combined blue light conditions were signifi- cantly different than the combined dark conditions that preceded the blue light conditions for both alpha and beta power, in the expected direction (alpha, p < 0.0001; beta, p < 0.0001). These effects are illustrated in the pair of pan- els labeled C in Figure 3. Data for the 10 lx conditions were significantly different, in the expected direction, than the data associated with the preceding dark conditions using both alpha power (p < 0.0001) and beta power (p < 0.0001), as illustrated in the pair of panels labeled D in Figure 3. Similarly, the data for the 40 lx conditions were significantly different, in the expected direction, than the data associated with the preceding dark conditions for both alpha power (p = 0.0002) and beta power (p < 0.0001), as shown in the pair of panels labeled E in Figure 3. Since there were no significant interactions between the variable dark versus light and any of the other variables in the first two ANOVAs, the aggregate alpha power means for dark and for light and the aggregate beta power means for dark and for light were determined. These averages are shown in the top pair of panels in Figure 3; the compari- son between light and dark for the alpha frequencies is shown in the left panel and for the beta frequencies in the right panel. To more precisely understand the effects of light spectra and light levels on nocturnal alertness, the aggregated mean alpha and beta power values for light and for dark were decomposed into smaller averages whereby, for example, the mean alpha power for red 3. Alpha power was higher for the dark condition preceding the blue-10 lx than for the blue-10 lx condition, although this difference (p = 0.007) did not reach the adjusted alpha criterion. The difference in alpha power was, how- ever, significantly higher for the dark condition preceding the blue-40 lx condition than for the blue-40 lx condition Page 6 of 11 (page number not for citation purposes) Page 6 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 , p Average relative (percent) power for alpha frequencies and beta frequencies for each lighting condition Figure 3 Average relative (percent) power for alpha frequencies and beta frequencies for each lighting condition. Results EEG Higher levels of EEG alertness were associated with light than with dark; see text for details. Each pair of panels (A through I) repre- sents the average relative (percent) power for alpha frequencies (α) and beta frequencies (β) obtained for a set of lighting con- ditions relative to those recorded during the corresponding set of preceding dark conditions. Error bars represent ± standard error of the mean (s.e.m.) for each condition. Asterisks represent significant differences (p < 0.00625). Panel A compares α and β obtained from all lighting conditions compared to α and β from the preceding dark conditions. Panels B and C compare α and β for the two light colors relative to α and β for the preceding dark conditions. Panels D and E compare α and β for the two light levels relative to α and β for the preceding dark conditions. Panels F through I compare α and β for the four combi- nations of light color and light level relative to α and β for the preceding dark conditions. Average re Figure 3 The findings for the blue light conditions were expected, but those for the red light conditions were not expected; a greater effect should have been seen for the 40 lx exposure than for the 10 lx exposure. This lack of sta- tistical significance may, in part at least, reflect the lower (p < 0.05) alpha power level associated with the dim con- dition preceding the R40 condition than with any of the other the dim condition. QRS. Given the results of the series of statistical analyses conducted for the EEG data, two repeated measures ANO- VAs (two [dark versus light] by two [first and second ses- sions within the same night] by two [first and second nights]) was performed using data from both the FFT and the QRS techniques. Only the main effect of sessions was statistically significant (F1,13 = 16.3, p = 0.004 using the FFT technique; F1,13 = 12.0, p = 0.005 using the QRS tech- nique) indicating, as might be expected, that heart rate was significantly lower during the second session (later at night) than during the first. Although there was no statis- tically significant effect of dark versus light, paired one-tail t-tests were conducted to determine if specific lighting conditions were statistically different than their previous dark condition. The first t-test showed a statistically signif- icant increase in heart rate after exposure to the blue-40 lx condition compared to the prior dark period for both techniques (FFT, p = 0.003; QRS, p = 0.04). The mean ± standard error of the mean (s.e.m.) heart rate in the blue- 40 lx condition was 64.4 ± 1.2 for the FFT and 63.6 ± 1.1 for the QRS. The mean ± s.e.m. heart rate in the dark pre- ceding the blue-40 lx condition was 61.8 ± 1.0 for the FFT and 62.3 ± 1.1 for the QRS. For the red-40 lx condition, only the QRS method showed a significant increase in heart rate relative to the previous dark condition (p = 0.04). The mean ± s.e.m. heart rate in the red-40 lx condi- tion was 61.9 ± 1.1 for the QRS, and in the dark preceding the red-40 lx condition it was 61.1 ± 1.1. No significant differences in heart rate were found using either technique at the lower level (10 lx) of either blue or red light. Average re Figure 3 Average re Figure 3 Average relative (percent) power for alpha frequencies and beta frequencies for each lighting condition Figure 3 Average relative (percent) power for alpha frequencies and beta frequencies for each lighting condition. Higher levels of EEG alertness were associated with light than with dark; see text for details. Each pair of panels (A through I) repre- sents the average relative (percent) power for alpha frequencies (α) and beta frequencies (β) obtained for a set of lighting con- ditions relative to those recorded during the corresponding set of preceding dark conditions. Error bars represent ± standard error of the mean (s.e.m.) for each condition. Asterisks represent significant differences (p < 0.00625). Panel A compares α and β obtained from all lighting conditions compared to α and β from the preceding dark conditions. Panels B and C compare α and β for the two light colors relative to α and β for the preceding dark conditions. Panels D and E compare α and β for the two light levels relative to α and β for the preceding dark conditions. Panels F through I compare α and β for the four combi- nations of light color and light level relative to α and β for the preceding dark conditions. Page 7 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 (p < 0.0001). These differences are illustrated in the left panels of H and I, respectively, in Figure 3. For beta power, the same ordering effects were observed, but in the oppo- site, expected direction; beta power was significantly lower for the dark condition preceding the blue-10 lx than for the blue-10 lx condition (p = 0.0006), and lower for the dark condition preceding the blue-40 lx condition than for the blue-40 lx condition (p < 0.0001), as illus- trated in the right panels of H and I, respectively, in Figure 3. Power in the alpha and beta frequencies were not sig- nificantly different for the red-40 lx condition and the pre- vious dark condition (panel G of Figure 3), whereas power in both, alpha and beta frequencies were significantly dif- ferent in the red-10 lx condition compared to the preced- ing dark condition (p < 0.0001 for alpha and beta) (panel F of Figure 3). Average re Figure 3 Although the ECG results are weaker than those from the EEG data, they do suggest that sufficient irradiance at the cornea of either red or blue light exposure can increase alertness at night. A repeated measures ANOVA (two [dark versus light] by two [first and second sessions within the same night] by two [first and second nights] by four [channels]) was per- formed using the AAC values computed from the alpha power with the eyes closed to the alpha power with the eyes opened (data not shown). Several maximum ampli- tude criteria were applied to the raw alpha power (47 μV, 75 μV, 100 μV) to minimize artifacts in the data (however, AAC for one subject could not be determined, so the ANOVAs included data for 13 subjects). As expected, the channels (F3,36 = 21.28, p < 0.0001) were statistically dif- ferent. The main effect of night (F1,12 = 5.61, p = 0.04) was also statistically significant. From this measure it would seem that subjects were more tired on the second night than on the first night. There was a statistically significant interaction between night and channel (F3,36 = 3.83, p = 0.02). This result reflects the fact that the differences in AAC among the four channels were smaller on the second night than on the first night. The interaction between light and channel (F3,36 = 3.21, p = 0.03) was also statistically significant. Parallel to the night by channel interaction, the differences in AAC among the four channels were smaller for the light condition than for the dark condi- tion. These significant interactions do not have any obvi- ous implication and since there was no main effect of light, the findings from the AAC will not be discussed fur- ther. PVT and KSS E h f h Each of the outcome measures obtained from the PDA (RT, TCRT, MTS, and KSS) were submitted to the same series of repeated measures ANOVAs as the ECG variables, with one additional factor, trial: (i.e., two [dark versus light] by two [first and second sessions within the same night] by two [first and second nights] by three [trials]). Three successive trials were conducted during every light and every dark condition, one at the beginning, one in the middle, and one at the end of the 60-minute light expo- sure or dark condition. One subject did not follow instructions; that subject's data were not included in the analyses. For KSS, the main effects of session (F1,12 = 46.14, p < 0.0001), trial (F2,24 = 22.06, p < 0.0001), and light (F1,12 = 34.98, p < 0.0001) were statistically significant, as was the session by light interaction (F1,12 = 11.19, p = 0.006). The three main effects demonstrate increasing sleepiness throughout the experiment. The second session was asso- http://www.biomedcentral.com/1471-2202/10/105 http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 ciated with higher values of KSS than in the first session; Paired two-tail t-tests showed that KSS values associated with the second trial and with the third trial were signifi- cantly higher than those in the first trial (p < 0.0004 and p < 0.0001, respectively) although values for the second trial were not significantly different than those associated with the third trial. KSS values were significantly higher (p < 0.0001) in the light exposure condition than in the dark, but since every light exposure condition followed a dark condition, even the significant main effect of light very likely reflects growing sleepiness by the subjects through- out the experiment. Interestingly, however, the significant interaction between session and light suggests that light, in fact, served as a countermeasure for fatigue: a two-tail paired t-test revealed that the difference in reported sleep- iness between session 1 and session 2 was significantly greater (p < 0.0001) in the dark than in the light. tion was based on 13 subjects. Using these data, a two [light spectra] by two [light levels] repeated measures ANOVA was performed; neither the main effects were sig- nificant (light color, F1,12 = 0.3, ns; light level F1,12 = 1.9, ns), nor was the interaction (F1,12 = 1.1, ns). However, melatonin concentrations were suppressed by 18% ± 15% (average ± s.e.m.) after exposure to the blue-40 lx condi- tion relative to the preceding dark condition, whereas neg- ative suppression values were determined for the other three lighting conditions (-62% ± 33% for red-10 lx, -39% ± 24% for red-40 lx, and -96% ± 82% for blue-10 lx). In other words, melatonin levels were significantly lower for the blue-40 lx condition than for the dark conditions pre- ceding the blue-40 lx exposures, supporting the literature [12] that this lighting condition was the strongest circa- dian stimulus. In fact, post hoc one-tail paired t-tests revealed a significantly higher level of melatonin suppres- sion for the blue-40 lx condition than for either the red-40 lx condition (p = 0.03) or the red-10 lx condition (p = 0.02). There was no significant difference between the blue-10 lx condition and the blue-40 lx condition (p = 0.09). http://www.biomedcentral.com/1471-2202/10/105 In terms of the performance measures, only the TCRT task showed a significant main effect for trial with response time (F2,24 = 6.06, p < 0.007) and with score (F2,24 = 8.43, p < 0.002). For both TCRT measures, performance was consistently worse as the trials progressed (i.e., response time increased and score decreased). Consistent with the KSS data, these results indicate that fatigue played an important role in this study. Moreover, the trial by night interaction was statistically significant for both TCRT measures (F2,24 = 4.98, p < 0.02, for response time; F2,24 = 4.08, p < 0.03, for score), suggesting that, consistent with the AAC results, fatigue played a greater role in the second night than in the first night; paired two-tail t-tests showed that the difference in TCRT for response time from the first trial to the third trial was significantly greater (p = 0.02) on second night than on the first night, but the difference in TCRT for score did not reach significance (p = 0.1). No effects of light exposure, either among the different light exposure conditions or relative to the previous dark con- ditions, were demonstrated by any of the performance measures. It must be noted that, because of the counterbalanced experimental design, the average suppression values used in these statistical analyses were determined from com- bined suppression values obtained at two different circa- dian times. In the counterbalanced experimental design, half the subjects would have been presented with the 10 lx conditions before the 40 lx conditions, and half the subjects would have been presented with the blue light conditions before the red light conditions. Thus, average suppression values for every lighting condition were based upon data obtained during both the early and the later exposure times in the sessions. Since the rate of mela- tonin synthesis changes throughout the night, melatonin suppression values will be differentially affected by the sample time. Specifically, the same light stimulus pre- sented early in the night will result in less suppression than when it is presented later in the night because the rate of melatonin synthesis is high in the early night, reaches a peak value in the middle of the night, and then decreases until the end of the night [27]. Melatonin Melatonin suppression for each lighting condition was calculated for every subject using the following formula: 1 - (melatonin in the light/melatonin in the dark). If light exposure is a strong stimulus to the circadian system, it will suppress melatonin levels below those measured in the previous dark condition, whereas if it is a weak stimu- lus, light exposure may not fully counteract the natural rise in melatonin levels that occurs during the early night- time, in which case negative suppression will be observed. The suppression values associated with three of the four lighting conditions were then determined from those obtained from all 14 subjects; melatonin levels could not be detected from one subject's saliva sample for the blue- 40 lx condition, so the average suppression for this condi- ECG As mentioned in the Methods section, the heart rate data were analyzed using two different techniques, FFT and Page 8 of 11 (page number not for citation purposes) Page 8 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) Authors' contributions g g J 14. Figueiro MG, Bullough JD, Bierman A, Fay CR, Rea MS: On light as an alerting stimulus at night. Acta Neurobiol Exp (Wars) 2007, 67(2):171-178. MGF participated in the conception and the design of the experiment, data collection, data analyses and interpreta- tion, and manuscript writing. AB participated in the data analyses and interpretation and reviewed the manuscript. BP participated in data collection, analyses, and drafted sections of the manuscript. MR participated in the concep- tion and design of the experiment, data interpretation and manuscript writing. All authors read and approved the final manuscript. ( ) 15. Saper CB, Cano G, Scammell TE: Homeostatic, circadian, and emotional regulation of sleep. J Comp Neurol 2005, 493(1):92-98. ( ) 16. Saper CB, Lu J, Chou TC, Gooley J: The hypothalamic integrator for circadian rhythms. Trends Neurosci 2005, 28(3):152-157. y ( ) 17. Saper CB, Scammell TE, Lu J: Hypothalamic regulation of sleep and circadian rhythms. Nature 2005, 437(7063):1257-1263. and circadian rhythms. Nature 2005, 437(7063):1257-1263. 18. Stone NJ: Environmental view and color for a simulated tele- marketing task. J Environ Psychol 2003, 23:63-78. 19. Hill RA, Barton RA: Psychology: red enhances human perform- ance in contests. Nature 2005, 435(7040):293. 18. Stone NJ: Environmental view and color for a simulated tele- marketing task. J Environ Psychol 2003, 23:63-78. g J y 19. Hill RA, Barton RA: Psychology: red enhances human perform- ance in contests. Nature 2005, 435(7040):293. ( ) 20. Elliot AJ, Maier MA, Moller AC, Friedman R, Meinhardt J: Color and psychological functioning: the effect of red on performance attainment. J Exp Psychol Gen 2007, 136(1):154-168. References 1. Badia P, Myers B, Moecker M, Culpepper J: Bright light effects on body temperature, alertness, EEG and behavior. Physiol Behav 1991, 50(3):583-588. ( ) 2. Boyce P, Beckstead J, Eklund N, Strobel R, Rea M: Lighting the graveyard shift: The influence of a daylight-simulating sky- light on the task performance and mood of nightshift work- ers. Light Res Technol 1997, 29(3):105-134. g ( ) 3. Cajochen C, Krauchi K, Danilenko KV, Wirz-Justice A: Evening administration of melatonin and bright light: interactions on the EEG during sleep and wakefulness. J Sleep Res 1998, 7(3):145-157. ( ) 4. Cajochen C, Zeitzer J, Czeisler C, Dijk D: Dose-response relation- ship for light intensity and ocular and electroencephalo- graphic correlates of human alertness. Behav Brain Res 2000, 115:75-83. Although there was some evidence that light mitigated the growing sleepiness in the experiment (i.e., the statistically significant session by light interaction), both the KSS and the PVT results did not support the inference that light is a robust countermeasure for subjective sleepiness or for performance decrement, thus suggesting that for this pro- tocol electrophysiological measures of alertness can be more sensitive than behavioral measures of alertness. 5. Campbell SS, Dawson D: Enhancement of nighttime alertness and performance with bright ambient light. Physiol Behav 1990, 48(2):317-320. ( ) 6. Daurat A, Foret J, Benoit O, Mauco G: Bright light during night- time: effects on the circadian regulation of alertness and per- formance. Biol Signals Recept 2000, 9(6):309-318. g p ( ) 7. Eastman CI, Liu L, Fogg LF: Circadian rhythm adaptation to sim- ulated night shift work: effect of nocturnal bright-light dura- tion. Sleep 1995, 18(6):399-407. 8. 8. Lowden A, Akerstedt T, Wibom R: Suppression of sleepiness and melatonin by bright light exposure during breaks in night work. J Sleep Res 2004, 13(1):37-43. Discussion Nocturnal alertness as measured by ECG and EEG is affected by light, but the results presented here suggest that a pathway other than the circadian system may affect light-induced nocturnal alertness because red light as well as blue light was effective. Heart rate measured with both the FFT and the QRS techniques increased significantly relative to the dark conditions for the higher level (40 lx) of both blue and red light, although the lower level (10 lx) of the two spectra was not effective. In terms of the EEG data, both red and blue light (combining both 10 lx and Page 9 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/10/105 BMC Neuroscience 2009, 10:105 http://www.biomedcentral.com/1471-2202/10/105 40 lx) reduced alpha power and increased beta power lev- els relative to their respective preceding dark conditions. Similarly, both 10 lx and 40 lx of light (combining both blue and red) had a significant impact on alpha and on beta relative to their respective preceding dark conditions. Although both 10 lx and 40 lx were effective for the blue light, only 10 lx, but not 40 lx of red light significantly affected the EEG recordings compared to their preceding dark conditions. This apparent dose intransitivity for the red light conditions remains unexplained and is some- what implausible, although it is conceivable that there is an optimum irradiance of red light for alertness (i.e., red- 10 lx), but this hypothesis seems rather unlikely and these results definitely demand further study. Russell Sage College are acknowledged for their support and contributions to the study. Dr. David Epstein, from the National Institute on Drug Abuse Intramural Research Program is greatly acknowledged for his comments to the manuscript. The authors are also very grateful to the reviewers and the associate editor for their comments and suggestions to the manuscript. Conclusion Based mainly on the EEG and ECG data, the present results are, to a limited extent, consistent with previous findings showing that light of sufficient corneal irradiance increases alertness at night [1-9]. There is previous com- pelling evidence that light-induced stimulation of the cir- cadian system increases alertness at night, but the present results suggest that this effect is not mediated only by the circadian system, implicating other mechanisms through which light can also increase nocturnal alertness. It is important then to determine if these inferred mechanisms are independent of the circadian system or interact with it by conducting light studies at different circadian times. J p ( ) 9. Figueiro M, Rea M, Boyce P, White R, Kolberg K: The effects of bright light on day and night shift nurses' performance and well-being in the NICU. Neonatal Intens Care 2001, 14(1):29-32. 10. Brainard G, Hanifin J, Greeson J, Byrne B, Glickman G, Gerner E, Rol- lag M: Action spectrum for melatonin regulation in humans: Evidence for a novel circadian photoreceptor. J Neurosci 2001, 21:6405-6412. 11. Thapan K, Arendt J, Skene DJ: An action spectrum for melatonin suppression: evidence for a novel non-rod, non-cone pho- toreceptor system in humans. J Physiol 2001, 535(Pt 1):261-267. p y J y ( ) 12. Rea M, Figueiro M, Bullough J, Bierman A: A model of phototrans- duction by the human circadian system. Brain Res Rev 2005, 50(2):213-228. ( ) 13. Cajochen C, Munch M, Kobialka S, Krauchi K, Steiner R, Oelhafen P, Orgul S, Wirz-Justice A: High sensitivity of human melatonin, alertness, thermoregulation and heart rate to short wave- length light. J Clin Endo Met 2005, 90:1311-1316. Acknowledgements Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 11 of 11 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Acknowledgements The study presented here was supported by the Office of Naval Research through the Young Investigator Program awarded to MGF. The authors would like to acknowledge Dr. Vodyanoy of the Office of Naval Research for his support. Dr. Christopher Steele of the Naval Research Medical Lab- oratory, John Bullough, Dennis Guyon, Bonnie Morgan, Chris Munson, and Jennifer Taylor of the Lighting Research Center, and Lauren Schramek of J p y ( ) 21. Roenneberg T, Wirz-Justice A, Merrow M: Life between clocks: daily temporal patterns of human chronotypes. J Biol Rhythms 2003, 18(1):80-90. ( ) 22. Thorne DR, Johnson DE, Redmond DP, Sing HC, Belenky G, Shapiro JM: The Walter Reed palm-held psychomotor vigilance test. Behav Res Methods 2005, 37(1):111-118. Page 10 of 11 (page number not for citation purposes) Page 10 of 11 (page number not for citation purposes) BMC Neuroscience 2009, 10:105 http://www.biomedcentral.com/1471-2202/10/105 http://www.biomedcentral.com/1471-2202/10/105 23. Lamond N, Dawson D, Roach GD: Fatigue assessment in the field: validation of a hand-held electronic psychomotor vigi- lance task. Aviat Space Environ Med 2005, 76(5):486-489. p , ( ) 24. Akerstedt T, Gillberg M: Subjective and objective sleepiness in the active individual. Int J Neurosci 1990, 52(12):29-37. J ( ) 25. American Electroencephalographic Society: Guidelines for stand- ard electrode position nomenclature. J Clin Neurophysiol 1991, 8:200-202. 26. Alloway CE, Ogilvie RD, Shapiro CM: The alpha attenuation test: assessing excessive daytime sleepiness in narcolepsy-cata- plexy. Sleep 1997, 20(4):258-266. p p ( ) 27. Meltzer JA, Zaveri HP, Goncharova II, Distasio MM, Papademetris X, Spencer SS, Spencer DD, Constable RT: Effects of working mem- ory load on oscillatory power in human intracranial EEG. Cereb Cortex 2008, 18(8):1843-1855.
W4391192875.txt	https://www.degruyter.com/document/doi/10.1515/9783839469125-034/pdf	de		8.3. Trutz: »[F]ür die Ewigkeit fixiert, festgehalten bis zum Tod«?	transcript Verlag eBooks	2,024	cc-by	18,417	232 Richard Slipp: Gedächtnis und Erzählen 8.3. Trutz: »[F]ür die Ewigkeit fixiert, festgehalten bis zum Tod«? »In much metafiction the reader is left with the impression that, since all fiction is a kind of parody of life, no matter how verisimilar it pretends to be, the most authentic and honest fiction might well be that which most freely acknowledges its fictionality.« – Linda Hutcheon51 8.3.1. Einleitung Christoph Heins 2017 erschienenem Roman Trutz verleihen gleich zwei der Hauptfiguren seinen Titel: Erster Namensgeber ist Rainer Trutz, ein angehender Schriftsteller vom vorpommerschen Land, der in den 1920er Jahren sein Glück in der Hauptstadt der instabilen Republik sucht; der zweite ist sein Sohn, Maykl, der seine Kindheit in der Sowjetischen Union verbringt, bevor er als Vollwaise in die DDR übersiedelt und eine Laufbahn als Archivar einschlägt. Was sich zwischen diesen Stationen im jeweiligen Leben der zwei Männer zuträgt, ist eine Geschichte des Leidens unter Repressalien, Verfolgung, Verhaftung und Zwangsarbeit. Der eher apolitische Rainer und seine Frau Gudrun, eine Gewerkschaftsangestellte und »Religiöse Sozialistin«, geraten durch satirischen Roman Rainers zur Zielscheibe zunächst der rechten Presse und dann rechter Schlägertrupps. In ihren Bemühungen, ein Visum für die Auswanderung zu erhalten, von unzähligen Auslandsvertretungen abgewiesen, geht das Paar dank der Freundschaft mit der sowjetischen Kulturfunktionärin Lilija Simonaitis ins Moskauer Exil. Dort wird Rainer dem Bau der U-Bahn zugeteilt, während Gudrun eine Stelle in einer Süßwarenfabrik bekommt. In Moskau lernen die jungen Eheleute Alina und Waldemar Gejm kennen; Letzterer ist ein Professor für Sprachwissenschaften, an dessen Gedächtnisexperimenten Trutzes zusammen mit ihrem Jungen Maykl und Gejms gleichaltrigem Sohn Rem teilnehmen. Nachdem Rainer wegen einer weit zurückliegenden, zweiseitigen Rezension in der Weltbühne zu fünf Jahren Gulag verurteilt und – nach dem qualvollen, aber überstandenen Marsch nach Workuta – gleich am Tag seiner Ankunft ermordet wird, gerät auch der inzwischen zwangsemeritierte Waldemar Gejm aufgrund seiner Forschungen zunehmend ins Visier der Staatsmacht. Die Gejms werden zunächst in dieselbe Siedlung wie Gudrun und Maykl Trutz deportiert, bis Waldemar schließlich ebenfalls zu einem Besserungsarbeitslager verurteilt wird, wo der für schwere körperliche Arbeit denkbarst ungeeignete Akademiker innerhalb kürzester Zeit bei einem Arbeitsunfall stirbt. Nach dem Hunger- und Erschöpfungstod seiner Mutter kommt Maykl zuerst in die Obhut der Gejms, dann in ein Waisenhaus nach Moskau und schließlich als junger Mann in die DDR, wo er Geschichte und Archivwesen studiert. Während sich sein eidetisches Gedächtnis anfangs von großem Vorteil für seine Berufswahl erweist, wird es ihm in seinen ersten romantischen Beziehungen und später auch professionell zum Verhängnis. Er ist es, der am Ende seines Lebens dem Erzähler die Geschichte der Familien Trutz und Gejm mitteilt und dem der Erzähler sein Buch widmet. Auf Maykls Beerdigung, mit der die Rahmenhandlung des Romans zu Ende geht, spielt das Fledermaus-Couplet: »Glücklich ist, wer vergisst, was doch nicht zu ändern ist«. 51 Hutcheon: Narcissistic Narrative, S. 49. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz 8.3.2. Fiktionalität (I): Die Rahmenerzählung als inszenierte Herausgeberfiktion »Obwohl das Machtwort darüber gefällt ist, machen sich viele Personen lächerlich, indem sie einen Schriftsteller zum Komplizen der Gefühle machen, die er seinen Figuren zuschreibt; und wenn er das Ich verwendet, sind fast alle versucht, ihn mit dem Erzähler zu verwechseln.« – Honoré de Balzac52 »Alle Eigenschaften des Sprechers dem Autor zuzuschreiben ist sicherlich naiv, aber ihm keine zuzuschreiben ist nur mit der Hilfe einer gewaltigen theoretischen Bornierung möglich.« – Fotis Jannidis53 Wie bereits kurz angedeutet, ist der eigentlichen Diegese von Trutz, d.h. den oben abgerissenen Handlungssträngen um die Familien Trutz und Gejm, eine Eingangssequenz von ca. fünfzehn Seiten vorgelagert, die in den frühen 2000er Jahren spielt und die Umstände des Kennenlernens des heterodiegetischen Erzählers mit dem Protagonisten Maykl Trutz schildert. Der erste Satz dieser Sequenz – und damit des Romans – ist einer, der nach dem Urteil mindestens eines Rezensenten »in die Literaturgeschichte eingehen« werde54 : »In diesen Roman geriet ich aus Versehen oder vielmehr durch eine Bequemlichkeit« (T 7). Anschließend berichtet der anonyme Erzähler, wie es auf einer Veranstaltung der Stiftung zur Aufarbeitung der SED-Diktatur zur unverhofften Begegnung mit Trutz und somit zum Stoff für einen Roman kam. Im Folgenden soll gezeigt werden, wie die Darstellung der Genese dieses Romans in der Rahmenerzählung einerseits eine Illusion der Authentizität erweckt und andererseits die eigene Fiktionalität andeutet, die in der Binnenerzählung dann schließlich offenlegt wird. Der soeben zitierte Rezensent, Carsten Otte, erläutert nicht genau, was ihn an diesem knappen, eher beiläufig anmutenden Erzählauftakt so begeistert, doch nimmt man den Satz näher unter die Lupe, lassen sich in ihm tatsächlich einige bezeichnende und teils paradoxe Interpretationsimpulse verdichtet ausmachen. Erstens wird, beginnend mit dem Verb »geraten«, das der Duden mit »ohne Absicht, zufällig an eine bestimmte 52 53 54 Aus dem Vorwort zu Le Lys dans la vallée, zitiert in Genette: Paratexte, S. 210. Jannidis: »Verstehen erklären«, S. 55. Carsten Otte: »Kein Klugscheißer, Mnemotechniker«, in: taz vom 29.6.2017; http://www.taz.de/ !5421540 (06.09.2023). Ähnlich kommentiert Rüdiger Bernhardt: »Die Eröffnung des Romans gehört zu jenen Sätzen, die in Zukunft erinnert werden […] Es wird in späteren Literaturgeschichtsschreibungen als einer der berühmtesten ersten Sätze bezeichnet werden«; Bernhardt: Der vergessene Mythos, S. 293. Bernhardt verfolgt in seiner 2022 gedruckten Interpretation des Prologs von Trutz generell einen ähnlichen Argumentationsaufbau wie in der vorliegenden Analyse der Rahmenerzählung; hiermit wird darauf aufmerksam gemacht, dass die obigen Ausführungen bereits Anfang 2021 vom Verfasser in leicht abgewandelter Form unter folgendem Titel veröffentlicht wurden: »[F]ür die Ewigkeit fixiert, festgehalten bis zum Tod«? Zu einem scheinbaren Wandel im Geschichts- und Erinnerungsverständnis von Christoph Hein im Roman Trutz (2017)«, in: German as a Foreign Language 1 (2021), S. 43–67; http://www.gfl-journal.de/1-2021/Slipp.pdf (06.09.2023). 233 234 Richard Slipp: Gedächtnis und Erzählen Stelle, irgendwohin gelangen« definiert,55 ein anfängliches Desinteresse des Erzählers gegenüber seinem Erzählprojekt suggeriert. Dieser Eindruck der Passivität wird durch die Substantive »Versehen« und »Bequemlichkeit« weiter verstärkt. Zweitens signalisiert die Erwähnung der literarischen Gattung »Roman« eine gewisse Distanz und Reflexion und lädt zunächst zu der Annahme ein, dass der Erzählauftakt und womöglich der ganze Eröffnungsabschnitt außerhalb der Fiktion zu situieren seien. Denn wenn hier über den vorliegenden Roman geredet wird, dann fühlt man sich als Leser – anders als bei einem Einstieg »in medias res« – wohl noch nicht mitten im Romangeschehen. So könnte bereits nach dem ersten Satz ein anfänglicher Verdacht entstehen, man habe es hier mit einem editorischen Prolog zu tun, der einer fertigen, vorgefundenen Geschichte vorangestellt wurde. Drittens – und gewissermaßen entgegen den ersteren zwei Beobachtungen – ist der Gebrauch des Präpositionalsatzes »in diesen Roman« auffällig: Der Erzähler erklärt nämlich nicht, wie er zu dem Roman bzw. zum Romanstoff kam, sondern wie er selber in einem Roman landete. Während er sich einerseits von der anschließenden Haupthandlung des Romans, deren heterodiegetischer Erzähler er ist, zu distanzieren sucht, thematisiert er gleichzeitig seine Anwesenheit als Romanfigur in der Rahmung und verdeutlicht damit den Status der Rahmung als Teil der Fiktion. Übrigens stellt Trutz das bislang einzige Beispiel eines »overt«56 oder personalisierten heterodiegetischen Erzählers bei Christoph Hein dar; in allen anderen längeren Prosatexten sind entweder homodiegetische (Ich-)Erzähler57 oder nicht personalisierte Erzählinstanzen58 am Werk. Die im ersten Satz des Romans angedeutete »Bequemlichkeit« wird dann vom Erzähler erläutert. Er habe nämlich gehofft, bei dem Vortrag in Berlin-Mitte eine Direktorin des Bundesarchivs ansprechen und sich so den längeren Weg nach Dahlem ersparen zu können (T 7). Dass es dem Erzähler in der Tat ursprünglich um andere Anliegen geht als das, was gleich zum Stoff seines zukünftigen Romans werden sollte, wird er nicht müde zu betonen. Das Thema der Veranstaltung, nämlich »deutsch-russische Verhältnisse im zwanzigsten Jahrhundert«, interessiere ihn »nur am Rande«, und er gesteht ein, schon im Voraus den Vortrag nur »scheinbar interessiert« zuhören zu wollen, denn er sei »mit anderen Dingen beschäftigt« und sein Ziel sei ja lediglich, sich deswegen Zugang zu einer »Person der oberen Etage des Bundesarchivs« zu erschaffen (T 7). Auch im Laufe des Vortrags ändert sich nichts an seiner Unbeeindrucktheit: »Die neuen Dokumente, die die Archivarin dem Publikum mit Overheadfolien präsentierte, erschienen mir belanglos. Es waren keine sensationellen Erkenntnisse, die eine Neubewertung der geschichtswissenschaftlichen Sicht auf das letzte Jahrhundert er- 55 56 57 58 Dudenredaktion: »geraten«, Duden online; https://www.duden.de/rechtschreibung/geraten_geli ngen_hingelangen (06.09.2023). Seymour Chatman: Story and Discourse. Narrative Structure in Fiction and Film, Ithaca, NY: Cornell UP 1978, S. 219f. Der fremde Freund, Horns Ende, Das Napoleon-Spiel, Landnahme, Von allem Anfang an, Frau Paula Trousseau, Glückskind mit Vater und Unterm Staub der Zeit. In Paula Trousseau und Glückskind wird streckenweise auch von einer Erzählinstanz in dritter Person erzählt. Der Tangospieler, Willenbrock, In seiner frühen Kindheit ein Garten, Weiskerns Nachlass, Verwirrnis und Guldenberg. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz forderlich machten, und was daran unbekannt war, überraschte nicht, sondern blieb im Rahmen des Erwartbaren.« (T 10). Erst bei der anschließenden Frage-Antwort-Runde wird die Aufmerksamkeit des Erzählers geweckt, als ein älterer Herr durch seine beharrlichen Korrekturen von Fehlern im Vortrag und sein auswendiges Zitieren von Quellen auffällt; die zwei Männer kommen dann nach dem Ende der Veranstaltung ins Gespräch.59 Die oben erwähnten »anderen Dinge«, mit denen der Erzähler zu diesem Zeitpunkt beschäftigt sei, seien Recherchen zu einem Rechtsfall mit politischer Brisanz, die er wie folgt darstellt: »Ich war seit mehr als einem Jahr dabei, die seltsamen Umstände des Todes eines Terroristen zu erhellen, der von einer Hundertschaft Grenzpolizisten ergriffen werden sollte, dabei durch eine Kugel ums Leben kam, doch war trotz mehrerer Prozesse nie aufzuklären, wer den tödlichen Schuss abgegeben hatte, der Gesuchte selbst oder einer der Beamten.« (T 7–8) Hinter dem nicht näher identifizierten Terroristen lässt sich ohne viel Phantasie Wolfgang Grams erkennen, dessen gewaltsamem Tod auf dem Bahnhof von Bad Kleinen die hier skizzierten Umstände ähneln; es handelt sich also um einen realen, vom wirklichen Autor Hein in seinem Roman In seiner frühen Kindheit ein Garten (2005) fiktionalisierten Stoff. Allerdings – obgleich der Erzähler von Trutz gegen Ende des Kapitels eine geplante Publikation zum Thema erwähnt (T 18) – identifiziert er sich an keiner Stelle als Schriftsteller und macht auch nicht klar, ob seine Beschäftigung mit dem Fall des Terroristentods literarischer, geschichtswissenschaftlicher oder sonst irgendeiner Natur ist; man erfährt lediglich, dass er ein »Mann ohne jede Amtsgewalt« (T 9) – also wohl selbst kein Anwalt oder Richter – sei. Wie bereits oben erwähnt, haben sich mehrere Kritikerinnen durch die Rahmenerzählung und insbesondere durch die Anspielung auf den Terroristen-Roman zu Rückschlüssen auf die Wahrheit der erzählten Begebnisse in Trutz, sowie auf die Identität zwischen Erzähler und Autor verleiten lassen. Ein dahingehender Kommentar von Claus-Ulrich Bielefeld wurde bereits zitiert;60 auf eine ähnliche Weise glaubt auch Jochen Schimmang feststellen zu können: »Der Erzähler dieses Romans ist ein Schriftsteller […]. Er hält sich weitgehend im Hintergrund und ist namenlos; wir dürfen ihn aber getrost Christoph Hein nennen.«61 Ebenso scheint Dietmar Jacobsen eine solche Gleichsetzung für eine gegebene Tatsache zu halten: »Es war ein Zufall […] der Christoph 59 60 61 Für das tatsächliche Stattfinden einer Veranstaltung in den frühen 2000er Jahren unter dem Titel »Feindliche Freunde« gibt es übrigens in den Pressemitteilungen und Bekanntmachungen der Bundesstiftung keinen Beleg, wenn auch die Stiftung zum betreffenden Zeitpunkt an einer gemeinsamen Geschichtskommission zu deutsch-russischen Beziehungen beteiligt war. Siehe »Bericht der Bundesregierung zum Stand der Aufarbeitung der SED-Diktatur«, S. 103; Bundesregierung, https://www.bundesregierung.de/Content/DE/_Anlagen/BKM/2013-01-08-bericht-aufarbei tung-sed-diktatur.pdf?__blob=publicationFile (06.09.2023). Siehe oben, Kapitel 8.1. Schimmang: »Als Stalin selbst zur Feder griff«. 235 236 Richard Slipp: Gedächtnis und Erzählen Hein zum Stoff für sein neues Buch verhalf. Im Prolog zu Trutz gibt uns der Berliner Autor (geb. 1944), […] Auskunft darüber, wie er an ihn kam«.62 Für Judith von Sternburg in der Frankfurter Rundschau lassen sich aufgrund der Anspielung auf den früheren HeinRoman sogar Spekulationen über die Entstehungsgeschichte von Trutz rechtfertigen: »Der Erzähler, erfährt man übrigens ungefragt, ist seinerseits eigentlich mit dem Fall Bad Kleinen beschäftigt, Thema im Roman ›In seiner Kindheit ein Garten‹ (2005) – das könnte ein Hinweis darauf sein, dass der Schriftsteller schon länger mit ›Trutz‹ befasst ist.«63 Auf die seltsame, hier offenbar für selbstverständlich gehaltene Annahme, man könne in Erzähltexten etwa zwischen von Lesenden »gefragten« und »ungefragten« Informationen unterscheiden, soll an dieser Stelle nicht näher eingegangen werden. Wenn aber auch die Schlussfolgerung Sternburgs bezüglich der Chronologie der Beschäftigung Heins mit dem Trutz-Stoff als fraglich eingestuft werden muss, wird ihr hier insofern Recht gegeben, als die Thematisierung der Recherchen des Erzählers zum möglichen Justizskandal um den Tod des Terroristen keineswegs beliebig oder zufällig ist; im Folgenden wird hierauf noch zurückzukommen sein. Dass der Erzähler in dieser Erinnerungssequenz anonym bleibt, kann einerseits als konsequent mit seiner Selbstinszenierung als bloßer bescheidener Vermittler einer Geschichte, die ihm von dem sich genauestens erinnernden Helden mitgeteilt wurde, gesehen werden. Sogar unter autodiegetischen Erzählern, d.h. Erzähler, die nicht nur Teil der Diegese sind, sondern auch ihre eigene Geschichte erzählen, ist Anonymität nicht ungewöhnlich; Erzählauftakte, bei denen sich der Protagonist namentlich identifiziert, wie etwa »Call me Ishmael«,64 oder gar verneinende Varianten wie »Ich bin nicht Stiller!«,65 bilden wohl eher die Ausnahme. Im Falle von Trutz wäre es aber sehr leicht denkbar oder sogar zu erwarten, dass der Erzähler seinen Namen verrät, betrachtet man den Ablauf der gegenseitigen Vorstellung des Erzählers und seines zukünftigen Romanhelden: 62 63 64 65 Dietmar Jacobsen: »Erinnern kann gefährlich sein«, in: literaturkritik.de 9 (September 2017); ht tp://literaturkritik.de/hein-trutz-erinnern-kann-gefaehrlich-sein,23637.html (06.09.2023). Jacobsen spricht zudem von einem Ich-Erzähler, »hinter dem man unschwer den Autor selbst erkennen kann.« An dieser Stelle muss angemerkt werden, dass einige wenige Rezensentinnen durchaus die Fiktionalität des Erzählrahmens sowie des Erzählanlasses erkannt haben, wie beispielsweise Carsten Otte, der feststellt: »[…] die Erzählperspektive und der sachliche Chronistentonfall sind höchst artifiziell. Nein, hier ist kein Autor am Werk, der in den Archiven auf eine spannende Geschichte gestoßen ist und der sich nun freut, die Lebensgeschichte einer außergewöhnlichen Familie zwischen zwei Buchdeckel zu pressen«; Otte: »Kein Klugscheißer, Mnemotechniker«; oder Christian Buß, der befindet: »Hein ist – dem Thema Erinnerung zum Trotz – ein lustvoller Fabulierer«; Christian Buß: »Hitler, Stalin und der kleine Trutz«, in: Spiegel Online vom 29.3.2017; http://www.spiegel.de/kultur/literatur/trutz-von-christoph-hein-ueber-eine-biograph ie-zwischen-hitler-und-stalin-a-1140568.html (06.09.2023). von Sternburg: »Ins Langzeitgedächtnis damit«; die Deduktionsarbeit der Rezensentin ist ohnehin überflüssig, da der Erzähler den Zeitraum seiner Bekanntschaft mit Trutz ausdrücklich in den Jahren 2003–2007 situiert. Herman Melville: Moby Dick; or, the Whale, Project Gutenberg; https://www.gutenberg.org/files/2701/2701-h/2701-h.htm (06.09.2023). Max Frisch: Stiller, Berlin: Suhrkamp 2017, S. 9. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz »›Trutz heiß ich, Maykl Trutz‹, sagte er. Ich gab ihm die Hand, stellte mich ebenfalls vor und fragte, wo man mehr über diese Wissenschaft erfahren könne, von der ich noch nie gehört hatte.« (T 15) Diese demonstrative Unterdrückung des eigenen Namens – sowie die Weigerung, seine Recherchen zum Tod des Terroristen in Verbindung mit einem Romantitel zu bringen – könnte signalisieren, der Autor wolle, dass sich Lesende nicht zwischen fiktionaler und faktualer Deutung dieser Rahmung entscheiden können oder müssen. Dass ihm in Trutz dies gelungen ist, ohne dass er dazu auch nur zum Modus der Autofiktion greifen muss, belegen die zahlreichen dahingehenden Bemerkungen, Spekulationen und Interviewfragen von selbst professionellen, literarisch gut versierten Leserinnen. Wenn auch das Anfangskapitel von Trutz in der Überschrift dieses Abschnitts und immer wieder im Text mit Begriffen wie »Rahmung«, »Rahmenhandlung«, »Erzählrahmen« u.Ä. umschrieben wird, bedarf diese aus theoretischer Sicht nicht ganz unproblematische Wortwahl einer kurzen terminologischen Klärung. Der Roman Trutz, dessen zwei Vergangenheitsebenen, d.h. die Eröffnungssequenz, die in den Jahren 2003–2007 spielt,66 und die Hauptdiegese, die die Zeit von ca. 1930 bis 2000 umfasst, von ein und demselben Erzähler präsentiert werden, entspricht nämlich vielen engeren Definitionen der Rahmenerzählung nicht. So insistiert zum Beispiel Andreas Jäggi darauf, »dass Rahmen- und Binnenerzählung von verschiedenen Erzählern erzählt werden müssen«,67 und Theoretiker wie William Nelles und Gérard Genette pflichten dieser Ansicht bei.68 Anderer Meinung zwar ist Gerrit Stratmann, der in dieser Forderung Jäggis »kein[en] notwendig[en] Zusammenhang zur Rahmung« entdecken kann,69 oder Tilman Köppe, der die Ansicht vertritt, dass schon »ein heterodiegetischer Erzähler eine ›Schachtelung‹ von Erzählebenen voraus[setzt]. Ein heterodiegetischer Erzähler ist Teil einer Rahmenerzählung, und was er erzählt, ist eine Binnenerzählung«.70 So gern sich die vorliegende Studie auf diese letzteren Positionen stützen würde, muss anerkannt werden, dass unter Theoretikern längst keine Einigkeit herrscht und dass besonders die sehr weite Auslegung Köppes den terminologischen Nutzen des Rahmenbegriffs zu gefährden scheint. Aus demselben Grund – d.h. der kontinuierlich gleichbleibenden Erzählinstanz – kann im Falle von Trutz noch weniger von einer seit Jahrhunderten verbreiteten und beliebten Variante der Rahmenform fachgerecht gesprochen werden, nämlich von der sogenannten Herausgeber- bzw. Manuskriptfiktion; bei diesem Kunstgriff präsentiert ein in der Rahmenhandlung erzählender, fiktiver Herausgeber einen vorgefundenen, von einer anderen fiktiven Person verfassten Text, der wiederum die Binnenerzählung bildet. Weniger zu beanstanden, insofern der Verfasser der »Rahmung« in Trutz sich auch als 66 67 68 69 70 Eine spätere Stelle im Roman macht deutlich, dass die Erzählergegenwart nicht (dem Ende von) diesem Zeitraum gleichzusetzen ist, sondern frühestens in der Zeit nach 2013 zu bestimmen ist; siehe S. 317 des Romans. Jäggi: Die Rahmenerzählung, S. 58. Nelles: Frameworks, S. 139–40; Genette: Die Erzählung, S. 147f. Gerrit Stratmann: Rahmenerzählungen der Moderne. Situation und Gestaltung einer Erzählform zwischen 1883 und 1928, Marburg: Tectum 2000. S. 26. Tilman Köppe: Erzählte Selbstrepräsentation im modernen Roman, Berlin: de Gruyter 2016. S. 80–81. 237 238 Richard Slipp: Gedächtnis und Erzählen Autor der Diegese identifiziert und eine »Schilderung der wahren oder erfundenen Umstände der Niederschrift«71 liefert, wäre wohl eine weniger enge Charakterisierung des Anfangskapitels nach Genette als »fiktionales Vorwort«.72 Doch geht es hier nicht darum, sich auf eine endgültige Zuordnung des Erzähleingangs in eine bestimmte Textsorte festzulegen, geschweige denn zwischen konkurrierenden Definitionsansätzen zu schlichten oder eigene Definitionen aufzustellen. Vielmehr wird es für hilfreich und interpretatorisch ergiebig gehalten, bei der Analyse von Trutz einige häufig identifizierte Merkmale und Funktionen von Rahmenerzählungen, Herausgeberfiktionen oder fiktionalen Vorworten im Blick zu behalten – ohne dass dabei an gut begründeten Einschränkungen der Anwendung solcher Kategorien auf diesen Fall vorbeigesehen wird. Anlass zur vorläufigen, bedingten Übernahme des Rahmen-Begriffs bietet Trutz in mehrfacher Hinsicht. Erstens ist die Rahmenform durch die bloße strukturelle Situierung der in der Vergangenheit spielenden Haupthandlung des Romans als Geschichte innerhalb einer Geschichte nahegelegt. Zweitens, insofern sich das Erzählte in der Diegese anscheinend (oder scheinbar?) auf ausführlichen Gesprächen mit dem Protagonisten auf der extradiegetischen Ebene beruht, kommt »eine mündliche Erzählsituation« – ein weiteres Abgrenzungsmerkmal Jäggis für Rahmenerzählungen73 – explizit zustande, und das kurz vor Ende der Eröffnungssequenz, d.h. just an der Grenze zwischen (postulierter) Rahmen- und Binnenerzählung: »›So‹, sagte Maykl Trutz, ›fangen wir an. Ich will nicht bis in die Antike zurückgehen, vorerst nicht. Ich fange mit meinem Vater an‹« (T 18). Folglich und drittens scheint aufgrund der Inszenierung der Binnenhandlung als keine vom Erzähler erfundene, sondern als eine gefundene Geschichte eine Anwendung – mit wesentlichen Vorbehalten – der Kategorie der Herausgeberfiktion angebracht. Besonders der letzte Punkt bedarf weiterer Erläuterung. Wenn auch der Erzähler in Trutz ja keinen fertigen Text, kein Manuskript vorfindet, stellt er ganz deutlich die langen Monologe Maykl Trutzʼ als die direkte Vorlage für die in der Folge eingebettete Erzählung hin. Insofern könnte man hier auch von einer von Jeffrey Williams postulierten Subkategorie von Erzählrahmen sprechen, nämlich vom »interview preface, which casts an oral rather than written source and therefore a dramatic scenario rather than an account of scholarly research.«74 Der Erzähler stellt das Szenarium wie folgt dar: »Ich besuchte Maykl Trutz acht Mal. Bei jedem Besuch sprach er vier Stunden, um dann plötzlich und ohne auf die Uhr zu schauen seinen Bericht abzubrechen, da er erschöpft sei und verbraucht, und mich rasch zu verabschieden« (T 18). Außerdem impliziert der Erzähler eine wortwörtliche Treue seiner Rekonstruktion von Trutzʼ Lebenserinnerungen, indem er – seinen Gesprächspartner anredend aber wohl gleichermaßen Leserinnen zugewandt – unübersehbar signalisiert, sich dabei keinesfalls nur auf sein eigenes Gedächtnis oder Notizen zu verlassen: 71 72 73 74 Genette: Paratexte, S. 166. Ebd., S. 190. Jäggi: Die Rahmenerzählung, S. 62. Williams: Theory and the Novel, S. 121 (Hervorhebung im Original). 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz »Er redete vier Stunden ohne Unterlass, ich hörte ihm zunehmend gebannt zu und schrieb in Stichpunkten mit, was er sagte. […] Ich fragte, ob ich ein kleines Aufzeichnungsgerät mitbringen dürfe, einen Rekorder, ich hätte nicht sein fabelhaftes Gedächtnis und beim Mitschreiben entgehe mir vieles.« (T 18) Die Zentralität der aus diesen Treffen resultierenden Aufnahmen für die geplante Veröffentlichung des Erzählers wird nochmals betont, als er an seinen Recherchen in den Archiven zu verzweifeln droht: »Die Suche war mühsam, zweimal unterbrach ich die Arbeit und war schon fast entschlossen, sie für immer einzustellen. Doch bei der Durchsicht meiner Gesprächsnotizen und einem wiederholten Anhören der Tonaufzeichnungen von Maykl Trutz wurde ich wieder verführt weiterzumachen.« (T 19) Indem der Erzähler in den oben zitierten und in weiteren Textstellen immer wieder das lückenlose Gedächtnis des älteren Herrn sowie die Kohärenz seiner Ausführungen unterstreicht, scheint er den Wahrheitsgehalt des Überlieferten versichern zu wollen; dies gipfelt im folgenden expliziten Plädoyer für die Glaubwürdigkeit seines Protagonisten: »Meine Zweifel gegenüber Trutz, die anfängliche Vermutung, es bei ihm mit einem jener Verwirrten zu tun zu haben, die eine fixe Idee beherrschte, die unter Zwangsvorstellungen litten und Verschwörungstheorien nachjagten, hatten sich bei meinem allerersten Besuch in Luft aufgelöst, dieser Mann hatte mein volles Vertrauen.« (Ebd.) Wie Andreas Jäggi konstatiert, werden »Unter den Wirkungsaspekten der Rahmenform […] am häufigsten die Beglaubigungsfunktion, die Möglichkeit der Lesersteuerung durch Kommentare der Rahmenerzähler und die distanzierende Wirkung der Rahmen genannt«.75 Dass der Prolog von Trutz eindeutig auf den erstgenannten Aspekt abzielt, konnte soeben gezeigt werden. Aber auch eine Distanzierungsgeste lässt sich in den Bemühungen des Erzählers erkennen, seine eigene Rolle herunterzuspielen und Maykl Trutz quasi als Urheber des Erzählten zu etablieren. Diese doppelte Funktion ist keineswegs widersprüchlich, sondern dient der anfänglichen Objektivitätsillusion und entspricht der von Hein (bzw. seinen Erzählern) konsequent eingenommenen Haltung als »Chronist ohne Botschaft«. Durch die wiederholte Betonung des Desinteresses des Erzählers an seinem späteren Erzählstoff, die Zufälligkeit des Treffens mit Maykl Trutz und somit die Inszenierung der Genese der Binnenhandlung als keine vom Erzähler erfundene, sondern als eine gefundene Geschichte, scheint dem Leser das angeboten zu werden, was Arata Takeda in Anlehnung an Lejeunes »pacte autobiographique« und »pacte romanesque« einen »editorischen Pakt« nennt; dieser »stützt sich auf die NichtIdentität zwischen Herausgeber und Urheber der Materialien«.76 Mit anderen Worten: Die Rahmenerzählung lässt sich – zumindest vorläufig – als Herausgeberfiktion charakterisieren. 75 76 Jäggi: Die Rahmenerzählung, S. 58. Arata Takeda: Die Erfindung des Anderen. Zur Genese des fiktionalen Herausgebers im Briefroman des 18. Jahrhunderts, Würzburg: Königshausen und Neumann 2008, S. 35–36. 239 240 Richard Slipp: Gedächtnis und Erzählen Und doch wird die »Binnenerzählung« von Trutz dann eben nicht von einem diegetischen Erzähler, nicht von dem sich erinnernden Protagonisten Maykl, sondern vom heterodiegetischen Erzähler erzählt, der zunehmend auktoriale Züge annimmt (was unten in seinen vielen Konsequenzen zu besprechen sein wird). Also wäre wohl »fiktionales Vorwort« eine passendere Bezeichnung für diese Rahmung, wobei, wie bereits angemerkt, ihre Fiktionalität in Rezensionen nicht selten übergangen wird – ja, einige übertragen sogar die vermeintliche Faktualität der Rahmung auf die Binnenhandlung. Wenn in der vorliegenden Studie zwar eine mangelnde Fiktionskompetenz (z.B. die Gleichsetzung von Erzähler und Autor) in manchen Kritikeraussagen moniert wird, ist die Tendenz, zumindest den Prolog von Trutz als faktuales Erzählen zu betrachten, vielleicht nicht allein etwa der Naivität zuzuschreiben, sondern ist ein Wirkungspotenzial, das der Rahmenform innewohnt. Um diese womöglich naheliegende, faktuale Lesart zu erklären, identifiziert Werner Wolf als eines der grundlegenden Merkmale vertrauter Rahmungen ihre Zuverlässigkeit: »Familiar framings are reliable. […] This also means, in the prototypical case of a preface (although in literature there are many deviations), that such framings are read, at least as a default option, as non-fictional. In a similar way we expect a picture frame […] to be solid and substantial and not a ›fictional‹, painted frame.«77 Diese Wirkung wird verstärkt durch die Möglichkeit, so eine vorangestellte Erzählsequenz quasi als Paratext und somit nicht als integralen Teil des »eigentlichen« Textes, d.h. des Romans, zu betrachten. Am Beispiel eines Romans aus dem neunzehnten Jahrhundert stellt Wolf ein weiteres Merkmal von »familiar framings« fest, nämlich dass sie auf einer diskreten, meistens höheren ontologischen Ebene situiert sind: »As a paratext, it is not only set off typographically from the main text but is also located on the superior level of the author who speaks of herself in the first person (as opposed to the following third-person narrative)«.78 Nun wird der Prolog in Trutz zwar nicht vom übrigen Text typographisch unterschieden, doch er setzt sich davon in anderen Hinsichten ab: Anders als die in drei Teile und insgesamt 31 Kapitel aufgeteilte Haupthandlung des Romans steht die Rahmung unter keiner Überschrift; außerdem folgt auf diese Sequenz eine leere Textseite und dann ein Zwischentitelblatt (»Erster Teil«). Die Rahmung von Trutz als Paratext lesen zu wollen, erfordert allerdings ein entschlossenes Hinwegsehen über die Tatsache, dass diesem fiktionalen Vorwort zwei Titelblätter und die Genrebezeichnung »Roman« vorausgehen. Indem textuelle Merkmale die Erwartung einer »klassischen« Rahmenerzählung oder gar einer Herausgeberfiktion erwecken, diese Erwartung jedoch letztlich nicht erfüllt wird, lässt sich eine weitere, im Falle von Trutz noch wesentlichere Funktion von solchen Rahmungsstrategien erkennen: nämlich um ein absichtliches Spiel mit der Grenze zwischen Fakt und Fiktion zu treiben. Dass z.B. Herausgeberfiktionen diese Wirkungsmöglichkeit schon immer besessen haben, stellen Lahn und Meister klar: »Erzähltexte dieses Typus wollen – meist zur Verwirrung der Leser – bewusst zwischen 77 78 Wolf: »Defamiliarized Initial Framings in Fiction«, S. 299 (Hervorhebung im Original). Ebd., S. 302. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz realer und fiktionaler Wirklichkeit oszillieren«,79 und Schaffrick und Willand sekundieren: »Insgesamt scheint sich die Produktion von Ambivalenzen durch den fiktiven Herausgeber als Teil des angesprochenen Verwirrspiels bis heute wenig verändert zu haben.«80 Nur macht Hein mit seinem Bruch der Konvention, d.h. seiner Nicht-Erfüllung der erwarteten Erzählsituation in der eingebetteten Erzählung, das reflexive Spiel womöglich noch transparenter. Dass so eine Variation dieser altbewährten Kunstgriffe, wie sie hier in Trutz vorliegt, im einundzwanzigsten Jahrhundert immer noch für Verwirrung zu sorgen vermag, mag Frank Zipfel zwar bezweifeln: »Seit den Anfängen des modernen Romans wird zuweilen im Paratext (oft im Vorwort) die Behauptung aufgestellt, daß die Erzählung nicht als ein vom Herausgeber verfaßter Text, sondern als Aufzeichnungen einer anderen Person zu verstehen ist. […] Allerdings ist fraglich, ob Leser sich durch solche Manöver (noch) täuschen lassen.«81 Doch widersprechen nicht nur die bereits zitierten Rezensentinnen, die einen wahren Hintergrund des Romans vermuten, sondern auch die Einstiegsbemerkung des ZDFKulturredakteurs Matthias Hügle in seinem Interview mit Christoph Hein auf der Leipziger Buchmesse, in der er die fiktive Rahmenhandlung mit der wirklichen Entstehungsgeschichte des Romans verwechselt zu haben scheint: »Sie kamen auf eine ganz interessante Weise zu diesem Thema, nämlich über Umwege. Erzählen Sie bitte kurz wie das kam«.82 Die Antwort des Autors darauf – oder vielmehr: seine höfliche Berichtigung, was die Prämisse der Fragestellung betrifft – ist ungewöhnlich deutlich und wirkt entgegen den meisten sonstigen, einen Wahrheitsanspruch untermauernden textexternen Äußerungen des Schriftstellers: »Das ist der Beginn des Romans, ich kann das erzählen, aber ich will darauf hinweisen: davor steht das Wort ›Roman‹, also auch der Beginn ist eine Erfindung, sage ich Ihnen gleich. Also es beginnt damit, dass der Erzähler einen Mann kennenlernt am Ende seines Lebens, der dann den Roman ihm erzählt […]«83 In einem weiteren Gespräch auf derselben Veranstaltung hat Hein diesen Einsatz eines Erzählrahmens im Dienste der Authentizitätsillusion nicht nur noch expliziter thematisiert, sondern ihn auch in den Kontext einer in seiner Prosa immer wiederkehrenden Erzählstrategie gesetzt: »[…] das ist ein Trick, um etwas zu erreichen, was im achtzehnten Jahrhundert noch ganz leicht möglich war. Im achtzehnten Jahrhundert konnte man, damit die Bücher 79 80 81 82 83 Lahn/Meister: Einführung in die Erzähltextanalyse, S. 100. Matthias Schaffrick und Marcus Willand (Hg.): Theorien und Praktiken der Autorschaft, Berlin: de Gruyter 2014, S. 74; Hervorhebung im Original. Zipfel: Fiktion, Fiktivität, Fiktionalität, S. 135. ZDF, »Das blaue Sofa«. Ebd. (Hervorhung R.S.); dass allerdings der Mann, den der Erzähler kennenlernt, also Maykl Trutz, den Roman eben nicht erzählt – zumindest nicht in Gänze –, wird in der Folge gezeigt. 241 242 Richard Slipp: Gedächtnis und Erzählen sich gut verkaufen, darüber schreiben ›Eine wahre Geschichte‹. Das geht heute nicht mehr, das glauben die Leute nicht mehr, also muss ich andere Tricks machen, indem ich zum Beispiel… in einem Roman84 habe ich eine Widmung reingeschrieben ›für‹ und dann den Namen der Hauptheldin hingeschrieben, sodass man glaubt, der ist der Hauptheldin gewidmet, [lachend:] alles erstunken und erlogen – nein, also habe ich eigentlich immer kleine Sachen, um den Punkt, dass es eine wahre Geschichte ist, auf eine andere Weise dem Leser zu vermitteln.«85 Übrigens scheint die Skepsis Heins, was die Wirkung einer nachdrücklichen Wahrheitsbeteuerung am Anfang eines Texts betrifft, unangebracht, denn nichts anderes als das hat er mit der umgekehrten Fiktionalitätsklausel in Glückskind mit Vater 86 eingesetzt, und diese wird keinesfalls von allen Lesenden als »Trick« durchschaut, wie im obigen Abschnitt dargelegt wurde. Man muss sich aber nicht auf Werk-Informationen epitextueller Art,87 also die öffentlichen Äußerungen des Autors zu seinem Text, verlassen, um den fiktionalen Charakter der Rahmenerzählung von Trutz zu belegen; auch eine nochmalige Betrachtung einiger Handlungselemente könnte sich als ergiebig erweisen. Zum Beispiel werden die erwähnten Recherchen des Erzählers zum Tod eines Terroristen mitnichten nur als einen beliebigen und an und für sich unwichtigen Anlass für den Besuch in der Bundesstiftung dargestellt, sondern es werden diesem Themenkomplex, d.h. dem tödlichen Schusswechsel zwischen Sicherheitsbeamten und dem gesuchten Terroristen sowie den anschließenden Untersuchungen und Prozessen, bereits ab dem dritten Absatz des Buches eineinhalb Textseiten (T 7–9) gewidmet – viel mehr als notwendig wäre, um etwa lediglich den Entstehungszeitpunkt des neuen Romans anzudeuten,88 oder bloß um ein »selbstreferentielle[s] Spiel«,89 bei dem sich der reale Autor Hein hinter dem Erzähler sichtbar mache, zu treiben. Vor allem geht es in den Passagen, die dieses andere Projekt des Erzählers betreffen, um die mangelnde Hilfsbereitschaft, die ihm bei seinem Gang durch die Archive begegnet: »Die ältere Archivarin, mit der ich sprach und der ich um den Bart ging, von dem tatsächlich etwas zu sehen war, meinte, ich würde ohne eine Genehmigung keine Einsicht bekommen, selbst wenn ich der Bundeskanzler wäre, auch bei dem hätte entweder der Bundestag oder der Generalbundesanwalt zuzustimmen.« (T 16) Der Erzähler beschreibt wenige Seiten darauf, wie sich die heimischen Behörden nun in der (für die BRD wohl weniger politisch brisanten) Causa Trutz/Gejm viel entgegenkommender zeigen: »Für diesen Fall gab es in den deutschen Archiven keinerlei Beschrän- 84 85 86 87 88 89 Gemeint ist Frau Paula Trousseau; siehe oben, Kapitel 7.4. Grandits: »Interview mit Christoph Hein«. »Der hier erzählten Geschichte liegen authentische Vorkommnisse zugrunde, die Personen der Handlung sind nicht frei erfunden«. Siehe oben, Kapitel 8.2.2. Vgl. Zipfel: »Fiktionssignale«, S. 103. Wie es Judith von Sternburg aus diesem intertextuellen Verweis schließt; Sternburg: »Ins Langzeitgedächtnis damit«. Bielefeld: »Lesestoff. Christoph Hein: Trutz«. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz kungen, ich bekam alles, um was ich bat« (T 19). Allerdings stößt der Erzähler jetzt beim erhofften Zugang zu Akten in den russischen Archiven auf Widerstände. So wird in der Rahmenhandlung nicht nur (zumindest gemutmaßte) übermäßige staatliche Gewalt, sondern auch und vor allem das nachfolgende offizielle Sich-nicht-erinnern-wollen bzw. Nicht-erinnert-werden-wollen thematisiert, das sich dann als ein zentrales Thema in der Haupthandlung von Trutz offenbart. Im dritten Teil des Romans, als Maykl Trutz als Forscher am Zentralarchiv in Potsdam arbeitet, bringt es sein Vorgesetzter Schmid auf den Punkt: »Der Sieger der Geschichte schreibt die Geschichte. Die Archive sollen nicht die Wahrheit liefern, sondern die dazu passende Wahrheit« (T 416). Indem der Erzähler gleich zum Auftakt an einen unrühmlichen Vorfall aus der jüngeren deutschen Geschichte, genauer: die womöglich außergerichtliche Exekution eines Flüchtigen und den daraus resultierenden Justiz- und Medienskandal, erinnert, bewirkt er, dass Leserinnen bei ihrer Lektüre über das Schicksal zweier Familien unter Diktaturen des zwanzigsten Jahrhunderts auch vom Bewusstsein des Weiterbestehens demokratischer und bürgerrechtlicher Defizite in der frisch vereinigten Bundesrepublik begleitet werden. Wie soeben angedeutet, wird auch, ohne dass falsche Gleichsetzungen betrieben werden, eine Parallele gezogen, was die staatliche Vergessenheits- und Verdrängungspolitik »damals« und heute, »dort« und hier anbelangt. Der systemübergreifende Charakter dieses Phänomens wird an einer späteren Stelle im Roman unterstrichen, als Maykl Trutz, der kurz davor die NS-Vergangenheit eines SED-Funktionärs entdeckt hat, sich mit Simon Wiesenthal in Wien trifft, um über eine anvisierte Mitarbeit an Wiesenthals Dokumentationszentrum zu sprechen; über seine derzeitigen Forschungsbedingungen bemerkt Maykl: »›In der DDR ist es schwierig. Es ist uns verboten…‹«, worauf der in Österreich tätige Wiesenthal ihm ins Wort fällt: »›Jaja, Freunde macht man sich damit nicht, nur treue Feinde. Hier ist das auch kein Zuckerschlecken‹« (T 401–402). In dieser thematischen Spiegelung auf den zwei Zeitebenen des Romans lassen sich also weitere Wirkungspotenziale von Rahmenerzählungen erkennen, nämlich, mit Andreas Jäggi gesprochen, »die Möglichkeit der Lesersteuerung durch Kommentare der Rahmenerzähler« wie auch »ästhetische Aspekte wie die der Kontrastwirkung oder das Spiel mit Parallelhandlungen«.90 In ähnlichem Sinne attestiert Werner Wolf der Rahmenform sowohl ästhetische Funktionen als auch die Fähigkeit, als Rezeptionsvorgabe oder Bezugsrahmen für die Deutung der eingebetteten Erzählung zu fungieren: »The framings of frame stories can also contribute to the aesthetic unity of the text as a whole (and thus fulfill yet another text-centred function) by other means, in particular by the establishment of thematic relationships between framing and framed texts, which is also a way, albeit often a covert one, of coding relevant frames of reference. […] In substance, such coherence can exist in relationships of causality or explanation (where the framing contains explanatory elements that are helpful for the reception of the framed text), but they can also operate along the more general lines of similarities and contrasts.«91 90 91 Jäggi: Die Rahmenerzählung, S. 58. Wolf: »Framing Borders in Frame Stories«, S. 197-8. 243 244 Richard Slipp: Gedächtnis und Erzählen Der Einsatz eines Erzählrahmens lässt also das Gegenwärtige der Vergangenheit deutlich hervortreten; gleichzeitig wird suggeriert, dass es im Roman – bei aller Konkretheit der geschichtlichen Bezüge – um tiefsitzende menschliche Denk- und Handlungsmuster im Verhältnis zwischen Gedächtnis und Macht, die nicht nur für eine einzelne historische Situation Gültigkeit besitzen. Während also der Erzähler einerseits die Beliebigkeit und die eigene Passivität in seiner Begegnung und Beschäftigung mit dem Fall Trutz behauptet, macht andererseits die Anspielung auf einen früheren Roman und dessen thematische Affinitäten mit dem aktuellen Roman die bewusste Erzählstrategie und somit die Konstruiertheit des Rahmens erkennbar. Übrigens kann, wie unten an passender Stelle ausführlicher erörtert wird, der intertextuelle Verweis an sich als Fiktionssignal gelten. Letztendlich und spätestens zu Anfang der Binnenerzählung wird aber klar, dass es sich bei der Rahmung von Trutz doch um keine Herausgeberfiktion handelt. Denn die Binnenerzählung wird eben nicht von einem diegetischen Erzähler, nicht von dem sich erinnernden Protagonisten Maykl, sondern vom heterodiegetischen Erzähler präsentiert, der zuweilen durchaus auktoriale Züge annimmt. Der Autor hätte seinem Erzähler ein Manuskript zukommen lassen können oder er hätte das Treffen mit Maykl Trutz als den Auslöser für dessen unvermittelte, d.h. von der Figur selbst erzählten Erinnerungen darstellen können – so wird der jeweilige Erinnerungs- und Erzählakt in den Rahmenerzählungen von Frau Paula Trousseau (2007) und Glückskind mit Vater (2016) inszeniert. Wenn mit Trutz so verfahren worden wäre, wäre aufgrund des angeblich fotografischen Gedächtnisses des Protagonisten mit einem monoperspektivischen autobiographischen Gedächtnisroman zu rechnen.92 Dass die durch die Vortäuschung einer Herausgeberfiktion erweckten Erwartungen jedoch nicht erfüllt werden, dass der Erzähler in der Binnenerzählung das Privileg des Selektierens, Ordnens, Kommentierens und nicht zuletzt des Fabulierens für sich selbst vorbehält, kommt mehr als einem Spiel mit den Grenzen zwischen Fakt und Fiktion gleich, sondern hat Konsequenzen für die erinnerungstheoretische Positionierung des Romans. 8.3.3. Fiktionalität (II): Auktoriales Erzählen, weitere Authentisierungsstrategien und deren gleichzeitigen Destabilisierung Trutz ist, wie oben bereits erwähnt, Heins bislang einziges Prosawerk von Buchlänge, das von einem heterodiegetischen persönlichen Erzähler93 erzählt wird. Mit anderen Worten: Der Erzähler erzählt in der dritten Person und ist nicht Teil der eigentlichen Diegese (d.h. der Haupthandlung um die Familien Gejm und Trutz); er ist aber zugleich keine entkörperlichte (und somit auch keine übermenschliche) Erzählinstanz, sondern er tritt als lebende Figur in der extradiegetischen Rahmung auf. Die Rahmung soll scheinbar erklären, wie der Erzähler zu dem Romanstoff kam, und soll obendrein, indem auf das eidetische Gedächtnis des sich erinnernden, quasi miterzählenden Maykl Trutz und die 92 93 Vgl. Neumann: Erinnerung – Identität – Narration, S. 210f. Stanzel: Theorie des Erzählens, S 24f. Zu der relativ gleichmäßigen Verteilung in Heins übrigen Werken zwischen homodiegetischen Ich-Erzählern und personalen Erzählsituationen (bzw. interner Fokalisierung), siehe oben. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz aufwendigen Archivbesuche des Erzählers verwiesen wird, für die Authentizität und Zuverlässigkeit der geschilderten Ereignisse bürgen. Die naheliegende Frage, die sich aus dieser fiktiven Genese des Romans ergibt, ist epistemischer Natur, nämlich: Was kann der Erzähler – den berichteten Umständen seines Bekanntwerdens mit dem Stoff entsprechend – tatsächlich wissen? Oder genauer: Welche Arten von Informationen kann er plausiblerweise bei seinen Gesprächen mit Trutz und seinen Archivrecherchen erfahren haben? Lesende dürften vielleicht erwarten, dass in der Binnenerzählung eine personale Erzählsituation vorherrschen, dass weitgehend aus der Perspektive des Protagonisten Maykl Trutz, der vorgeblichen Hauptquelle der Geschichte, erzählt werde, womöglich angereichert und untermauert mit historischen Hintergrunddetails, also mit den aus den Akten gewonnenen Erkenntnissen. Doch schon eine kursorische Betrachtung des Erzählten lässt durchblicken, dass – trotz des bescheidenen Auftretens des Erzählers, trotz allen Herunterspielens der eigenen Rolle auf die einer neutralen Vermittlungsinstanz – die dargebotenen Schilderungen »in vielerlei Hinsicht die Menge und die Art der für einen realen Erzähler verfügbaren Informationen übersteigen«, um mit Frank Zipfel zu sprechen.94 Außerdem, indem der Erzähler entgegen den in der Rahmung sich selbst gesetzten Beschränkungen einen allwissenden, auktorialen Gestus annimmt und immer wieder mit bewertenden und metanarrativen Kommentaren in den Vordergrund tritt, zeigt er sich als »Herr des Erzählens«,95 also als der, der allein für das Selektieren, Ordnen und Vervollständigen, wenn nicht gar für das Erfinden der Handlungselemente zuständig ist. Die Nichtdeckungsgleichheit des Erzählten mit den Erinnerungen des Protagonisten Maykl Trutz wird schon aufgrund der bloßen Tatsache etabliert, dass es über den ersten Teil des Romans hinaus, d.h. in etwa den ersten zweihundert Seiten, von Ereignissen handelt, die sich vor Maykls Geburt zutragen. Da der jüngere Trutz schon als Kleinkind seinen Vater zum letzten Mal sieht und im Alter von neun Jahren seine Mutter verliert, muss auch als unwahrscheinlich gelten, dass ihm zu irgendeinem Zeitpunkt von der Heimat Rainers, dessen Junggesellentagen oder dem Eheleben seiner Eltern aus erster Hand berichtet wurde. Doch auch von der Sicht der Hauptfigur dieses Abschnitts, Rainer Trutz, ist die Erzählperspektive weitgehend abgekoppelt. Die besonders in den ersten Kapiteln dominierende Null-Fokalisierung lässt sich bereits in den ersten zwei Absätzen der Binnenerzählung erkennen, in denen in eines Theodor Fontanes würdiger Vogelperspektive wie auch Satzlänge Ausgangsschauplatz und -situation des Protagonisten geschildert werden: »Rainer Trutz, Maykls Vater, hatte als Neunzehnjähriger sein Heimatdorf Busow verlassen, eine kleine Siedlung an der Bahnstrecke, die von Ducherow und Kamp über eine eingleisige, handbetriebene Drehbrücke nach Swinemünde führte, und war nach Berlin gegangen, da der väterliche Bauernhof seinem zwei Jahre älteren Bruder Frieder übereignet worden war und ihm der Sinn nicht danach stand, sein Leben mit Feldarbeit und Viehzucht zu verbringen. In seinem Dorf und in der weiteren 94 95 Zipfel: »Fiktionssignale«, S. 112. Vgl. Scheffel: Formen selbstreflexiven Erzählens, S. 60. 245 246 Richard Slipp: Gedächtnis und Erzählen Umgebung gab es keine Arbeit, die ihn lockte, zumal in der gesamten nördlichen Region die Arbeitslosigkeit höher war als im restlichen Deutschen Reich. Das einzig größere Bauprojekt in diesem deutschen Randbezirk war ein in Planung befindliches Brückenbauwerk, eine zweigleisige Hubbrücke nach dem Vorbild der Marstallbrücke, die aber anders als die Lübecker Konstruktion nicht dem Autoverkehr und den Fußgängern dienen, sondern ausschließlich Zügen der Reichsbahn vorbehalten sein sollte. Die neue Brücke war seit längerer Zeit ein beständiges Thema der regionalen Zeitung, die Bevölkerung beteiligte sich mit teilweise deftigen Leserbriefen lebhaft an der Diskussion, die einen erwarteten von der Brücke Arbeitsplätze und eine steigende Zahl von Feriengästen, andere befürchteten, das Bauwerk würde die Küstenlandschaft verschandeln und ein in schwindelerregender Höhe dahinrasender D-Zug sei eine fortwährende Gefahr für Leib und Leben nicht allein der Reisenden, sondern auch der unter der Bahnstrecke ansässigen Mitbürger.« (T 25) Wenn auch Befindlichkeiten des Protagonisten hier wiedergegeben werden, ist die Wahrnehmung ganz offensichtlich nicht an ihn gebunden, da auch von der geographischen und wirtschaftlichen Lage der Gegend sowie weit ausholend von einem umstrittenen Großbauprojekt erzählt wird, wobei der Vergleich mit einem Bau im über 200 Kilometer entfernten Lübeck gemacht wird. Dies alles übersteigt mit Sicherheit den Wissensstand eines Bauernjungen, der, wie man der Exposition entnehmen kann, in seinem bisherigen Leben selten über Anklam hinaus gekommen war. Dass der Erzähler außerdem Zugang zu den Gedanken und Gefühlen weiterer Figuren hat, bei der Innensicht jedoch nicht verharrt, zeigt sich an früher Stelle in der Beschreibung von Rainers Außenseitertum innerhalb der eigenen Familie: »Sich mit einem Buch zurückzuziehen galt als Faulenzerei und wurde gerüffelt, selbst die erforderliche Lektüre für die Schule und das Lernen der englischen und französischen Vokabeln wurde daheim ungern gesehen. Da sein älterer Bruder bereits nach der achten Klasse von der Schule abgegangen war und keinerlei Interesse an Büchern oder Musik aufbrachte, sondern von früh an sich auf dem Hof nützlich machte und dem Vater zur Seite stand, wirkten Rainers Vorlieben für Literatur und die Künste auf seine Eltern besonders befremdlich, ihr jüngerer Sohn war in ihren Augen lebensuntauglich. […] Den Eltern wie seinem Bruder schienen alle Berufswünsche von Rainer nur Tagträumereien eines Menschen zu sein, der der Arbeit aus dem Wege zu gehen suchte und seine Zeit stattdessen mit Büchern verbringen wollte. Rainer entschied sich daraufhin, das Elternhaus zu verlassen und für immer nach Berlin zu gehen.« (T 26–27, Hervorhebung R.S.) Die olympische Sicht wechselt sich dann zunehmend mit einer internen Fokalisierung durch die Figur Rainer, wie es am Ende des ersten Kapitels am Einsatz von erlebter Rede zu erkennen ist; kurz nach seiner Ankunft in Berlin fällt Rainer Trutz dem Schwindel eines unseriösen Arbeitgebers zum Opfer und überlegt sich dann seine Alternativen: »Nein, ins Dorf zurückzugehen, das kam für ihn nicht in Frage, und wenn Berlin und die deutschen Großstädte in verschmähten, würde er nach Amerika auswandern, nach New York, um dort sein Glück zu machen. Er könnte als Schiffsjunge oder Küchenkraft auf einem Überseedampfer anheuern, und in Amerika würde es ihm irgendwie gelin- 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz gen, unbemerkt von Bord zu kommen und, legal oder illegal, amerikanischen Boden zu betreten. […] So oder so, er würde es schaffen, er würde in einer großen deutschen Stadt oder im Ausland seinen Weg finden, und dann würden seine Eltern und der Bruder eine bunte Ansichtskarte von ihm bekommen, über die sie nur staunen konnten.« (T 34–35) Solche Passagen sind aber übersichtlich in Zahl und Umfang, und scheinen für krisenhafte Augenblicke im Leben des Protagonisten vorbehalten zu sein. Ein ähnlicher Übergang in erlebte Rede befindet sich etwas später, am Anfang des zweiten Teils des Romans, als der gerade im Moskauer Exil angekommene Rainer sich noch einmal über seine Zukunftsperspektiven – diesmal allerdings deutlich weniger optimistisch – sinniert: »Dann ging er, tief in Gedanken versunken, weiter bis zum Troitzki-Tor. Er würde an der Metro mitarbeiten, nach der Schicht nachmittags oder nachts todmüde ins Bett fallen, unfähig, noch irgendetwas zu tun, das Schreiben wäre für ihn vorbei. In sieben Monaten hätte er ein Kind, für das er sorgen müsste, mit dem zusammen er in ihrem einzigen Zimmer leben würde, so dass er keinen Moment Ruhe und Muße zum Nachdenken oder gar Schreiben finden würde.« (T 167) Auch ist Rainer nicht die einzige Figur, in deren Innenleben in Passagen interner Fokalisierung Einblick gewährt wird; im Laufe des Romans rücken streckenweise auch Gudrun, vor allem wenn von ihrem Arbeitsalltag in der Süßwarenfabrik berichtet wird (z.B. ab S. 170), Waldemar Gejm (S. 229f.) und Maykl Trutz (im gesamten dritten Teil, ab S. 379) stärker in den Fokus des Erzählens und fungieren gewissermaßen als Reflektorfiguren. So kann mit Genette von einer variablen internen Fokalisierung gesprochen werden.96 Die Innenweltdarstellung, d.h. »der Einblick in die Psyche Dritter«, charakterisiert Frank Zipfel als eine »Besonderheit des sogenannten Erzählerwissens«, die »sicherlich das am meisten besprochene Fiktionssignal« sei.97 Eine weitere Überschreitung des natürlich Wissbaren eines Erzählers – und ganz konkret des Erzählers von Trutz, der immer wieder zu verstehen gibt, seine Informationen ausschließlich aus seinen Sitzungen mit Maykl Trutz und Archivakten bezogen zu haben –, stellt die Wiedergabe von Gesprächen dar, an denen keine der Hauptprotagonisten teilgenommen haben können. Hierzu zählen nicht nur die Handlungen und vertraulichen Unterredungen von Randfiguren (wie etwa die zwischen dem Rektor der Moskauer Staatlichen Universität und dem Redakteur der Prawda auf S. 267–268), sondern auch die auf Russisch geführten und teils ausführlich mitgeteilten Dialoge,98 die zwar in Anwesenheit von Rainer und Gudrun stattfinden, von denen sie jedoch nichts oder nur wenig verstehen, da sie zum betreffenden Zeitpunkt in der Diegese der Sprache noch nicht mächtig sind (beispielsweise auf S. 184–196). Solche Informationen sind für Zipfel »an sich schon transgressiv gegenüber dem, was ein realer Erzähler wissen 96 97 98 Genette: Die Erzählung, S. 121. Zipfel: »Fiktionssignale«, S. 112. Im Roman werden diese Diskussionen in Deutsch wiedergegeben; es wird aber explizit darauf hingewiesen, dass Russisch gesprochen wird und dass das Ehepaar die Gespräche nicht verfolgen kann. 247 248 Richard Slipp: Gedächtnis und Erzählen kann« und zählen somit zu den Beispielen »›übernatürliche[r]‹ Wissensfülle«.99 Wie der Fiktionstheoretiker an anderer Stelle bemerkt, macht die Ausübung des auktorialen Privilegs der Allgegenwärtigkeit wie des der Innensicht die Fiktionalität der Erzählsituation erkennbar: »Ein Erzähler, der ›nur‹ durch Wände sehen kann, gehört genauso in den Bereich der Fiktion wie einer, der in die Köpfe schauen kann«.100 Eine narratoriale bzw. auktoriale Perspektive kommt auch zum Vorschein in gelegentlichen, kurzen Prolepsen, die deutlich erkennen lassen, dass nicht etwa »in actu«, d.h. im »Jetzt und Hier des erzählenden Geschehens«,101 sondern aus einer beträchtlichen zeitlichen Distanz rückblickend erzählt wird. Ein frühes Beispiel im Roman betrifft den Zufall, dem Rainer seine Freundschaft mit einer Frau verdankt, die ihm und seiner Frau in der Folge immer wieder behilflich sein sollte: »Rainer Trutz sagte noch Jahre später, in seinem Leben sei ihm nie etwas Besseres zugestoßen als der Zusammenstoß mit Lilijas Auto« (T 38). In vielen dieser Fälle geht der zeitliche Parameter der Perspektive einher mit dem ideologischen Parameter,102 denn nicht selten lässt der Erzähler zugleich durchblicken, dass er ja, anders als die Figuren, weiß, wie die Geschichte weiter verläuft, zum Beispiel später im Roman, als die nun vaterlose Familie Trutz in eine Arbeitssiedlung deportiert und dort in ihre neue Behausung eingewiesen wird: »Gudrun Trutz sollte bis zu ihrem Tod in diesem Zimmer wohnen« (T 346); oder wenn vom Unfalltod Waldemar Gejms in einem Arbeitslager, das nur Monate später aufgelöst wurde, bitter ironisch erzählt wird: »Tscheljabinsk erhielt nicht den Status Geschlossene Stadt und das Tscheljabinsker Besserungsarbeitslager ITL wurde im Oktober 1951 geschlossen, was Gejm jedoch nicht mehr erlebte« (T 364–365). So ein mit narratorialer zeitlicher Perspektive verbundener »freie[r] Umgang mit der Zeit«, diese Fähigkeit des Erzählers, »beliebig die Zeitebenen [zu] wechseln und auch spätere Entwicklungen vorweg[zu]nehmen« – um einige Formulierungen Wolf Schmids zu bedienen103 – dient gewiss nicht nur der Ironie und der Steuerung der Lesererwartungen, sondern auch dazu, den in der Geschichte vertieften Leser noch einmal daran zu erinnern, dass hier ein souveräner, ordnender Erzähler am Werk ist. Auch gibt es bewertende Erzählerkommentare, die zwar flüchtig aber dennoch unübersehbar sind und die besonders im Angesicht der sonstigen Seltenheit ihresgleichen bei Christoph Hein auffallen. Dies kann sich, wie in der Schilderung des Gemütszustands Rainers und Gudruns, nachdem sie sich für die Auswanderung aus Deutschland entscheiden, in Form eines subjektiven, vorausdeutenden Einschubs äußern: »Obgleich sie gut gegessen hatten, deckten sie Tisch mit den Köstlichkeiten, öffneten die Weinflasche und sprachen über eine Zukunft in einem fernen, ihnen unbekannten und sie beängstigenden Land. Sie erhofften viel, viel zu viel von dem neuen Land und waren zugleich niedergeschlagen, da sie nicht wussten, wie man sie in Russland 99 100 101 102 Zipfel: »Fiktionssignale«, S. 112. Zipfel: Fiktion, Fiktivität, Fiktionalität, S. 147. Stanzel: Theorie des Erzählens, S. 278. Schmid: Elemente der Narratologie, S. 123f.; »Ideologisch« ist hier nicht im engeren Sinne zu verstehen, sondern meint »das subjektive Verhältnis eines Beobachters zu einem Ereignis« im Allgemeinen (ebd.). 103 Schmid: Elemente der Narratologie, S. 137–138. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz aufnehmen würde, wovon sie leben sollten, da sie die Sprache nicht kannten, die kyrillische nicht lesen konnten und künftig vom Wohlwollen anderer Leute abhängig sein würden.« (T 123, Hervorhebung R.S.) Dieses »viel zu viel« ist zweifellos auf den Erzähler zurückzuführen, der, wie in den obigen Beispielen, sein Wissen um den Ausgang des Erzählten andeutet. Als weitere Erzählerwertung, wenn sie sich auch hinter Figurenrede versteckt, könnte die dramatische Ironie eingestuft werden, die an Stellen wie der folgenden erzeugt wird: »Mein Gott, Rainer, was sind wir für Glückspilze!« (T 143); dass mit ihrer Flucht aus Berlin nach Moskau das Paar nur vom Regen in die Traufe kommen wird, ahnen Leserinnen bereits an diesem Punkt in der Erzählung gewiss schon längst. Auf eine weitere Spur des bewertend kommentierenden Erzählers sei hier noch hingewiesen. Lilija Simonaitis erfährt – aus zweiter Hand – vom Verbot antifaschistischer Filme, Bühnenstücke und Bücher, das im Zuge des Nichtangriffspaktes auf einer Sitzung leitender sowjetischer Kulturfunktionäre bekanntgegeben wurde, und teilt dies Waldemar Gejm und Gudrun Trutz mit. Trotz ihrer eigenen Abwesenheit wird diese Sitzung über mehrere Seiten relativ detailliert geschildert, und vor allem die durchaus bildhafte und subjektiv anmutende Beschreibung der Gestik und Mimik des präsidierenden Volkskommissars ist wohl viel weniger Lilija als dem Erzähler zuzuschreiben: »Der alte, müde Mann habe sich schwerfällig von seinem Sitz erhoben, mit dem trägen Blinzeln eines Krokodils über die versammelte Runde geschaut und dann, bevor er in den Sitzungssaal der Volkskommissare zurückkehrte, mit heiserer Stimme gekrächzt: ›Sofort, Genossen, unverzüglich. Stalin erwartet es von uns, ich verlange es von euch.‹« (T 287) Ob nun auch die auffallende Ähnlichkeit zwischen der Wortwahl des Funktionärs (»Sofort, Genossen, unverzüglich«) bei der Verkündung des Inkrafttretens des Verbotes und den Worten Günter Schabowskis im November 1989 als einen weiteren gewollten Eingriff des Erzählers, etwa als Thematisierung aktuellerer historischer Ereignisse, anzusehen ist, sei dahingestellt. Dass viele Leserinnen eines gewissen Alters an dieser Stelle – noch dazu in einem Roman vom »DDR-Schriftsteller« Christoph Hein – unwillkürlich an diese legendär gewordene Pressekonferenz denken müssen, ist jedenfalls leicht vorstellbar. Eine letzte Bemerkung zur Offenlegung der Fiktionalität auf der »discours«-Ebene gilt solchen Erzählerkommentaren, die vordergründig beglaubigend wirken sollen, die aber bei näherer Betrachtung vielmehr die durchgehende Präsenz eines erfindenden Erzählers verraten. Repräsentativ hierfür sind Passagen, in denen etwa eine bestimmte Ereignisabfolge mit einem Verweis auf die sorgfältigen Archivrecherchen des Erzählers eingeleitet und belegt wird; zum Beispiel steht vor der Schilderung einer Episode in der fortschreitenden Zersetzung von Waldemar Gejms Forschungsgruppe folgende Anmerkung: »Zwei Monate später erlitt seine Arbeitsgruppe einen Verlust, der sie fast noch heftiger entsetzte. Nach den mittlerweile freigegebenen Akten der beiden Moskauer Ar- 249 250 Richard Slipp: Gedächtnis und Erzählen meearchive ergab meine nachträgliche Rekonstruktion der Hintergründe dieses Ereignisses den folgenden Ablauf: […].« (T 236) Während hier zwar eine Art Verbürgung der Wahrheit der Geschichte geleistet wird, gibt der Erzähler seinen Lesenden in zweierlei Hinsicht Anlass zur Skepsis: Erstens, indem er unter Gebrauch eines Possessivartikels stärker als sonst in den Vordergrund tritt und den Akt des ordnenden und ergänzenden Erzählens (»meine nachträgliche Rekonstruktion«) thematisiert, räumt der Erzähler einen gewissen Grad an Subjektivität oder gar Unzuverlässigkeit seiner Darstellung ein; zweitens stechen diese Erzählereinschübe aufgrund ihrer unregelmäßigen Verteilung hervor, d.h. es wird vor und nach ihnen über Dutzende von Seiten hinweg andere Handlungsereignisse ohne solch demonstratives Anführen von Quellen detailliert geschildert, was Leserinnen zu berechtigten Fragen nach dem vom Erzähler implizierten Wahrheitsstatus dieser letzteren, unbelegten Ereignisse einlädt. Da dies uns aber allmählich weg von der Betrachtung der Fiktionalität auf der Ebene des Erzählens in Richtung Fiktivität auf der Ebene des Erzählten hinführt, wird im nächsten Abschnitt auf Erzählerkommentare wie den oben zitierten noch einmal eingehender zurückzukommen sein. 8.3.4. Fiktivität der Ereignisse I: Der Erzähler als »Herr des Erzählten« Neben den vielen Indizien für einen scheinbar allwissenden, Einblick in die Gedankenwelt aller Figuren gewährenden Erzähler lassen sich in Trutz auch zahlreiche Fiktionssignale auf der Ebene der »histoire« aufzeigen, d.h. erzählte Ereignisse und andere Elemente, die nach textexternen sowie -internen Kriterien als widersprüchlich, unwahrscheinlich oder gar unmöglich eingestuft werden müssen und die somit ihre eigene Fiktivität bloßlegen. Schon bei den Hauptprotagonisten könnte argumentiert werden, dass ihr fiktiver Status nicht etwa im Dienste eines Realitätseffekts kaschiert, sondern noch stärker in den Vordergrund gerückt wird als es für andere Figuren im Roman und für Hein’sche Figuren im Allgemeinen der Fall ist. Anders als etwa Nikolai Bucharin oder Simon Wiesenthal, die im Roman auch auftreten, sind Rainer Trutz und Waldemar Gejm sogenannte »native objects«104 der Geschichte, d.h. sie existierten in der realen Welt nicht. Zudem könnte nach Zipfel schon der sprechende Name Trutz als ein Faktor gelten, der die Fiktivität der Figur transparent macht: »Schließlich können Elemente, die in überdurchschnittlichem Maß unwahrscheinlich sind, als Fiktionssignale fungieren. Hierzu gehören Namen, die ihre Träger in irgendeiner Weise charakterisieren, besonders wenn sie gehäuft auftreten«.105 Auf Heins wiederholten Einsatz einer leicht erkennbaren »Nomen-est-omen«-Technik (z.B. Willenbrock, Haber, Stolzenburg) wurde in der vorliegenden Studie bereits hingewiesen; zur Namensgebung beim vorerst neuesten Beispiel sowie in seinem Schaffen generell machte Hein in einem Gespräch folgende Bemerkung: 104 Zu diesem vom amerikanischen Soziologen Talcott Parsons geprägten Begriff siehe Zipfel: Fiktion, Fiktivität, Fiktionalität, S. 91–92. Für Figuren, die auf realen Personen beruhen, schlägt Parsons den Begriff »immigrant objects« vor. 105 Zipfel: »Fiktionssignale«, S. 109. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz »Von Trutz kann man viel ableiten: Trotz oder trutzig. Mir sind die Namen enorm wichtig. Vorher kann ich gar nicht schreiben. Wenn ich merke, der Name einer Figur stimmt nicht, habe ich schon ganze Kapitel wieder verworfen«.106 Auch ein intertextueller Verweis auf Grimmelshausens Trutz Simplex soll hier nicht unerwähnt bleiben.107 So dargestellt, könnte die Klassifizierung dieser Figuren als »fiktiv« als zu offensichtlich gelten, um der Rede wert zu sein. Da aber Trutz und Gejm als mal mehr, mal weniger prominente Akteure auf ihren jeweiligen Tätigkeitsfeldern dargestellt werden, und da diese Tätigkeitsfelder sich in reale, zeitlich und räumlich verortbare Zusammenhänge einordnen lassen – d.h. in die Literaturszene der Weimarer Republik bzw. die Moskauer Intelligenz und Kulturelite der 1930er Jahre – sticht ihre Fiktivität viel deutlicher hervor, als es bei Figuren, die etwa in einer unbenannten Süßwarenfabrik oder in einem Archiv in der Provinz arbeiten, der Fall ist. Dass um 1930 in Berlin kein Schriftsteller mit dem Namen »Trutz« lebte, steht fest. Aber auch, dass sich etwa eine reale Vorlage verschlüsselt hinter diesem fingierten Namen verstecken könnte, scheint ausgeschlossen zu sein; dagegen spricht, dass die schriftstellerische Karriere der Figur Trutz und die Beschreibung seiner zwei Romane auffallende Ähnlichkeiten mit dem literarischen Werdegang und den Werken solch prominenter Autoren wie Erich Kästner und Hans Fallada aufweisen. Aufgrund der Fiktivität der Hauptfigur müsste nach Rudolf Hallers Argumentation sogar das Berlin des Romans zu den »fiktiven Gegenständen« des Textes zugeordnet werden, denn, wie er in einem von Fiktionstheoretikern oft angeführten Zitat klarstellt: »Das London Sherlock Holmesʼ ist zwar dem realen London gleichartig, aber ist in der von Doyle erfundenen Geschichte, strenggenommen, nur so benannt wie das tatsächliche London. […] Ein London, in dem Mr. Holmes in der Baker Street wohnt, existiert nicht, sowenig wie das Haus, in dem er wohnt, die Straßen, in denen er geht usw.«108 Man muss allerdings nicht so weit gehen wie Haller, der alle Gegenstände quasi zu »native objects« oder rein fiktiven Gegenständen erklärt und eine Unterscheidung in fiktionalen Texten zwischen Erfundenem und Nicht-Erfundenem für nicht zulässig hält. Weniger absolutistisch und wohl hilfreicher ist Thomas Pavels Vorschlag einer dritten Kategorie, nämlich die der »surrogate objects«; für Zipfel besteht der Vorteil von Pavels Modifizierung der Parsons’schen Theorie darin, dass man nun »zwischen native objects (originär fiktiven Objekten), immigrant objects (aus der Realität übernommene Objekte) 106 Claudia von Duehren: »Christoph Hein: ›Mein Werk soll ja unterhalten‹«, in: Berliner Zeitung vom 08.05.2017; http://www.bz-berlin.de/kultur/literatur/christoph-hein-mein-werk-soll-ja-unte rhalten (06.09.2023). 107 Zu diesem Bezug äußerte sich der Autor selbst: »Ich wurde für Glückskind mit Vater ausgezeichnet – der Nachfolgeroman, der ein Jahr später erschien, heißt Trutz, und da ist schon im Titel ein Bezug auf Grimmelshausen. Ich habe auch im Text einen indirekten Bezug: Da Courasche als eine indirekte Antwort auf seinen alten Roman Simplicius Teutsch entstand, habe ich in Trutz einen Bezug auf einen anderen Roman genommen, aus ähnlichen Gründen, um auf etwas hinzuweisen. Da gab es also einen mehr als direkten Bezug zu Grimmelshausen«; Claudia Christophersen: »Christoph Hein: ›Ich bin kein Prediger‹«, ndr.de 09.11.2017; http://web.archive.org/web/20171112090734/ww w.ndr.de/kultur/Grimmelshausen-Preis-fuer-Christoph-Hein,journal1066.html (06.09.2023). 108 Rudolf Haller: Facta und Ficta. Studien zu ästhetischen Grundlagenfragen, Stuttgart: Philipp Reclam jun. 1986, S. 82; Hervorhebung im Original. 251 252 Richard Slipp: Gedächtnis und Erzählen und der Zwischenkategorie surrogate objects (aus der Realität entlehnte, jedoch signifikant abgewandelte Objekte) unterscheiden« könne.109 Im Falle des Schauplatzes des ersten Teiles von Trutz kann analog dem oben zitierten Beispiel Hallers – jedoch mit der Kategorie der »surrogate objects« im Blick – argumentiert werden: Ein Berlin, in dem ein Schriftsteller Rainer Trutz lebt oder gelebt hat, existiert nicht. Aber was noch stärker für eine Betrachtung des Handlungsortes als »surrogate object« spricht, ist, dass ein Berlin (und erst recht das Berlin der Weimarer Republik), in dem die Schriftsteller Erich Kästner und Hans Fallada nicht leben und wirken – denn dies ist die naheliegende Implikation, die sich aus der Existenz eines fiktiven Schriftstellers ergibt, dem zwei Bücher zugeschrieben werden, die sich Hauptwerken dieser realen Autoren weitestgehend ähneln – nicht mit unserem (literatur-)geschichtlichen Wissen über die reale Stadt vereinbar ist. Auf die angedeuteten Parallelen zwischen Rainer Trutzʼ zwei Romanen und Veröffentlichungen Kästners und Falladas wird unten in dem Abschnitt zu Intertextualität näher eingegangen. Vor allem aber lässt die Schilderung der Deportation Rainer Trutzʼ nach Workuta, die nach langen Fahrten in Zügen und auf der Ladefläche eines Lasters mit einem noch entbehrungsreicheren und gefährlicheren Marsch endlich zum Ziel führt, wo ein gewaltsamer Tod bereits am ersten Tag auf den Protagonisten wartet, lässt schließlich – trotz des durch eine Fülle an Details erzeugten Realitätseffekts – die eigene Fiktionalität durchscheinen. Diese Episode, die für viele (auch eher kritische) Rezensentinnen, zu den stärksten des Romans zählt,110 erstreckt sich über fünfzehn Seiten und zeichnet sich durch eine präzise Angabe der zeitlichen Verhältnisse und des jeweiligen Ortes aus, anfangend mit der Abfahrt aus Moskau: »Pünktlich um sechs Uhr verließ der Jaroslaw-Express den Kursker Bahnhof. In Jaroslaw endete die Reise, die Gefangenen wurden von den Soldaten in einen Warteraum geführt, sechs Stunden später bestiegen sie einen weiteren Zug und vierzehn Stunden später einen dritten, der sie bis zur Endstelle in Kotlas brachte.« (T 298) Weitere Absätze beginnen mit orientierenden Informationen wie »Nach zwei Wochen […]« (T 299), »Zehn Tage später erreichten sie die Pechora […]« (T 300), oder »In der sechsten Woche ihrer Reise erreichten sie die Usa […]« (T 304), bis die Schilderung der Reise wie folgt zu Ende geht: »Am siebzehnten November, dreiundfünfzig Tage nach der Abfahrt vom Kursker Bahnhof, erreichte die Gruppe ihren Bestimmungsort, das Lager Rudnik Eins« (T 305). Die Genauigkeit dieser Chronik wird durch einen erst gegen Ende des Berichts eingeschobenen Hinweis des Erzählers auf seine Archivrecherchen erklärt und bekräftigt: »Nach den Akten des seit 2004 zugänglichen Archivs ITL in Tscheljabinsk ergab sich nach der Fahrt über die Usa folgendes Bild für den weiteren Transport dieser Gruppe: […]« (T 304). 109 Zipfel: Fiktion, Fiktivität, Fiktionalität, S. 97–98; Hervorhebung im Original. 110 Vgl. z.B. Albrecht: »Die Geschichte eines Jahrhunderts«; Schimmang: »Als Stalin selbst zur Feder griff«; von Sternburg: »Ins Langzeitgedächtnis damit«; Carsten Otte: »Kein Klugscheißer, Mnemotechniker«. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz Relativiert wird allerdings der Authentizität beanspruchende Erzählstil durch den Einsatz über Strecken von szenischer Darstellung; Gespräche zwischen Rainer, seinen Mitdeportierten und ihren Wächtern werden hier in zitierter Rede unter fast komplettem Verzicht auf Erzählereinschübe wiedergegeben (T 301–303 und 308–313). Zipfel ordnet solche Szenen in den Bereich des »detailgenauen Erzählens« zu, das, wenngleich damit eine Wirklichkeitsillusion erzielt werden kann, dennoch als Fiktionssignal gilt, da »man sich nur die Mühe machen muss, die Illusion von Wirklichkeitsdarstellung zu erzeugen, wenn nicht tatsächlich Wirklichkeitsdarstellung betrieben wird«.111 Hinzu kommt natürlich die Tatsache, dass Informationen über die Inhalte, die Beteiligten, oder gar die Existenz dieser Gespräche auf dem Marsch nach Workuta sicherlich das Wissen des Erzählers und alles, was er aus den Beständen eines Archivs erfahren haben könnte, übersteigt. Die Fiktionalität der ganzen Erzählsequenz wird aber auf eine viel direktere Weise bloßgelegt. Nachdem Rainer Trutz kurz nach seiner Einweisung in eine Baracke von drei Zimmermitbewohnern ausgeraubt wird, erscheint ein vierter Mithäftling in seinem Zimmer, der auch den letzten Besitz des Neuankömmlings für sich haben will: »›Es ist mein allerletzter Tabak‹, wiederholte Rainer. Die Hand mit dem Tabakpäckchen führte er unwillkürlich hinter den Rücken. ›Sag ich doch, du führst dich schlecht ein, Sechsvierzehn-Achtundsiebzig. Fick deine Mutter‹, schrie der Mann, schwang seinen polierten Holzknüppel und traf ihn mitten auf die Stirn. Rainer fiel um. […] Vier Stunden später kamen die anderen Zimmerbewohner von ihrer Schicht und der Abendmahlzeit zurück und fanden den ihnen am Vormittag angekündigten neuen Mitbewohner reglos auf der Erde, rings um seinen blutenden Kopf lagen Kippen und Zigarettenasche. In dem ungeheizten Zimmer war der Körper von Rainer Trutz bereits erkaltet.« (T 312–313) Als der Leiter des Lagers beim Verfassen seines wöchentlichen Berichts auf den Fall eines Häftlings aufmerksam wird, der laut den Unterlagen an einem Arbeitsunfall gestorben sein soll, obwohl er noch nicht lange genug im Lager gewesen war, um gearbeitet zu haben, ist er besorgt, dass diese Unstimmigkeit – zusammen mit der bereits ungewöhnlich hohen Zahl an tödlichen Unfällen im Lager – unwillkommenes Aufsehen in der NKWDZentrale erregen könnte. Nach kurzer Überlegung fällt ihm eine Lösung ein: »Plötzlich hellte seine Miene auf und mit einem schmalen Lächeln griff er nach dem Lagerbuch in der Ablage, schlug die letzten Eintragungen auf, strich die Lagernummer Sechsvierzehn-Achtundsiebzig hinter dem Namen Rainer Trutz mehrmals durch und setzte ein Kreuz dahinter und ein Fragezeichen, womit dieser Deportierte in seinem Lager nie angekommen, sondern an einem unbekannten Tag auf dem Marsch durch die Tundra, beim Überqueren der Pechora oder bei der Fahrt auf der Usa verstorben war.« (T 315–316) 111 Zipfel: »Fiktionssignale«, S. 110–111. 253 254 Richard Slipp: Gedächtnis und Erzählen Zum guten Schluss tauscht der Offizier dann auch die Lagernummer von Rainer Trutz mit der eines lebenden Deportierten im Lagerbuch und in ihren Personaldokumenten aus, gießt sich einen Wodka ein und denkt zufrieden über sein Handeln nach. Somit werden auch die so detailreich beschriebenen letzten Wochen im Leben Rainer Trutzʼ, seine Odyssee nach Workuta und die Umstände seines Mordes, als eine Erfindung des Erzählers entlarvt. Denn, wie der Erzähler selber klarstellt, kann er – trotz eines erneuten Hinweises auf seine Recherchen im Archiv – schwerlich über dokumentarische Belege für diesen Hergang verfügen: »Nach der korrigierten Aktenlage war der Deportierte Trutz in Workuta nie angekommen, sondern irgendwo auf dem Weg dorthin verstorben. […] Im Lager Rudnik Eins jedenfalls gab es keine Spuren von ihm, lediglich in der Effektenkammer einen halb gefüllten Rucksack dieses Deportierten und einen Koffer, in dem sich drei russische und fünf deutsche Bücher befanden sowie ein paar handschriftliche Manuskriptseiten, die ich bei meinem Besuch des Archivs in Tscheljabinsk, fast auf den Tag genau dreiundsiebzig Jahre und fünf Monate nach der Ankunft des Deportierten Rainer Trutz in dem Lager, in die Hand bekam und als einen ersten Entwurf seines dritten Romans identifizieren konnte.« (T 316–317) Auch das geschilderte »Korrigieren« der Akte selbst kann natürlich nur vom Erzähler gemutmaßt werden und muss als von ihm erfunden gelten, und das, nicht nur weil zitierte und erlebte Rede eingesetzt werden, also Dialoge und Gedankenvorgänge, zu denen er ja keinen Zugang hat. Zudem wird durch die Thematisierung der Fälschung von Unterlagen – entgegen der wiederholten Inszenierung des Erzählten als eine teilweise Rekonstruktion auf der Basis von Ergebnissen der Archivbesuche des Erzählers – die Frage der generellen Zuverlässigkeit solcher Quellen in den Raum gestellt. Die Vermischung des Erzählten mit der Berufung auf Akten und historische Quellen scheint einerseits die Illusion der Authentizität zu verstärken, verdeutlicht aber gleichzeitig, wo die Einschränkungen eines solchen einseitig textuellen Zugangs zur Geschichte sind sowie wo der Erzähler hinzuerdichtet oder gänzlich fabuliert. Weitere logische Widersprüche auf der Ebene der Geschichte machen die Fiktivität der Ereignisse noch transparenter. Das Bekanntwerden des Bruchs des deutsch-sowjetischen-Nichtangriffspakts wird zunächst wie folgt geschildert: »Am 22. Juni 1941, am Geburtstag von Gejms Frau Alina, beendeten Sirenen, die in ganz Moskau aufheulten, dieses beschauliche Familienleben. In den frühen Morgenstunden dieses Tages hatten deutsche Truppen die Grenzen zur Sowjetunion von der Ostsee bis zum Schwarzen Meer überschritten und rückten, unterstützt von Tausenden von Flugzeugen und Panzern, am ersten Kriegstag meilenweit vor. Im Rundfunk hatte der stellvertretende Chef des Rates der Volkskommissare zu einem neuen Vaterländischen Krieg aufgerufen und die Bürger der Sowjetunion zu einem Schulterschluss mit der Regierung, mit der Partei und dem Genossen Stalin aufgefordert.« (T 331–332) Dem Erzählerbericht zufolge entscheidet sich dann Alina Gejm – noch am Morgen –, dass Mann und Kinder von der Arbeit und Schule zu Hause bleiben sollen, während sie 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz Lebensmittel besorgt, um der vermutlich anstehenden Rationierung zuvorzukommen. Es wird als eine Tatsache dargestellt, dass die Sirenen bzw. der daran anschließende Aufruf Molotows den Auslöser für die Handlungen der Familienmitglieder an diesem Vormittag bilden. Doch dann unterbricht der Erzähler seinen Bericht mit einigen geschichtswissenschaftlichen Bemerkungen, die seine bisherige Darstellung des ersten Kriegstages radikal in Zweifel ziehen: »An dieser Stelle verlangt die Verpflichtung des Chronisten einen Hinweis auf die Forschungen des Historikers Alexej Khairetdinov, der energisch bestreitet, dass am Tag des Kriegsbeginns in Moskau frühmorgens Sirenen aufheulten. Er verweist diese Berichte in das Reich der ebenso populären wie unhaltbaren Legenden, die seinerzeit in den sowjetischen Filmen verbreitet wurden wie in den Theaterstücken und vaterländischen Romanen. Seinen Forschungen zufolge haben die Moskauer bis zur Mittagsstunde nichts von dem Überfall der deutschen Wehrmacht gewusst. Erst um zwölf Uhr mittags hätte Molotow die entsprechende Erklärung der sowjetischen Regierung verlesen, die dann viermal vom Radiosprecher Lewitan wiederholt worden sei. […] Ein Sirenenalarm, der auf eine tatsächlich bevorstehende Gefährdung hinwies, ertönte in Moskau erst einen Monat nach jenem 22. Juni, als sich deutsche Flugzeuge zum ersten Mal der Hauptstadt näherten.« (T 332–333) Der sich über ca. eineinhalb Seiten erstreckende historiographische Exkurs geht dann in die Berücksichtigung anderer Quellen über, die »ungeachtet des Khairetdinov’schen Einwands ein Aufheulen der Sirenen« bezeugen (T 333). Der Erzähler schenkt allerdings diesen Gegenstimmen wenig Glauben, sondern paraphrasiert erneut und ausführlich die Argumente Khairetdinovs, die Erinnerungsverzerrungen und »false memories« thematisieren;112 die Zeugnisse seien nämlich »[…] ausnahmslos alle erst Monate oder Jahre später zu Papier gebracht worden, zu einer Zeit, da ihre Urheber durch die Darstellung dieses Ereignisses in den vaterländischen Filmen, Theaterstücken und Büchern derart indoktriniert waren, dass sie die eigenen Erinnerungen verfälschten und ihre Schilderung jenes Tages ungewollt und unwissentlich mit der offiziellen patriotischen Geschichtsschreibung in Übereinstimmung zu bringen suchten, was ein bekanntes psychopathologisches Phänomen und häufig zu verzeichnen sei, […] zumal wenn es ein Ereignis von nationaler oder religiöser Bedeutung betrifft.« (T 333) Dieser kommentierende Einschub ist bei mindestens einem Leser, nämlich einem Amazon-Rezensenten, auf Befremden und Unverständnis gestoßen: »[…] die Diskussion, ob zu Kriegsbeginn am Morgen in Moskau die Sirenen heulten, oder ob erst am Mittag Molotow eine Rundfunkansprache hielt, ist drollig. Aber warum tritt Hein gerade da ganz bewusst aus seiner ›Erzähler‹-haltung und trägt zu einer Historikerdiskussion bei, sonst nicht? Und warum so leichtfüßig?«113 112 113 Vgl. Schacter: Searching for Memory, S. 250–251. »Harold in Italy«: »Leider missglückt«. 255 256 Richard Slipp: Gedächtnis und Erzählen Abgesehen davon, dass hier nicht etwa Christoph Hein, sondern der Erzähler in den Vordergrund tritt, kann überzeugend gezeigt werden, dass diese geschichts- und erinnerungstheoretischen Überlegungen keinesfalls nur einer belanglosen Digression des Erzählers in historische Details gleichkommen. Vielmehr dienen sie erzählstrategischen Funktionen. Wenn der Erzähler divergierende Vergangenheitsversionen präsentiert, soll dies einerseits wohl seine ständige Bemühung um Objektivität (»die Verpflichtung des Chronisten«) andeuten; wenn er das Phänomen der Erinnerungsverfälschung anführt, dann anscheinend, um eine Version zuungunsten der anderen zu privilegieren. Andererseits aber werden Lesende zugleich indirekt aufgefordert, auch der gesamten Darstellung des Erzählers ein gewisses Maß an Skepsis entgegenzubringen, denn bei der hier vorgelegten Familienchronik handelt es sich ja um eine aus einer zeitlichen Distanz von fast acht Jahrzehnten rückblickend erzählten – oder besser: nacherzählten – Geschichte. Zudem stammt vermutlich vieles, was der Erzähler von Maykl Trutz erfahren haben soll, nicht aus erster Hand, sondern wurde auch diesem irgendwann von anderen Menschen mitgeteilt. Seine Überlegungen schließt der Erzähler mit den folgenden Worten ab: »Trotz meiner umfänglichen Recherchen konnte ich keinen eindeutigen und unstrittigen Nachweis des morgendlichen Sirenenalarms erbringen noch diesen zweifelsfrei ausschließen und muss mich daher mit einer Ungewissheit zufriedengeben und den Leser mit einem vorerst ungeklärten Fakt behelligen.« (T 333) Dass er nur an dieser einen Stelle eine Ungewissheit über die Faktizität seiner Schilderung einräumt, wo doch andernorts im Text auffallende Widersprüche bestehen und so viel von seinem übrigen Bericht allein aus epistemischen Gründen – man denke an die Gedankenwiedergabe der Figuren oder andere Indizien einer »übernatürlichen Wissensfülle«114 des Erzählers – als erfunden gelten muss, kann durchaus als ironischer Kommentar gelesen werden. Doch diese narratoriale Besprechung der »Sirenen-Frage« bleibt nicht über der Ebene der Geschichte schwebend, sondern scheint in der Folge in die weitere Darstellung der Ereignisse radikal einzugreifen. Im unmittelbaren Anschluss an das soeben zitierte Fazit des Erzählers setzt er seinen Bericht fort, und gleich wird der nächste Widerspruch erkennbar: »Gudrun Trutz erfuhr in der Schokoladenfabrik vom Kriegsbeginn. Sie hatte Maykl im betriebseigenen Kindergarten abgegeben, ihr war die nervöse Stimmung aufgefallen, aber erst in ihrer Brigade erfuhr sie den Grund für die Aufregung. In der Mittagstunde wurde in der Staatlichen Süßwarenfabrik Roter Oktober mit dem schrillen Signal für Notfälle überraschend die Arbeit unterbrochen, alle Arbeiterinnen lauschten schweigend der Rede des Außenministers Molotow, die über Lautsprecher in alle Produktionshallen übertragen wurde.« (T 333–334) Auch Gudruns Kolleginnen scheint die Nachricht unvorbereitet zu treffen, denn »[e]inige der Frauen weinten, andere schluchzten auf während der Rede […]« (T 334). Wenn aber 114 Vgl. Zipfel: »Fiktionssignale«, S. 112. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz die Sirenen »in ganz Moskau aufheulten«, wie es noch kaum zwei Seiten früher im Text heißt, ist es schwer vorstellbar, dass auch Gudrun – um von der gesamten Belegschaft ihrer Fabrik ganz zu schweigen – dies nicht mitbekommen, sondern erst um Mittag vom Ausbruch des Krieges erfahren haben soll. Man könnte den Eindruck gewinnen, der Erzähler habe sich durch den eigenen Exkurs über die historische Streitfrage mitten im Fluss des Erzählens umstimmen lassen. Klar ist jedenfalls, dass dieses repetitive Erzählen115 desselben Sachverhalts, d.h. des ersten Tages des Krieges zwischen Nazi-Deutschland und der Sowjetunion, zwei sich widersprechende und gegenseitig ausschließende Versionen erzeugt. So etwas kommt zwar nicht selten bei repetitivem Erzählen vor, wenn Ereignisse etwa aus verschiedenen Perspektiven geschildert werden, d.h. wenn das, was Vera und Ansgar Nünning »kontradiktorisches (bzw. inkompatibles) multiperspektivisches Erzählen« nennen, vorliegt;116 dass aber inkonsistente Versionen von ein und demselben heterodiegetischen Erzähler stammen und quasi als gleich glaubwürdige Tatsachenberichte präsentiert werden, zieht seine Zuverlässigkeit als Chronist in Zweifel. Man könnte hier in Anlehnung an Jean-Pierre Palmier eher von »unentscheidbar-repetitivem Erzählen« sprechen.117 Also entpuppen sich die geschichtlichen Anmerkungen des Erzählers, die dem Anschein nach seine gewissenhaften Bemühungen verbürgen sollen, einen möglichst historisch genauen Bericht abzulegen, letztlich als eine weitere Demonstration seiner Souveränität als »Herr des Erzählten«.118 Insofern der Erzähler in diesen Passagen nicht nur seinen eigenen Akt des Erzählens thematisiert, sondern dann auch mittels eines unlösbaren Widerspruchs auf der »histoire«-Ebene die Konstruiertheit des Erzählten explizit macht, verlässt er den Bereich der bloßen Metanarration und begibt sich in das der Metafiktion.119 Ja, man könnte präzisierend behaupten, der Erzähler wage sich auf ein postmodernes Terrain, das wohl nicht oft mit Christoph Heins Prosa in Verbindung gebracht wird, nämlich das der historiographischen Metafiktion. Denn, zumindest an solchen Stellen wie den oben behandelten, ist der Hein-Text jenen Romanen nicht unähnlich, die – mit Linda Hutcheon gesprochen – hochgradig selbstreflexiv sind und dennoch paradoxerweise auf historische Ereignisse und Persönlichkeiten als Grundlage Anspruch erheben.120 In weiteren Ausführungen zu historiographischer Metafiktion und zur Abgrenzung dieser Gattung von der des historischen Romans schreibt Hutcheon Folgendes: 115 116 117 Vgl. Genette: Die Erzählung, S. 74. Nünning/Nünning: »Multiperspektivität aus narratologischer Sicht«, S. 58. Vgl. Jean-Pierre Palmier: Gefühlte Geschichten. Unentscheidbares Erzählen und emotionales Erleben, Paderborn: Wilhelm Fink 2014, S. 270–274. 118 Vgl. Scheffel: Formen selbstreflexiven Erzählens, S. 60. 119 Vgl. Birgit Neumann und Ansgar Nünning: »Metanarration und Metafiction«, in: Handbook of Narratology, hg. von Peter Hühn, John Pier, Wolf Schmid, Jörg Schönert, Berlin: de Gruyter 2009. S. 204–211. 120 Hutcheon: A Poetics of Postmodernism, S. 5; bei Hutcheon lautet es im Original: »those wellknown and popular novels which are both intensively self-reflective and yet paradoxically lay claim to historical events and personages.« 257 258 Richard Slipp: Gedächtnis und Erzählen »First, historiographic metafiction plays upon the truth and lies of the historical record. […] certain known historical details are deliberately falsified in order to foreground the possible mnemonic failures of recorded history and the constant potential for both deliberate and inadvertent error. The second difference lies in the way in which postmodern fiction actually uses detail or historical data. Historical fiction (pace Lukács) usually incorporates and assimilates these data in order to lend a feeling of verifiability […] to the fictional world. Historiographic metafiction incorporates, but rarely assimilates such data. More often, the process of attempting to assimilate is what is foregrounded […]. As readers, we see both the collecting and the attempts to make narrative order. Historiographic metafiction acknowledges the paradox of the reality of the past but its textualized accessibility to us today.«121 Auf eine ähnliche Weise dienen in Trutz das Zitieren von Quellen und das Verweisen auf Archivbesuche sowohl in der »Workuta«- wie auch der »Sirenen«-Episode nur bedingt einer Beglaubigung des Erzählten. Vielmehr werden die Tücken und Unzulänglichkeiten thematisiert, die sich aus der Angewiesenheit eines die Vergangenheit rekonstruierenden Erzählers auf schriftlich Überliefertes ergeben. Auch der sogenannte »Gejm-Pflug« gehört in die Kategorie der Fiktionssignale. Bei dieser »grandiose[n] Erfindung« des deutschstämmigen Vorfahren Waldemar Gejms geht es um ein landwirtschaftliches Gerät, das »im ganzen russischen Reich für Aufsehen gesorgt und bei der Bekämpfung des Hungers einen Meilenstein dargestellt habe« (T 349). Gleichzeitig ist der Gejm-Pflug eines der Objekte im Roman, die offenbar so authentisch wirken, dass manche Leserinnen an deren Existenz in der außertextuellen Wirklichkeit zu glauben versucht sind, wie Sternburg attestiert: »Das ist eine Stelle, an der die Leserin nachschaut, ob es nicht doch einen Gejm-Pflug gibt (nein) […].«122 Aber signifikanter als die einfache Fiktivität des Pfluges ist die entscheidende Funktion, die er auf der Ebene der Geschichte einnimmt und die wiederum einer Bloßlegung der Fiktionalität dient. Als Gejm samt Familie nicht zuletzt wegen seiner deutschen Herkunft zur Zwangsarbeit nach Kasachstan deportiert werden soll, erfährt der zuständige Offizier von der Verwandtschaft des vor ihm stehenden Mannes mit dem Erfinder des von ihm geschätzten Pflugs: »Der Oberleutnant lächelte Gejm an und fragte: ›Der Gejm-Pflug? Sie sind der Gejm-Pflug? […] Tatsächlich? Nun, dieser Gejm-Plug hat meinem Vater vor drei Jahren die Kolchose gerettet‹« (T 349). Der schlagartig viel freundlicher gesinnte Oberleutnant lässt sich sodann überreden, Gejm als Dorfschullehrer nach Korkino zu schicken, wohin Gudrun und Maykl Trutz kurze Zeit davor deportiert worden waren. Dieser »Deus-ex-machina«-artige Eingriff in das Schicksal der Figuren mag zwar die Gutgläubigkeit der Leserinnen etwas strapazieren, für das Weltverständnis des Vernunftmenschen Waldemar Gejm aber stellt er eine fast inakzeptable Herausforderung dar. Als der junge Rem Gejm von dem eventuell lebensrettenden Zufall lernt und, in Anspielung auf eine früher (wohl halb scherzhaft) geäußerte Ansicht seines Vaters, die Menschen wären besser mit Zahlen als mit Namen gekennzeichnet, ihn darauf aufmerksam macht, dass der Offizier sonst nie von der berühmten Verwandtschaft der Familie erfahren hätte, reagiert der ältere Gejm folgendermaßen: 121 122 Ebd., S. 114. von Sternburg: »Ins Langzeitgedächtnis damit«. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz »Waldemar Gejm nickte und sagte: ›Ja, schön, wir werden Maykl wiedersehen. Ein glücklicher Zufall. Und du hast recht, mein vorwitziger Knabe, in dieser Welt geht nicht alles logisch zu. Gelegentlich regiert der blinde Zufall, und diesmal war es für uns ein glücklicher Zufall. Wer hätte ahnen können, dass ein Moskauer Milizionär sich Urlaub nimmt, um den Pflug von deinem Urururgroßvater nachzubauen. Das ist grotesk, Rem, das ist aberwitzig, aber offensichtlich war es so. Es gibt keine andere Erklärung. Schön für uns, aber nicht logisch. Logisch gesehen ist das widersinnig und bizarr. Nicht hinnehmbar. Aber wir sehen Maykl und seine Eltern wieder, da wollen wir der Welt diesen logischen Unsinn verzeihen.‹« (T 351) Da ein Urteil über die Wahrscheinlichkeit und Glaubwürdigkeit eines Ereignisses eine Frage der subjektiven Interpretation ist, und da der Zufall sehr wohl eine Rolle im realen Menschenleben spielt, wäre es übereilt, den Griff zum Gejm-Pflug als rettende Instanz alleine an sich zum Fiktionssignal zu erklären. In der Tat ist das schicksalsbestimmende Potenzial des Zufälligen ein immer wiederkehrendes Thema in Trutz und man könnte argumentieren, dass erst recht ein Roman, der alles Willkürliche ausschlösse und sämtliche Handlungen und Ereignisse nur nach Logik und Kausalität geschehen ließe, alles andere als realistisch wirken würde. Doch der Status dieser Sequenz um die Rolle des Gejm-Pflugs bei der verhinderten Workuta-Deportation wird durch eine spätere Szene im Roman vollständig verunklart. Als sich die alten Männer Maykl Trutz und Rem Gejm Ender der 1990er Jahre ein letztes Mal treffen und versuchen, die gegenseitigen Lücken in ihrer gemeinsamen Geschichte zu schließen, nennt Rem eine ungeklärte Frage, die ihn nicht mehr loslässt, nämlich, warum sein Vater geschont und nicht in das berüchtigte Arbeitslager geschickt wurde: »Alle Männer der Forschungsgruppe, die nicht in Moskau umgebracht wurden, kamen nach Workuta. Ausnahmslos alle oder fast ausnahmslos. Ihre Frauen und Kinder wurden nach Sibirien geschickt. Die Ausnahme war mein Vater. Er kam nicht nach Workuta, ihn schickten sie mit uns, mit seiner Familie nach Korkino. Warum? Schließlich leitete er das Institut, war der spiritus rector der Arbeitsgruppe. Wieso haben sie ausgerechnet ihn nicht in das gefürchtete Workuta geschickt? Ich weiß es bis heute nicht, ich kann es nur vermuten.« (T 466) In der früheren Szene aber weiß der junge Rem noch sehr gut, was seinen Vater vor der Arbeit im Lager (zeitweilig) bewahrte. Diesen offensichtlichen inhaltlichen Widerspruch, der kaum hundert Seiten nach der Szene mit dem Gejm-Pflug auftaucht, als eklatanten Fehler des (die eigene genaue Forschungsarbeit hervorhebenden) Autors aufzufassen, scheint wenig plausibel.123 Noch schwieriger wäre es anzunehmen, der von seinem Vater in der Kunst der Mnemotechnik geschulte Rem Gejm habe den so entscheidenden Vorfall in seiner Familiengeschichte so gründlich vergessen (und dass ihm sein Freund Maykl bei diesem Rätsel nicht weiterzuhelfen weiß). Die Inkonsistenz ergibt nur dann einen Sinn, wenn man die eine – wohl die frühere – Episode als pure Erfindung des Erzählers betrachtet, und die andere spätere Episode als eine Schilderung, die mehr oder 123 Wenn auch der Roman nicht frei von einigen kleineren Fehlern ist, wie die widersprüchlichen Zeitangaben auf S. 115 bzw. 132. 259 260 Richard Slipp: Gedächtnis und Erzählen weniger direkt auf den von Maykl Trutz mündlich mitgeteilten Erinnerungen beruht. So wird explizit suggeriert, dass – trotz der häufig beteuerten Authentizität – der Erzähler sich keineswegs durchgehend an seine Archivrecherchen und Gesprächsaufzeichnungen hält, sondern wesentliche Teile seiner Erzählung fabuliert. 8.3.5. Fiktivität der Ereignisse (II): Intertextualität Schon in der Rahmenerzählung von Trutz liegt eine Art Intertextualität vor, auf die oben eingegangen wurde: Recherchen, denen der Erzähler zum Zeitpunkt seines Kennenlernens mit Maykl Trutz nachgeht, können – trotz der Auslassung des Titels – als klare Anspielung auf den Stoff des früheren Hein-Romans In seiner frühen Kindheit ein Garten verstanden werden. Als mögliche Funktionen dieser Allusionen des Erzählers auf das andere Werk wurden einerseits die teilweise Verwischung der Grenzen zwischen Fiktion und Wahrheit, andererseits die Implizierung thematischer Affinitäten zwischen den zwei Texten und somit die Generierung von einigen universellen, oder zumindest systemübergreifenden Interpretationsimpulsen genannt, was das Themenkomplex kollektives Erinnern und Verdrängen betrifft. Auch an den im Folgenden diskutierten Beispielen von Intertextualität kann eine erzähltechnische und interpretatorische Funktionsvielfalt aufgezeigt werden. Da man es im ersten Teil des Romans beim Protagonisten Rainer Trutz mit einem Schriftsteller im Berlin der späten 1920er Jahre zu tun hat, wäre es nicht weiter verwunderlich, wenn Lesende auch in diesen Passagen Bezüge zum realen Literaturbetrieb und zu realen literarischen Persönlichkeiten und Werken konstatieren würden. Die Frage, ob nun alle solche Echos als vom Autor intendiert oder als eher rezeptionsseitig einzustufen sind – soweit es überhaupt möglich ist, diese Kategorien sauber auseinanderzuhalten –, ist wohl hinfällig: Da das liberale Nachtleben, die schillernde Kulturszene und die politischen Turbulenzen im Berlin der Weimarer Republik dank der vielfachen literarischen und filmischen Verarbeitung fast zu festgeprägten Bildern in der Vorstellung wohl der meisten Leserinnen geworden sind, kann von einer Einkalkulierung solcher bereits existierenden Assoziationen in die Darstellung der Stadt und der Epoche ausgegangen werden. Bis zu einem gewissen Grad dient die Aktivierung solcher Assoziationen gewiss der Erzählökonomie: Mit der Ausnahme von Rainers Dialogen mit seinem Verleger Verhelst und der Schilderung eines Tumults vor der wiedereröffneten Piscator-Bühne verzichtet der Erzähler bei seiner Konstruktion des künstlerischen Milieus der »gerühmten und aufregenden Weltstadt« (T 27) weitgehend auf detaillierte szenische Darstellung zugunsten summarischen Erzählens, das den Rezipientinnen zur Konkretisierung mit vorhandenem Wissen (bzw. populären Annahmen) über Berlin in der Weimarer Republik anregt. Allerdings ist ebendiese womöglich wenig originell anmutende Behandlung des Stoffes bei manchen Rezensentinnen auf Kritik gestoßen, wie etwa bei Jochen Schimmang, der nach einer Zusammenfassung einiger einschlägiger Handlungsepisoden befindet: »So sprunghaft, pulsierend und klischeebeladen geht es zumindest im ersten Teil des Romans auch weiter.«124 Eine weitere mögliche Funktion des Verweisens auf schon 124 Schimmang: »Als Stalin selbst zur Feder griff«. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz Dagewesenes, die in dieser Kritik übersehen wird, erhält erzähl- und erinnerungstheoretische Relevanz: Dadurch könnte der Erzähler nämlich andeuten wollen, dass die Vergangenheit nie erzählt bzw. erinnert werden kann, ohne dass Prätexte über diese Vergangenheit dabei automatisch mitgedacht werden. In der Folge beschränkt sich die Analyse allerdings auf solche Fälle von Intertextualität, die weniger dem weiten Verständnis des Begriffs als universales Merkmal von allen literarischen Texten oder gar allen sprachlichen Äußerungen entsprechen, als vielmehr seiner engeren Bedeutung als ein bewusstes, intendiertes und markiertes literarisches Verfahren, bei dem zwei oder mehr literarische Texten in Dialog miteinander gesetzt werden – als das also, was Manfred Pfister in Anlehnung an Genette die konkret »greifbare Anwesenheit eines Textes in einem anderen« nennt.125 Ferner wird aber argumentiert, dass die intertextuellen Beziehungen – vor allem in ihrer Deutlichkeit – auch der Offenlegung der Fiktionalität des Erzählten dienen. Die unmittelbarsten Bezüge zu anderen Texten, Figuren und realen Autoren sind weniger in der Schilderung von Rainer Trutzʼ Werdegang126 oder seines Umgangs mit Schriftstellerkollegen zu finden als in den Passagen, die seine eigenen zwei Romane nacherzählen und kommentieren. Die Beschreibung der Thematik von der ersten Buchveröffentlichung Trutzʼ lässt an solch klassische Großstadtromane der Weimarer Republik wie Alfred Döblins Berlin Alexanderplatz, Hans Falladas Kleiner Mann, was nun? oder Erich Kästners Fabian denken: »Seine Erlebnisse in dem Obdachlosenquartier im Gewerkschaftshaus und dem Asyl in der Krausnickstraße, die wenigen Tage, in denen er als Bettelbruder und Klinkenputzer für die Annoncen-Expedition Chipper unterwegs war, bildeten die Grundlagen für den Roman […]. Ohne die politischen Ereignisse auch nur zu erwähnen, gelang Rainer Trutz dennoch ein genaues und einfühlsames Buch über das Alltagsleben in Berlin, zehn Jahre nach dem Ende des Weltkriegs. Der Zeitgeist, die politischen Verunsicherungen, die Folgen des Wirtschaftschaos und der Reparationsforderungen der Siegermächte wurden in seinem Roman mit den genau skizzierten Auswirkungen auf den kleinen Mann deutlich, ohne dass er eine politische Botschaft vermitteln wollte, denn er hatte gar keine.« (T 49) Besonders die Parallelen mit Kästners Fabian sind auffallend und lassen den Leser mit einiger Zuversicht auf eine intendierte Intertextualität schließen. Grund zu dieser Annahme gibt schon die unüberhörbare Verwandtschaft zwischen den Namen der jeweiligen Hauptfiguren bzw. den Titeln der zwei Werke: Trutzʼ Titelheld heißt nämlich »Ferdinand«. Diese Namensähnlichkeit ist ein intertextueller Bezug für sich und kann zugleich auch als eine Markierung der Intertextualität betrachtet werden, folgt man Ulrich Broich: »Als erste von zahlreichen Möglichkeiten für eine Markierung, die ausschließlich im äußeren Kommunikationssystem erfolgt, soll die Wahl von Namen genannt wer125 126 Manfred Pfister: »Konzepte der Intertextualität«, in: Intertextualität. Formen, Funktionen, anglistische Fallstudien, hg. von Ulrich Broich und Manfred Pfister, Tübingen: Max Niemeyer 1985. S. 1–30; hier: S. 17. Wenn es auch vielleicht nicht ganz uninteressant ist, dass Rainer Trutzʼ Heimatdorf, Busow, ein Ortsteil von Ducherow, der Heimat Johannes Pinnebergs in Kleiner Mann, was nun, ist. 261 262 Richard Slipp: Gedächtnis und Erzählen den«.127 Fasst man die jeweilige Genese der zwei Buchtitel, d.h. des realen sowie des fiktiven Romans, ins Auge, zeigen sich weitere Parallelen: Im Falle von Kästners Roman lautete der vom Verlag aufgedrängte Zusatz zum einfachen Titel Fabian – nachdem mehrere vom Autor gewünschte Titel abgelehnt worden sein sollen128 – »Die Geschichte eines Moralisten«; auch der Roman von Trutz erhält von seinem Verleger eine Erweiterung und heißt dann: »Außer ordentlich und tadelig: Ferdinand« (T 55). Außerdem kommt zum Titel beider Werke schließlich die Genrebezeichnung »Ein Großstadtroman« hinzu (T 56). Auf der inhaltlichen Ebene der jeweiligen Texte geht es mit den Ähnlichkeiten weiter. Genau wie die Streifzüge des Kästner’schen Romanhelden mit seinem Freund Labude durch Berliner Nachtlokale einen beträchtlichen Fokus der Diegese des Romans bildet, schickt auch Rainer Trutz »seinen Helden Ferdinand auf eine beschwerliche, aber glückliche Odyssee durch das Berlin Ende der zwanziger Jahre, und die beiden Besuche Ferdinands im Romanischen Café, im Bassin für Nichtschwimmer wie auch im Schwimmerbassin waren die turbulenten Höhepunkte des Textes […].« (T 50) Andere Sätze über den Protagonisten Ferdinand bescheinigen ihm eine gewisse, an Fabian erinnernde Lebensuntüchtigkeit, und auch seine Behandlung (sowie die anderer Figuren) durch den Erzähler, d.h. vor allem der Verzicht auf Pathos, auf eine »parteipolitische Festlegung« und auf auktoriale Bewertung zugunsten der Beobachtung und Berichterstattung,129 der dem fiktiven Roman attestiert wird, ist ganz im Sinne der Ästhetik der Neuen Sachlichkeit und könnte genauso gut dem Wahlberliner Erich Kästner gelten: »Er selbst war so unbedarft wie sein Held, und ebendas machte den Reiz dieses kleinen Romans aus, da der Autor sich nie klüger gab als sein Ferdinand, sich nicht über ihn belustigte, sondern ihn einfach und kommentarlos durch die Stadt und auf den zumeist krummen Wegen begleitete. Die Figuren lebten, und die Sprache Berlins konnte er, da er seit seiner Ankunft in der Stadt die ihm fremden Laute ebenso erstaunt wie aufmerksam wahrgenommen hatte, genau und mit einem feinen Humor wiedergeben.« (T 49–50) 127 128 129 Ulrich Broich: »Formen der Markierung von Intertextualität«, in: Broich und Pfister (Hg.), Intertextualität, S. 31–47; hier: S. 41. Zur Geschichte der Titelgebung für Kästners Roman siehe das »Nachwort des Herausgebers« in Erich Kästner: Der Gang vor die Hunde, hg. von Sven Hanuschek, Zürich: Atrium 2013: »Die größten Probleme hatte der Lektor […] mit dem Titel. Alle Vorschläge seines Autors missfielen ihm – Kästner nannte ›Saustall‹, ›Saustall ohne Herkules‹, ›Jugend im Vacuum‹. […] Nach Beendigung seiner Lektüre schlug Lang [ein Verlagsmitarbeiter – R.S.] dann ›Fabian, die Geschichte eines Moralisten‹ vor. Weller [Kästners Lektor – R.S.] teilt Kästner diesen Vorschlag bereits handschriftlich im Postskriptum seines ersten Briefes vom 10. Juli 1931 mit, mit dem er auf das Manuskript reagiert. Der Autor selbst war mit keinem der Vorschläge so recht zufrieden« (S. 284–285). Zu Kriterien des neusachlichen Erzählens siehe Sabine Becker: »Neue Sachlichkeit im Roman«, in: dies. (Hg.), Neue Sachlichkeit im Roman. Neue Interpretationen zum Roman der Weimarer Republik, Stuttgart: Metzler 1995, S. 7–26. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz Wenig später heißt es, dass Rainer Trutz »die Touristen im großen Saal wie die Berühmtheiten im kleineren Schwimmerbassin ironisch und bissig, aber liebevoll zeichnete« (T 49–50), was sich fast als Variierung der immer wieder zitierten Charakterisierung der Figur Fabian durch Jörg Drews als »zarter Ironiker« liest.130 Auch Aspekte der Veröffentlichungsgeschichte und Rezeption des realen Romans widerspiegeln sich im Falle des fiktiven Romans. Wie Kästner mit Fabian, so handelt sich auch Trutz mit seinem Ferdinand dank der freizügigen Darstellung von Sexualität einige Schwierigkeiten mit den Sittenwächtern ein. Der Verleger Verhelst wittert diese Probleme schon bei seinem ersten Gespräch mit Trutz, als Ersterer das Manuskript des Schriftstellerneulings zur Veröffentlichung annimmt. Verhelst charakterisiert den Roman wie folgt: »Witzig, etwas frech, etwas sehr anzüglich, na ja, das könnte Ärger geben bei den feinen Magazinen für die gehobenen Damen« (T 54); und etwas später, als der Verleger seinen Autor zu der Titeländerung überreden will, bemerkt er: »Oder glauben Sie, die Damen aus den Literatursoireen werden Ihr Buch lesen? Nein, dazu ist zu viel Unanständiges drin, da werde ich froh sein können, wenn wir keinen Ärger mit der Zensur bekommen. Sehr keck, mein junger Freund« (T 55). Schließlich werden bei der Veröffentlichung des Romans in Fortsetzungen auf Drängen eines Zeitungsredakteurs dann tatsächlich Stellen gestrichen und der Abdruck vorzeitig zu Ende geführt: »Die Zeitung hatte Verhelst vier Tage vor Weihnachten frühmorgens aus dem Bett geklingelt, um ihm mitzuteilen, dass die Redaktion entschieden habe, den Abdruck umgehend abzubrechen, man werde bereits am nächsten Tag mit dem Abdruck eines geeigneteren Machwerks beginnen. Die Briefe empörter Leser hätten sich in einer der Chefredaktion nicht mehr zumutbaren Anzahl erhöht, Abonnenten hätten mit der Kündigung gedroht, wenn dieser obszöne und geradezu zotige Text auch noch an den Weihnachtsfeiertagen, an denen man mit der Familie zusammen die heiligen Tage verbringe und gewöhnlich auch gemeinsam die Tagesnachrichten lese, noch immer in ihrem Blatt zu finden sei.« (T 66) Auch an Kästners Fabian wurden mit der widerstrebenden Einwilligung des Autors Kürzungen erotischer Passagen vorgenommen, sodass der Roman erst 2013 in einer rekonstruierten, vollständigen Fassung unter dem angeblich ursprünglich vom Autor präferierten131 Titel Der Gang vor die Hunde erschien. In »Fabian und die Sittenrichter«, einem zunächst als Nachwort geplanten Beitrag in der Weltbühne, hat sich Erich Kästner bekanntermaßen mit antizipierten Reaktionen auf die Darstellung von Sexualität in seinem Roman auseinandergesetzt.132 Bei Rainer Trutzʼ zweitem Buch, Kleine Stadt, Sonntagmorgen, gelten die Anspielungen auf einen realen Text bei näherem Hinsehen offenbar dem 1931 erschienenen Roman 130 Jörg Drews: »Erich Kästner«, in: Kindler Klassiker. Deutsche Literatur, Zusammengestellt von Hermann Korte, Stuttgart: Metzler 2015, S. 343. Außerdem kommen die Attribute »bissig und schonungslos« fast immer in den Klappentexten neuerer Auflagen des Romans vor. 131 Vgl. das »Vorwort des Verfassers« (1950) in Erich Kästner: Fabian, Die Geschichte eines Moralisten, Frankfurt a.M.: Büchergilde Gutenberg 1966: »Der ursprüngliche Titel, den, samt einigen krassen kapiteln, der Erstverleger nicht zuließ, lautete ›der Gang vor die Hunde‹« (S. 5–6). 132 Erich Kästner: »Fabian und die Sittenrichter«, in: Die Weltbühne 43 (27. Oktober 1931), S. 642–643. 263 264 Richard Slipp: Gedächtnis und Erzählen Bauern, Bonzen und Bomben von Hans Fallada.133 Allerdings ist es bestreitbar, ob auf der inhaltlichen Ebene so schlüssige Parallelen sich ziehen lassen, wie es bei Ferdinand/Fabian der Fall ist. Zwar handelt es sich beim realen wie beim fiktiven Roman um die Milieustudie einer deutschen Kleinstadt, in der die politischen Verhältnisse in der Republik, z.B. Machtkämpfe zwischen Links und Rechts, thematisiert werden und ein Bürgermeister als eine der Hauptfiguren auftritt. Doch weist die Handlung von Kleine Stadt, Sonntagmorgen, die der Erzähler in größerem Detail als die von Ferdinand nacherzählt, wesentliche Unterschiede zum Prätext auf. Während Falladas Roman vor dem realen Hintergrund der Protestaktionen der schleswig-holsteinischen Landvolkbewegung den Boykott einer Stadt (dessen Bürgermeister Sozialdemokrat ist) durch die örtlichen Bauern behandelt, ist der Stoff von Trutzʼ Kriminalroman der (fiktive) »Mord an einem jüdischen Buchhalter während der Inflationsjahre, in den der Bürgermeister seines fiktiven Städtchens und zwei Handwerker verwickelt waren, die alle drei wenige Jahre später Mitglieder der nationalsozialistischen Partei wurden« (T 83–84). Man könnte aufgrund dieser Abweichungen mit einigem Recht zu dem Schluss kommen, eine Nebeneinanderstellung des Fallada-Textes und des fiktiven Romans sei weit hergeholt – allerdings nur bis der Erzähler eine Kritikerreaktion auf Trutzʼ Buch erwähnt und paraphrasiert, die der Wirklichkeit unverkennbar entlehnt ist. Bei Trutz heißt es nämlich: »In der zweiten Dezemberwoche erschien zu seiner großen Überraschung und Freude in seiner geliebten Weltbühne eine überaus positive Kritik, und besonders beglückte ihn, dass einer der von ihm am meisten geschätzten und bewunderten Mitarbeiter des Blattes seinen Roman rezensiert hatte. Es sei die beste Schilderung der deutschen Kleinstadt der letzten Jahrzehnte, schrieb er, trotz ihrer Kaltschnäuzigkeit stecke in dieser Wirklichkeitsschilderung echte Menschenliebe. Und im beschließenden Urteil hieß es, dieser deutsche, dieser urdeutsche Roman sei so beängstigend echt, dass es einen grausen würde.« (T 92) Zu Falladas Bauern, Bonzen und Bomben erschien in der Weltbühne vom 7. März 1931 eine Rezension unter dem Namen Ignaz Wrobel – einem bekannten Pseudonym Kurt Tucholskys –, in der der Roman auf ähnliche Weise gepriesen wird (die offensichtlichsten Parallelen zur fiktiven Rezension in Trutz sind hervorgehoben): »Falladas Buch ist die beste Schilderung der deutschen Kleinstadt, die mir in den letzten Jahren bekannt geworden ist. […] Die Technik ist simpel; es ist der brave, gute, alte Naturalismus, das Dichterische ist schwach, aber der Verfasser prätendiert auch gar nicht, ein großes Dichtwerk gegeben zu haben. Ein paar Stellen sind darin, an denen schlägt ein Herz. Nein, ein großes Kunstwerk ist das nicht. Aber es ist echt … es ist so unheimlich echt, dass es einem graut. […] Ich empfehle diesen Roman jedem, der über Deutschland Bescheid wissen will. […] Hier ist Deutschland – hier ist es.«134 133 134 Hans Fallada: Bauern, Bonzen und Bomben, Hamburg: Hoffmann und Campe 2006. Ignaz Wrobel (Kurt Tucholsky): »Bauern, Bonzen und Bomben«, in: Die Weltbühne 14 (07.03.1931), S. 496. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz In seiner Anlehnung an Tucholsky überschreitet der Erzähler in Trutz die Grenze von bloßer Anspielung – wie sie etwa bei der Wiedergabe des Inhalts der zwei Romane Rainer Trutzʼ vorliegt –, hin zur Paraphrase, wenn nicht gar zum direkten Zitat; neben der fast identischen Wortwahl an mindestens zwei Stellen fällt auch die Verwendung ähnlicher rhetorischer Stilmittel (z.B. der Repetitio: »Hier ist Deutschland – hier ist es« vs. »dieser deutsche, dieser urdeutsche Roman«) ins Auge. Tucholsky beendet seine Rezension von Bauern, Bonzen und Bomben mit einem Wort der Warnung an Fallada angesichts der politischen Schwierigkeiten, die auf ihn zukommen könnten: »Wenn sie dich kriegen, Hans Fallada, wenn sie dich kriegen: sieh dich vor, dass du nicht hangest!«. Es befindet sich in der fiktiven Rezension, oder besser: in ihrer Zusammenfassung durch den Erzähler von Trutz, kein direktes Pendant zu dieser düsteren Vorhersage. Doch das an dieser Stelle Weggelassene wird kurz darauf in Rainers und Gudruns erzählter Wirklichkeit beinahe vollgezogen: Es ist schließlich Rainers Veröffentlichung von Kleine Stadt, Sonntagmorgen, die die Verfolgung des Paares durch rechte Zeitungen und nationalsozialistische Schlägertrupps erst in Gang setzt. Der unabweisbare Vorlagecharakter von Tucholskys Rezension erfüllt hier dieselbe Funktion wie an früherer Stelle die Namensähnlichkeit zwischen »Ferdinand« und »Fabian«, nämlich, dass er nicht nur Bestandteil eines intertextuellen Bezugs (oder, im Fall der Tucholsky-Paraphrase, praktisch einen eigenständigen Bezug) bildet, sondern gleichzeitig als Markierung der Intertextualität dient, d.h. als ein Wink an Lesende, dass die Anspielungen auf Falladas Roman bewusst und intendiert sind. Allerdings könnte man einwenden, dass es sich hier um eine denkbar schwache Markierung handele, denn, während der »Grad der Bewußtheit des intertextuellen Bezugs beim Autor«135 kaum zur Debatte stehen kann, ist es alles anders als selbstverständlich, dass der durchschnittliche Rezipient von Heins Roman mit der inzwischen über neunzig Jahre alten Rezension Tucholskys vertraut ist und somit das Zitat als solches erkennen würde. Demnach hätte man es hier mit einem »esoterischen Prätext« zu tun, der »nur für eine coterie als intendiertes Leserpublikum unmittelbar kommunikativ relevant werden« könnte.136 Und doch spricht einiges dafür, die Markierung der Intertextualität hier als so deutlich einzustufen, wie es mit der Aufrechterhaltung der Fiktion verträglich wäre. Trutz soll schließlich der Autor von zwei Romanen sein, die zwei realen literarischen Texten so analog sind, dass sie die Existenz letzterer Texte in der fiktiven Welt auszuschließen scheinen; eine eindeutige Intertextualitätsmarkierung in Form einer namentlichen Erwähnung von Fallada oder Kästner – oder Tucholsky – würde diese Illusion zerstören. Außerdem könnte die Vorbereitung der Leserinnen durch frühere, stärker markierte intertextuelle Bezüge (z.B. auf In seiner frühen Kindheit ein Garten und Fabian) die Notwendigkeit einer erneuten, gleichermaßen deutlichen Markierung verringern; Broich stellt Folgendes fest: »Ein Autor kann darüber hinaus bestimmte intertextuelle Bezüge zunächst im werkimmanenten Kommunikationssystem markieren und dann, wenn das Intertextualitätsbewußtsein des Lesers geschärft ist, auf eine Markierung auf 135 136 Siehe Pfister: »Konzepte der Intertextualität«, S. 27; die Bewusstheit des interkulturellen Bezugs beim Autor wie beim Leser macht für Pfister das Kriterium der kommunikativen Relevanz aus – eins seiner sechs »Kriterien für die »Intensität intertextueller Verweise«. Ebd. 265 266 Richard Slipp: Gedächtnis und Erzählen dieser Ebene verzichten«.137 Und immerhin ist der intertextuelle Bezug auf Tucholsky insofern schon markiert, als der Erzähler die Urteile über Trutzʼ Roman nicht etwa als seine eigenen Ansichten ausgibt, sondern explizit einen Fremdtext – eine Rezension in einer Zeitschrift, die ja auch in der außertextuellen Welt existierte – zitiert. Selbst viele derjenigen Leserinnen, denen nicht bewusst ist, dass dieser fiktionalen Rezension eine reale Vorlage zugrunde liegt, werden bei der Erwähnung eines »geschätzten und bewunderten Mitarbeiter[s]« der Weltbühne vielleicht an Kurt Tucholsky denken müssen. Oben wurde bereits argumentiert, dass ein Weimarer Berlin, in dem Erich Kästner und Hans Fallada nicht existieren – oder zumindest durch einen fiktiven Protagonisten um zwei ihrer bekanntesten Werke gebracht werden138 –, eine signifikante Abweichung von dem realen geschichtlichen Berlin darstellt und somit als »surrogate object« und Fiktionssignal aufzufassen ist. Ferner kann man behaupten, dass der transparente Aufbau (zumindest von Teilen) der fiktiven Welt aus klar erkennbaren literarischen Vorgängern einen weiteren Hinweis auf die Artifizialität des Erzählten darstellt. In gleicher Weise argumentiert Monika Lindner: »Die Thematisierung der eigenen Intertextualität bringt es mit sich, daß der Text den Blick weglenkt vom Entwurf einer möglichen Lebensweltlichkeit und hin auf sich selbst als ein durch andere Texte konditioniertes Konstrukt«.139 Auch Frank Zipfel zählt Intertextualität zu den Elementen eines Textes, die dessen Fiktionalität erkennbar machen: »Auch in den Bereich des fiktionsverdächtig Unwahrscheinlichen gehören Geschichten, die offensichtlich nach ästhetischen Kriterien konstruiert sind. […] Ähnliches gilt für das Phänomen der strukturellen Intertextualität, da es recht unwahrscheinlich erscheint, dass reale Ereignisse genaue Parallelen zu literarischen Vorlagen ausbilden.«140 Es soll hier nicht über die Tatsache hinweggesehen werden, dass bei Zipfel ausdrücklich von struktureller Intertextualität die Rede ist; es ließe sich darüber streiten, inwieweit man es in Trutz mit dieser Form der Intertextualität zu tun hat, denn das jeweilige inhaltliche Muster der Prätexte wird in Heins Roman nicht in der Diegese, sondern quasi in Metadiegesen, d.h. auf der Ebene von Fiktionen innerhalb der Romanfiktion, widergespiegelt. Dennoch wird mit den intertextuellen Bezügen offensichtlich ein Spiel mit der Fiktionalität betrieben, insofern einerseits die Wiedererkennung der Verweise durch die 137 138 Broich: »Formen der Markierung«, S. 42. Die Vorstellung, dass besonders die Existenz von Trutzʼ erstem Roman die Existenz von Kästners Fabian in der erzählten Welt des Romans ausschließt, wird weiter durch eine Schilderung Hanuscheks bekräftigt, nach der der reale Autor Kästner besorgt gewesen sein soll, dass man ihm mit einem ähnlichen Stoff zuvorkommen könnte, und er deswegen seinen Roman schnell zu Ende schrieb; siehe Kästner: Der Gang vor die Hunde: »Eine Woche gab er sich für den restlichen Text, bedrängt von der Schreckensnachricht, dass Hermann Kesten und Ernst Glaeser ›genau denselben Roman‹ schrieben […]« (S. 283). 139 Monika Lindner: »Integrationsformen der Intertextualität«, in: Broich/Pfister, Intertextualität, S. 116–135; hier: S. 130. 140 Zipfel: »Fiktionssignale«, S. 109–110. 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz Lesenden vorausgesetzt wird, während andererseits von ihnen verlangt wird, im Interesse der Kohärenz der erzählten Welt für die Dauer der Lektüre die Nicht-Existenz der literarischen Vorlagen sich vorzustellen. 8.3.6. Poetologische Reflexionen im Text; Schlussbemerkungen Eigentlich ist bereits in den obigen Ausführungen zur Intertextualität, zur Metanarration sowie -fiktion und zu der Rahmenerzählung im Roman Trutz immer auch implizit von literarischer Selbstreflexivität die Rede gewesen. Wenn an dieser Stelle diesem übergreifenden Anliegen ein eigener, abschließender Abschnitt gewidmet wird, so geschieht dies mit einem nun engeren Blick auf das explizite Vorhandensein von »literaturnahen Motiven und Figuren«141 und auf die Thematisierung von Akten des Schreibens und Lesens literarischer Werke. Mit Michael Scheffel wird hier »reflektieren« (einschließlich entsprechender Nomen und Adjektive) sowohl im Sinne von »(wider)spiegeln« als auch »betrachten« verstanden.142 So könnten z.B. die Schriftstellerfigur Rainer Trutz und seine zwei Romane, die ja jeweils ein Buch im Buch sind, als Spiegelungen des Erzählers, des Erzählens und der Erzählung innerhalb des Erzählten angesehen werden; Erzählerkommentare über die Entstehung und Beschaffenheit dieser fiktiven Romane (und folglich über die eigene Tätigkeit) entsprechen der zweitgenannten Bedeutung von »Reflexion« – ebenso wie Überlegungen des Erzählers oder der Figuren über das Wesen, die Funktionsweisen und das Potenzial von Literatur dies tun. Verweilen wir noch etwas bei Rainer Trutzʼ literarischer Produktion und kehren zu einer Passage zurück, die im oberen Abschnitt im Kontext der intertextuellen Bezüge auf bekannte Prosawerke der Weimarer Republik bereits zitiert wurde; hier wird das Augenmerk nunmehr auf Parallelen in diesem Erzählerkommentar mit dem vom realen Autor proklamierten (und von Kritikerinnen immer wieder angeführten) dichterischen Selbstverständnis als »Chronist ohne Botschaft« bzw. »ohne Hass und Eifer« gerichtet. Der Erzähler charakterisiert den Erzählgestus in Trutzʼ erstem Roman wie folgt: »Ohne die politischen Ereignisse auch nur zu erwähnen, gelang Rainer Trutz dennoch ein genaues und einfühlsames Buch über das Alltagsleben in Berlin, zehn Jahre nach dem Ende des Weltkriegs. Der Zeitgeist, die politischen Verunsicherungen, die Folgen des Wirtschaftschaos und der Reparationsforderungen der Siegermächte wurden in seinem Roman mit den genau skizzierten Auswirkungen auf den kleinen Mann deutlich, ohne dass er eine politische Botschaft vermitteln wollte, denn er hatte gar keine. Er selbst war so unbedarft wie sein Held, und ebendas machte den Reiz dieses kleinen Romans aus, da der Autor sich nie klüger gab als sein Ferdinand, sich nicht über ihn belustigte, sondern ihn einfach und kommentarlos durch die Stadt und auf den zumeist krummen Wegen begleitete.« (T 49–50) 141 Vgl. Dorea Dauner: »Literarische Selbstreflexivität«, Diss., Universität Stuttgart 2009, S. 58 und 60; https://elib.uni-stuttgart.de/bitstream/11682/5349/1/DissDaunerPublish.pdf (06.09.2023). 142 Scheffel: Formen selbstreflexiven Erzählens, S. 47. 267 268 Richard Slipp: Gedächtnis und Erzählen Wenn zwar, wie oben gezeigt wurde, Trutz im Gegensatz zum übrigen Schaffen Heins143 nicht ganz frei von auktorialen Kommentaren ist und neben dem »kleinen Mann« auch die »großen« politischen Umstände deutlicher in den Fokus rücken, ist es leicht zu erkennen, dass auch Heins Erzähler mit seiner Hauptfigur Rainer Trutz so verfährt, wie es dieser seinerseits mit seinem Ferdinand tut. Es ist ein bestimmendes Merkmal Hein’scher Prosa, dass historische Ereignisse und ihre Konsequenzen zuallererst, wenn nicht gar ausschließlich, über das alltägliche Dasein des (vermeintlich) unpolitischen Individuums angedeutet und gedeutet werden.144 In diesem, mit Joachim Lehmann gesprochen, »Aufscheinen der ›großen‹ Geschichte in der ›kleinen‹«145 lässt Hein gewiss den andernorts vielfach belegten Einfluss Walter Benjamins erkennen,146 und besonders dessen Auffassung der Arbeit des dem historischen Materialismus verpflichteten Chronisten, der »die Ereignisse hererzählt, ohne große und kleine zu unterscheiden«.147 Auch Graham McKnight verweist auf Heins Benjamin-Rezeption, führt aber zugleich diese Erzählstrategie auf die DDR-Provenienz des Schriftstellers zurück: »Banale Gespräche zwischen fiktiven Charakteren aus dem Alltag erlaubten Autoren in der DDR, Weltpolitik widerzuspiegeln und zu zeigen, daß das Privatleben dieser Figuren eine mikrokosmische Repräsentation dessen ist, wie Geschichte Änderungen oder Stagnation in der Gesellschaft in einem Maße bewirkt, das in den Standardwerken der (politischen) Geschichte kaum Berücksichtigung findet.«148 Wider diese verlockend einfache Erklärung ließe allerdings sowohl die Beobachtung einwenden, dass Heins Texte auch drei Jahrzehnte nach der Wende weiterhin die »große Geschichte« den »kleinen« Geschichten unterordnen, als auch die intertextuelle Andeutung in der eingangs zitierten Stelle auf eine Affinität dieser Erzählhaltung mit der Ästhetik der Neuen Sachlichkeit. Während Rainer Trutzʼ zwei Romane einen Fall von »produktionsbezogener« Reflexivität darstellen, bietet der Text auch, indem Waldemar Gejms Lektüre literarischer Tex- Weitere Ausnahmen in dieser Hinsicht bilden vor allem die Erzählungen in Nachtfahrt und früher Morgen und Vor der Zeit; auch in neueren Romanen Heins, wie Verwirrnis und Guldenberg, treten auktoriale Kommentare häufiger auf. 144 Im vorliegenden Kapitel wurde bereits das Beispiel des Aufstands des 17. Juni erwähnt, der in mehreren Texten Heins nur indirekt vorkommt; auch emblematisch für diese Erzählhaltung ist am Schluss von Von allem Anfang an Tante Magdalenas Gleichsetzung des Krieges mit einer Ohrfeige, die sie von der Mutter ihres gefallenen Verlobten abbekommen hatte: »Wenn ich etwas vom Krieg höre und von heldenhaftem Kampf, tut mir noch heute die Backe weh« (VaAa 195–196). 145 Joachim Lehmann: »Christoph Hein – Chronist und historischer Materialist«, in: Arnold (Hg.) Text+Kritik 111, S. 44–56; hier: S. 48. 146 Zu Heins Benjamin-Rezeption, siehe: Fischer: Christoph Hein; Lehmann: »Christoph Hein – Chronist und ›historischer Materialist‹«; Zekert: Poetologie und Prophetie; Graham Jackman: »The Fear of Allegory. Benjaminian Elements in Christoph Hein’s the Distant Lover«, in: New German Critique 66 (1995), S. 164–192; ders.: »›Nur wo er spielt, ganz Mensch‹«; Kiewitz: Der stumme Schrei, S. 199f. 147 Walter Benjamin: »Über den Begriff der Geschichte«, in: ders., Sprache und Geschichte. Philosophische Essays, hg. von Rolf Tiedemann, Stuttgart: Reclam 1992, S. 141–154; hier: S. 142. 148 McKnight: »Geschichte und DDR-Literatur«, S. 203. 143 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz te thematisiert wird, ein Beispiel »rezeptionsbezogener« Reflexivität.149 Der Sprachwissenschaftler, der nach seiner Zwangsemeritierung einen Job in der Garderobe des Moskauer Maly-Theaters annimmt, entdeckt für sich nach früherer Skepsis die Welt der Literatur: »Seine wissenschaftliche Arbeit hatte er völlig eingestellt, er las stattdessen in seiner freien Zeit die großen russischen und französischen Romane, auch Lyrik aus beiden Ländern, worauf er in seiner Universitätszeit keine Minute verschwendet hatte, da ihm diese Künste damals banal und beliebig erschienen.« (T 326) Worin genau diese lange gehegte Skepsis bestanden hatte, verdeutlicht Gejm in einem Wortwechsel mit Kollegen am Beispiel von Gogols »Die Nase« und Tolstois Anna Karenina: »Wann, so hatte er einmal an seiner Universität einen Professor der Literaturwissenschaft gefragt, der sich in einer Kollegenrunde über Gogol und Tolstoi ausgelassen hatte, als seien diese Männer Aristoteles oder Newton vergleichbar, wann, bitte, hat dieser Kollegienassessor Kowaljov gelebt, in welcher russischen Stadt wurde seine Nase, eine lächerliche, gewöhnliche, russische Nase, Staatsrat? Und woher weiß das geschätzte Genie, was sich eine Anna Karenina und ein Herr Wronskij, die vermutlich nie lebten, was diese sich auf einem Bahnsteig in Moskau oder Sankt Petersburg ins Ohr flüsterten? Hatten diese Autoren, von denen gewiss keiner ein Meister der Mnemonik war, ein so außerordentliches Gedächtnis, dass sie deren Gespräche, die Hunderte von Seiten füllen, derart genau und Wort für Wort zitieren konnten?« (T 326–7) Was hier von Gejm beanstandet wird, ist nichts anderes als die »›übernatürliche‹ Wissensfülle«150 des (auktorialen) Erzählers, von der in dem vorliegenden Kapitel bereits die Rede war. Somit dient auch diese Passage dazu, den Leserinnen von Trutz die Fiktionalität der eigenen Erzählsituation zu signalisieren, denn der Erzähler leistet in diesem Roman genau dasselbe wie die Erzähler Gogols und Tolstois, nämlich »Gespräche, die Hunderte von Seiten füllen, […] genau und Wort für Wort« zu zitieren, was übrigens ironischerweise ja auch für die Gespräche und Gedanken Gejms, die an dieser Stelle wiedergegeben werden, gilt. Gleichzeitig aber treibt diese Darstellung von Gejms Literaturverständnis und vor allem von seiner Unfähigkeit, sich auf eine willentliche Aussetzung der Ungläubigkeit einzustellen, seine übrige Charakterisierung im Roman als reinen Vernunftmensch voran. Eine derartig satirische Überzeichnung der Haltung Gejms verhindert übrigens, dass Lesende andere seiner Positionen – zum Beispiel was die potentielle Unfehlbarkeit und Vollständigkeit des menschlichen Gedächtnisses angeht – etwa mit denen des Autors gleichsetzen. Man fühlt sich hier an zwei weitere lesende Figuren in Hein-Texten erinnert, wenn auch diese weniger eine Skepsis denn eine allzu große Gutgläubigkeit gegenüber fiktionalen Texten an den Tag legen. Gemeint sind Daniels Großmutter in Von allem Anfang an, für die ihre belletristischen Lektüren »nicht weniger wahr und verpflichtend als die Ermahnungen der Propheten aus der Heiligen Schrift« sind (VaAa 136), und 149 Vgl. Dauner: Literarische Selbstreflexivität, S. 95f. 150 Zipfel: »Fiktionssignale«, S. 112. 269 270 Richard Slipp: Gedächtnis und Erzählen Thomas in Horns Ende, der die versteckten erotischen Hefte seines Vaters als dessen eigene, wahre Memoiren, die darin enthaltenen fingierten Dokumente (Fotos, Theaterkarten usw.) als eine Sammlung von Andenken an eine frühere Liebesaffäre missdeutet (HE 148–156). Man kann diese liebevolle Verspottung der Naivität der Figuren gewiss als eine indirekte Warnung an die Leserinnen, auch den vorliegenden Text nicht einer allzu wortwörtlichen Lesart zu unterziehen151 ; dies scheint sogar expliziten Ausdruck in den Worten Tante Magdalenas in Von allem Anfang an zu finden, die ermahnt: »[…] in den Romanen werde das Leben im Allgemeinen viel zu dramatisch beschrieben, so dass sie für den eigenen Alltag wenig nutzen brächten und man sich hüten sollte, sie für bare Münze zu nehmen oder sich gar von ihnen leiten zu lassen« (VaAa 135).152 Nicht zuletzt aber machen solche Stellen den Lesenden ihre Rolle bei der Konstruktion von Bedeutung bewusst und fordern sie zum Nachdenken nicht nur über das Verhältnis zwischen Fiktion und Realität, sondern auch über den Akt des Lesens im Allgemeinen auf. Dass das Lesen und Deuten fiktiver Texte, ebenso wie das Erinnern an Vergangenes, immer nach gegenwärtigen Bedürfnissen erfolgt, zeigt sich am neuen Erfahren der Literatur durch den einst skeptischen Waldemar Gejm; konfrontiert mit Zufällen und Schicksalsschlägen, die nicht in sein wissenschaftliches Weltbild passen, entdeckt er ein unvermutetes Potenzial in den Texten: »[…] nun war ihm diese Lektüre ein willkommener Zeitvertreib, und zu seiner eigenen Überraschung war er von der Lebensgeschichte einer französischen Madame namens Bovary, die vermutlich ebenso realistisch war wie jene zum Staatsrat aufgestiegene russische Assessorennase, zu Tränen gerührt, was ihn überraschte, befremdete und verstörte.« (T 328) Insofern der lesende Gejm ungeachtet seines nach wie vor ausgeprägten Bewusstseins der Fiktivität der in Flauberts Roman erzählten Geschichte dennoch so stark vom Schicksal der Hauptfigur betroffen wird, werden mögliche, wichtige Interpretationshinweise an die realen Leser*innen von Trutz erkennbar. Diese betreffen vor allem einige Funktionen des ständigen Bekenntnisses des Textes zur Fiktionalität. Denn, wenn im Roman immer wieder signalisiert wird, dass das Erzählen fiktional und das Erzählte fiktiv ist, dann soll – analog der tief greifenden Wirkung der Lektüre auf Gejm – die Erzählung deswegen nicht als minder wertvoll gelten; im Gegenteil wird suggeriert, dass die fiktionale Literatur einiges in der (Re-)Konstruktion von Vergangenheit zu vollziehen vermag, was einer den verifizierbaren Tatsachen vorgeblich treuen Geschichtsschreibung versagt bleibt. Hutcheon charakterisiert, Frank Kermode paraphrasierend, diese Ur-Funktionen menschlichen Erzählens, wie folgt: 151 152 Christl Kiewitz hat gezeigt, wie dieses Beispiel aus Horns Ende zur Kritik an einer Leserhaltung dient, die – besonders »unter den Bedingungen der Zensur« – versucht, Texte auf der Suche nach »Aussagen über die Realität« zu dekodieren; Christl Kiewitz: Der stumme Schrei, S. 205. Eine vergleichbare, wenn viel zynischere Einstellung lässt sich bei Dr. Spodeck in Horns Ende feststellen: »Ich lese sie [Bücher] nicht mehr, dafür fehlt mir die Geduld. Ich bin es überdrüssig, erfundenen Figuren nachzugehen und den Gesprächen des Papiers zuzuhören, diesen angestrengten, künstlichen Gebärden vorgeblichen Lebens« (HE 9). 8. Ein Jahr, zwei »Jahrhundertromane«: Glückskind mit Vater und Trutz »[…] fictions are indeed man’s way of dealing with the discrete brute facts of chaotic reality. He constructs ordered worlds, mental structures, which humanize time by giving it the form of narrative plots. […] The novel is not a copy of the empirical world, nor does it stand in opposition to it. It is rather a continuation of that ordering, fictionmaking process that is part of our normal coming to terms with experience.«153 Die Leistung des weder an Aktenbefunde noch an die Monoperspektive eines Protagonisten gebundenen Erzählers von Trutz geht über eine bloße Trostfunktion hinaus und besteht vor allem in seiner Fähigkeit, mithilfe dichterischer Erfindung das Geschichtliche zu vervollständigen, zu erklären, Sinn zu stiften, sich mehrere parallele Realitätsversionen vorzustellen und das zuweilen Unfassbare in Worte zu fassen. 153 Hutcheon: Narcissistic Narrative, S. 88–89. 271
https://openalex.org/W2038737929	https://bmcneurosci.biomedcentral.com/counter/pdf/10.1186/1471-2202-14-S1-P360	English	null	Plasticity of network dynamics as observed experimentally requires heterogeneity of the network connectivity pattern	BMC neuroscience	2,013	cc-by	1,217	POSTER PRESENTATION POSTER PRESENTATION Huertas et al. BMC Neuroscience 2013, 14(Suppl 1):P360 http://www.biomedcentral.com/1471-2202/14/S1/P360 Open Access Open Access Plasticity of network dynamics as observed experimentally requires heterogeneity of the network connectivity pattern Marco A Huertas1, Marshall Hussain Shuler2, Harel Z Shouval1* From Twenty Second Annual Computational Neuroscience Meeting: CNS2013 Paris, France. 13-18 July 2013 slowly decays to base level at the time of reward, 2) a sustained decrease (SD) in firing rate until the time of reward and 3) a population firing rate that initially decays and later peaks (P) at the time of reward [1,2]. In a previous computational approach [3,4] we proposed a model that accounts for the mechanism responsible for the SI type of response that allowed the synaptic weights of the lateral connectivity between neurons in an excita- tory population to learn the correct values over several Recent in vivo experiments in rats [1,2] have shown that when visual stimulation is paired with a reward signal, neurons in the visual cortex (V1) can exhibit population responses that correlate well with the expected time of the reward. This indicates that V1 neurons have the ability to learn to report the expected times in the sec- onds range. Data from extracellular recordings suggest that there are at least three different types of responses: 1) a sustained increase (SI) in population firing rate that Figure 1 A. Responses of excitatory population in network model that can report the time of reward (cyan line): SI (black), P (red) and SD (green). The network is initially driven by 400 ms of simulated input from LGN corresponding to full-field stimulation (magenta lines) and background noise. B. Population firing rate. Figure 1 A. Responses of excitatory population in network model that can report the time of reward (cyan line): SI (black), P (red) and SD (green). The network is initially driven by 400 ms of simulated input from LGN corresponding to full-field stimulation (magenta lines) and background noise. B. Population firing rate. Correspondence: Harel.Shouval@uth.tmc.edu 1Department of Neurobiology and Anatomy, University of Texas Medical School, Houston TX, 77030, USA Full list of author information is available at the end of the article © 2013 Huertas 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. * Correspondence: Harel.Shouval@uth.tmc.edu 1Department of Neurobiology and Anatomy, University of Texas Medical School, Houston TX, 77030, USA Full list of author information is available at the end of the article © 2013 Huertas et al; licensee BioMed Central Ltd. Author details 1D f 1Department of Neurobiology and Anatomy, University of Texas Medical School, Houston TX, 77030, USA. 2Department of Neuroscience, The Johns Hopkins University, Baltimore MD, 21205, USA. Plasticity of network dynamics as observed experimentally requires heterogeneity of the network connectivity pattern Marco A Huertas1, Marshall Hussain Shuler2, Harel Z Shouval1* From Twenty Second Annual Computational Neuroscience Meeting: CNS*2013 Paris, France. 13-18 July 2013 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. © 2013 Huertas 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. Page 2 of 2 Page 2 of 2 Huertas et al. BMC Neuroscience 2013, 14(Suppl 1):P360 http://www.biomedcentral.com/1471-2202/14/S1/P360 Huertas et al. BMC Neuroscience 2013, 14(Suppl 1):P360 http://www.biomedcentral.com/1471-2202/14/S1/P360 Huertas et al. BMC Neuroscience 2013, 14(Suppl 1):P360 http://www.biomedcentral.com/1471-2202/14/S1/P360 trials such that the population firing-rate decayed slowly (due to reverberations) until the time of reward. Here we present an expanded network model that can account for the three types of responses observed in the in vivo experiments (Figure 1). The model consists of two populations of integrate-and-fire neurons, one exci- tatory and the other inhibitory. We use our previously proposed plasticity rule [4] to learn the excitatory effica- cies in the model while keeping the inhibitory connec- tions fixed. We also assume a sparse and heterogeneous connectivity pattern. Under these assumptions the three response types (SI, SD, P) emerge naturally from the learning rule applied to the synaptic weight. This model also exhibits heterogeneity within the different response types, as observed experimentally. The model also exhi- bits significant robustness to variations of static synaptic weights to and from the inhibitory population and to the presence of noise that generates realistic spike statistics. 1Department of Neurobiology and Anatomy, University of Texas Medical School, Houston TX, 77030, USA. 2Department of Neuroscience, The Johns Hopkins University, Baltimore MD, 21205, USA. References References 1. Shuler MG, Bear MF: Reward timing in the primary visual cortex. Science 2006, 311(5767):1606-9. 2. Chubykin AA, Roach EB, Bear MF, Shuler MG: A cholinergic mechanism for reward timing within primary visual cortex. Neuron 2013, 77(4):723-735. 3. Gavornik JP, Shouval H: A network of spiking neurons that can represent interval timing: mean field analysis. J Comput Neurosci 2011, 30(2):501-132. 4. Gavornik JP, Shuler MG, Loewenstein Y, Bear MF, Shouval HZ: Learning reward timing in cortex through reward dependent expression of synaptic plasticity. Proc Natl Acad Sci USA 2009, 106(16):6826-31. doi:10.1186/1471-2202-14-S1-P360 Cite this article as: Huertas et al.: Plasticity of network dynamics as observed experimentally requires heterogeneity of the network connectivity pattern. BMC Neuroscience 2013 14(Suppl 1):P360. 1. Shuler MG, Bear MF: Reward timing in the primary visual cortex. Science 2006, 311(5767):1606-9. 1. Shuler MG, Bear MF: Reward timing in the primary visual cortex. Science 2006, 311(5767):1606-9. 2. Chubykin AA, Roach EB, Bear MF, Shuler MG: A cholinergic mechanism for reward timing within primary visual cortex. Neuron 2013, 77(4):723-735. 3. Gavornik JP, Shouval H: A network of spiking neurons that can represent interval timing: mean field analysis. J Comput Neurosci 2011, 30(2):501-132. 4. Gavornik JP, Shuler MG, Loewenstein Y, Bear MF, Shouval HZ: Learning reward timing in cortex through reward dependent expression of synaptic plasticity. Proc Natl Acad Sci USA 2009, 106(16):6826-31. 4. Gavornik JP, Shuler MG, Loewenstein Y, Bear MF, Shouval HZ: Learning reward timing in cortex through reward dependent expression of synaptic plasticity. Proc Natl Acad Sci USA 2009, 106(16):6826-31. 4. Gavornik JP, Shuler MG, Loewenstein Y, Bear MF, Shouval HZ: Learning reward timing in cortex through reward dependent expression of synaptic plasticity. Proc Natl Acad Sci USA 2009, 106(16):6826-31. doi:10.1186/1471-2202-14-S1-P360 Cite this article as: Huertas et al.: Plasticity of network dynamics as observed experimentally requires heterogeneity of the network connectivity pattern. BMC Neuroscience 2013 14(Suppl 1):P360. doi:10.1186/1471-2202-14-S1-P360 Cite this article as: Huertas et al.: Plasticity of network dynamics as observed experimentally requires heterogeneity of the network connectivity pattern. BMC Neuroscience 2013 14(Suppl 1):P360. References 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: • Convenient online submission
https://openalex.org/W3036525653	https://europepmc.org/articles/pmc7299996?pdf=render	English	null	Simulating dissolved 90Sr concentrations within a small catchment in the Chernobyl Exclusion Zone using a parametric hydrochemical model	Scientific reports	2,020	cc-by	6,554	Simulating dissolved 90Sr concentrations within a small catchment in the Chernobyl Exclusion Zone using a parametric hydrochemical model PEN Yasunori Igarashi 1 ✉, Yuichi Onda 2, Jim Smith 3, Sergey Obrizan4, Serhii Kirieiev5, Volodymyr Demianovych5, Gennady Laptev6, Dmitri Bugai7, Hlib Lisovyi6, Alexei Konoplev1, Mark Zheleznyak1, Yoshifumi Wakiyama1 & Kenji Nanba1 Strontium-90 (90Sr) is the major long-lived radionuclide derived from the Chernobyl accident, and is still being detected in the heavily contaminated catchments of the Chernobyl Exclusion Zone. This study examines the long-term decrease in the dissolved-phase 90Sr concentration and the concentration– discharge (90Sr-Q) relationship in stream water since the accident. We show that the slow decline in 90Sr follows a double-exponential function, and that there is a clear relationship between 90Sr and Q. This study is the first to reveal that the log(90Sr)-log(Q) slope has been gradually decreasing since the accident. This trend persists after decay correction. Thus, it is not caused by the physical decay of 90Sr and environmental diffusion, but implies that the concentration formation processes in stream water have been changing over a long period. We propose a hydrochemical model to explain the time- dependency of the 90Sr-Q relationship. This paper presents a mathematical implementation of the new concept and describes the model assumptions. Our model accurately represents both the long-term 90Sr trend in stream water and the time-dependency of the 90Sr-Q relationship. Although this paper considers a small catchment in Chernobyl, the conceptual model is shown to be applicable to other accidental releases of radionuclides. It has been 34 years since the Chernobyl nuclear power plant (CNPP) accident in Ukraine (Fig. 1), when large amounts of radionuclides were released into the environment. At present, high levels of dissolved-phase strontium-90 (90Sr) are still being detected in the small catchment streams inside the Chernobyl Exclusion Zone (CEZ) (Fig. 2b). The main long-term source of exchangeable and available 90Sr in the CEZ soils is micron-sized “fuel particles” (FP) that are gradually dissolving. These are derived from the accidental release from CNPP Unit 4 on 26 April, 1986, and were formed by the mechanical destruction of nuclear fuel. The dissolution rate of FP in CEZ soils varies from several percent to several tens of percent of the activity inventory per year, depending on the degree of oxidation of the particle UO2 matrix and the ambient geochemical conditions (e.g. soil solution pH and oxygen availability)1–6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports OPEN OPEN Simulating dissolved 90Sr concentrations within a small catchment in the Chernobyl Exclusion Zone using a parametric hydrochemical model PEN The 90Sr from small streams in the CEZ is discharged into the main Pripyat river, which is a critical component of the Dnieper river–reservoir system, one of the largest surface water systems in Europe7. Through this river system, 90Sr is also transported to the Kiev metropolitan area in Ukraine. Therefore, predicting and evaluating the radionuclide flux within the water system, and using models to evaluate the poten- tial radiological consequences to the downstream population, remain important tasks. 1Institute of Environmental Radioactivity, Fukushima University, Fukushima, 960-1296, Japan. 2Center for Research in Isotopes and Environmental Dynamics, University of Tsukuba, Tsukuba, 305-8572, Japan. 3School of Earth & Environmental Sciences, University of Portsmouth, Portsmouth, PO1 3QL, UK. 4The Chornobyl Radiation and Ecological Biosphere Reserve, Chernobyl, 07270, Ukraine. 5Chernobyl Ecocentre, State Agency of Ukraine on Exclusion Zone Management, Chernobyl, 07261, Ukraine. 6Ukrainian Hydrometeorological Institute, Kiev, 03028, Ukraine. 7Institute of Geological Sciences, Kiev, 01601, Ukraine. ✉e-mail: y-igarashi@ipc.fukushima-u.ac.jp Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ Figure 1. Location of Sakhan river catchment at the Chernobyl Exclusion Zone in northern Ukraine with 90Sr inventory in 1997. Flow situation at the Sakhan river mouth in July 2017 (C) and February 2018 (D). igure 1. Location of Sakhan river catchment at the Chernobyl Exclusion Zone in northern Ukraine with 90Sr nventory in 1997. Flow situation at the Sakhan river mouth in July 2017 (C) and February 2018 (D). Figure 2. Time series of observed discharge (Q: mm/day) (A), dissolved-phase 90Sr concentration (Bq/m3) (B). igure 2. Time series of observed discharge (Q: mm/day) (A), dissolved-phase 90Sr concentration (Bq/m3) (B). The annual-scale dissolved 90Sr concentration in the CEZ rivers is known to exhibit a rapid decrease in recently deposited activity followed by slower transfers during subsequent years, and this can be adequately expressed by an exponential function8–10. At the same time, the 90Sr concentration varies widely over the course of a year. For instance, the 90Sr concentration increases with increasing water levels in the Pripyat river during the snowmelt season11,12. The short-term temporal variations in 90Sr concentration in the CEZ rivers have been expressed using two- and three-dimensional numerical simulations13–15. Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 Theory d Based on the clear relationship between 90Sr and Q from Chernobyl24–26, we assumed that the 90Sr-Q relationship in Chernobyl rivers could be described by the parametric C-Q model16–23. As the C-Q relationship, which is primarily determined by the solutes produced from mineral weathering/leaching, is governed by geochemical processes and climate, it is assumed to be in a steady state. It is also assumed that the radionuclide concentrations in the local streams are affected by weathering/leaching processes1–5 in the environment. For instance, the radi- oactive half-life of 90Sr is 28.79 years, which is too fast to assume a steady state in the C-Q relationship. Thus, the time series of observed dissolved 90Sr concentrations should be corrected by the long-term diminishing trend. The long-term diminishing trend of 90Sr concentrations (Cref: Bq/m3) in the stream is expressed as follows: α λ β λ = − + × . + − + × . C t k t k t ( ) exp[ ( ) /365 25] exp[ ( ) /365 25] (1) ref 1 2 (1) α λ β λ = − + × . + − + × . C t k t k t ( ) exp[ ( ) /365 25] exp[ ( ) /365 25] ref 1 2 where t (days) is the number of days after the Chernobyl accident (26/4/1986), λ (1/yr) is the physical decay rate (= 0.024) of 90Sr, and α, β (Bq/m3), k1, and k2 (1/yr) are empirically determined (radionuclide-specific) constants. An exponential model from previous studies8–10 was used to describe the decrease in 90Sr concentrations over decadal time-scales. The constants α, β, k1, and k2 were set so as to minimize the root mean square error (RMSE) between the annual averaged values and the predicted values using the ‘optim’ package in the R software27. The annual averaged values were used to avoid excessive fluctuations from non-averaged data in the model validation stage. Igarashi model. This is a new conceptual model that derives a C-Q relationship by developing the idea of a vertical distribution of the lateral flow of water across the vertical profile of soil water chemistry near the river19,22. The key assumption in our model is that the distribution of the lateral flow of soil22 and the vertical profile of radi- onuclides in the soil provides a stream discharge and determines the radionuclide concentrations in a stream. Simulating dissolved 90Sr concentrations within a small catchment in the Chernobyl Exclusion Zone using a parametric hydrochemical model PEN The differential equations governing the water and sedi- ment transport, and the calculation of radionuclide concentrations using the distribution coefficient (Kd) between sediment and water, have been used in short-term simulations.h The relationship between dissolved ions and/or other substances and the discharge rate (C-Q relationship) is a general phenomenon that has been widely observed for soluble ions and dissolved organic carbon in various river water systems worldwide16–23. The tight coupling between 90Sr and the stream discharge rate (Q) in CEZ rivers can be explained by the empirical and theoretical background and parametric modelling developed in previous hydrochemical studies24–26. Based on these studies, the variations in stream chemistry with respect to discharge Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ can be explained by the variations in water and solute fluxes with respect to depth for a representative soil pro- file19,22. Here, the source of 90Sr observed in the CEZ rivers is the oxidized/non-oxidized FP released by the acci- dent2–5. The 90Sr dissolved from the FP is thought to migrate into the soil before being transported by the water flux, finally reaching the river water. The difference between 90Sr in Chernobyl and the dissolved ions observed in other catchments is that the soil solute concentration profile (in this case, vertical 90Sr concentration profile in soil) changes over time. Thus, the 90Sr-Q relationship is assumed to change with time. Therefore, the goal of this study is to examine the long-term 90Sr concentration formation process by focusing on the 90Sr-Q relationship and to develop a conceptual model and mathematical implementation that explain the observed data in the field. The analysis was performed using long-term field measurement data collected biweekly from the Sakhan catchment in the CEZ (Fig. 1). The results of this study will not only inform better conceptual models for accident-derived radionuclide concentrations in rivers, but also provide new concepts regarding the dissolved ion concentration formation process in rivers. Theory d The radionuclide concentration profile in the soil is then represented by the following modified equation: c z t C dz t ( , ) exp (2) ref =       (2) where c(z, t) is the concentration as a function of depth z and time t since fallout on the surface (26/4/1986). Cref is the long-term diminishing trend of 90Sr concentration, and d is a model coefficient. The z-axis has negative values below the ground surface, and intersects the ground surface at z0. Thus, the 90Sr concentration in the stream is defined by an integral expression, which can be solved analytically to give: = +       C Q t C bt bt d bQ a ( , ) (3) d bt ref / (3) This type of power-law function also represents an empirical method of describing the log(C)-log(Q) rela- tionship. It has been clarified mathematically that the relationship between 90Sr and Q in a stream obeys a log-log relationship. In this study, the model parameters were calibrated against observed 90Sr concentration data. The objective function implemented in the Markov chain Monte Carlo modelling is the standard log-likelihood func- tion. Details can be found in the Supplementary Information. Results L t Long-term decline and seasonal fluctuation of 90Sr concentration in CEZ streams. The long- term trends of 90Sr and Q are shown in Fig. 2(a,b). Unfortunately, data for Q and the 90Sr concentration were not available for the four years following the accident. Thus, we focused on the long-term time series in 90Sr and Q. Firstly, we found that the long-term data indicate large seasonal variations in Q (Fig. 2a). High values of Q were observed from March–May each year, which is defined as the snowmelt season, with the highest Q recorded on April 13, 2013 (Q = 4.2 mm/day). After the snowmelt season, Q began to decrease and stayed relatively low dur- ing the summer. The lowest Q was recorded between August 4 and December 5, 2015 (Q = 0.004 mm/day). 90Sr concentrations exhibit a long-term decline following a double-exponential function. We applied the exponential function to the 90Sr concentration data measured from 1990–2016 (Fig. 2b). The fitted parameters of the exponen- tial model are α = 9357 (Bq/m3), β = 2500 (Bq/m3), k1 = 0.254 (1/yr), and k2 = 0.002 (1/yr). A large seasonality was also observed in the long-term decrease in 90Sr concentrations. High 90Sr values were observed during the snowmelt season, and 90Sr decreased during the summer. Here, the mean observed 90Sr concentrations in stream Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ Figure 3. Response of dissolved-phase 90Sr concentrations (A) and 90Sr/Cref (B) to discharges rate. (1), (2), and (3) for each panel indicate the period used for aggregation, 1991–2000, 2001–2010, and 2011–2016, respectively. Grey dots indicate the observed values. Black dots and bars represent averages and quantiles for observed 90Sr, divided into 10 classes by the x-axis. Dashed lines express the best-fitted power-law regression lines for each period. Figure 3. Response of dissolved-phase 90Sr concentrations (A) and 90Sr/Cref (B) to discharges rate. (1), (2), and (3) for each panel indicate the period used for aggregation, 1991–2000, 2001–2010, and 2011–2016, respectively. Grey dots indicate the observed values. Black dots and bars represent averages and quantiles for observed 90Sr, divided into 10 classes by the x-axis. Dashed lines express the best-fitted power-law regression lines for each period. water decreased from ~3340 (Bq/m3) in the period 1990–1995 to ~1450 (Bq/m3) from 2012–2016. Using the observed 90Sr concentrations and Q, the wash-off of 90Sr through the stream discharge (from 1993–2016) was estimated to be ~5.0 (kBq/m2). Results L t This is only 1% of the initial 90Sr inventory across the whole watershed. Response of 90Sr concentration to stream discharge rate. The relationship between 90Sr concen- tration and Q is shown in Fig. 3. A power-law function (y = px q) was fitted for each period (Fig. 3), and the parameter p was found to decrease according to the physical decay of 90Sr and diffusion in the environment; the power-law exponent q (slope of log(90Sr)-log(Q)) also decreased over time (Fig. 3, a1–a3). This study is the first to show the time-dependency of the 90Sr-Q relationship from long-term field observations in the Sakhan catch- ment. When the 90Sr concentration was corrected according to its long-term attenuation (90Sr/Cref), the constant parameter p was found to range from 1.41–1.73, but the power-law exponent q continued to decrease even after the attenuation correction (Fig. 3, b1–b3). This result means that the time-dependency of the 90Sr-Q relationship is not caused by the long-term attenuation of the 90Sr concentration, but by the changing concentration formation processes over a long period of time. Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ Interestingly, the normalized surface wash-off coefficient for dissolved-phase 90Sr (Nl(90Sr)) during summer rainfall events is almost twice that of the snowmelt season1. This is because 90Sr exchange between wash-off liquid and topsoil cannot occur when the topsoil layer is frozen29. However, as reported in previous studies14,25,26, high 90Sr concentrations appear in high-Q periods (i.e. snowmelt season, heavy rainfall). Here, the discharge process could be the key to understanding the formation of solutes in the river. As shown by Freed et al.25,26, there is a clear correspondence between the stream flow and the groundwater level, and in the Chernobyl area, which is characterized by small differences in elevation within the basin, small increases in the ground- water level would cause large increases in the area of saturated soils. A relatively large contribution from the near-surface layer could then recharge the discharge level, leading to high dissolved-phase 90Sr concentrations in the snowmelt season. In addition, the effect of Nl(90Sr) on the 90Sr concentration in the river could be limited, because this region is too flat for the wash-off liquid to reach the river30. Frequent sampling could reveal a link between heavy rainfall events and 90Sr wash-off during the summer, therefore, requires further study.i yf g q y Another key finding of this study besides the clear 90Sr-Q relationship is the time-dependency of the 90Sr-Q relationship from long-term field observations (Fig. 3a). It is also worth noting that the time-dependency of the 90Sr-Q relationship is preserved after the attenuation correction of 90Sr (Fig. 3b). Clearly, the log(90Sr/Cref)-log(Q) slope during the first 10-year period (1991–2000) is greater than that in the last six years (2011–2016). If the 90Sr concentration in the stream is determined by the interaction between water and solutes in the soil, the time-dependency of the 90Sr-Q relationship is presumably the result of a temporal change in the solute concen- tration in the soil19,22. This is because the water flow pathways in the soil do not change over timescales of a few decades. The solute concentration in the soil, which is the source of 90Sr in the stream water, is thought to have changed over time just after the accident. In fact, the vertical profile of the 90Sr concentration in the soil changes with time. This is the result of leaching from the initially deposited FP and subsequent downward movement in the soil1,31. Various previous studies have discussed the C-Q relationship. www.nature.com/scientificreports/ initial sharp drop in concentration is represented by a parameter value of 0.09 (1/yr)9. The long-term attenuation component calculated from a study of 90Sr in five Belarusian rivers was also estimated to be 0.09 (1/yr)10. From our results, the long-term reduction in 90Sr concentrations in the Sakhan catchment (k1 = 0.27 (1/yr)) is three times higher than the rates reported for other rivers only 90 km away. Other rivers in Belarus are also located in the Belarusian Chernobyl area, and it is unlikely that k has been affected by climatological differences. This finding may be attributed to the fact that the main source of 90Sr in the Sakhan catchment soils is non-oxidized FP that is dissolving relatively slowly;2–5 this forms the “Western trace” of Chernobyl contamination across the Sakhan watershed. The dissolution rate of 90Sr from non-oxidized FP is 7–10 times slower than from oxidized FP, and model estimates2 show that, in 2016, the main portion of radionuclides (up to 47% at pH = 6.0) was still within the particle matrix in the Western trace, whereas the basic portion of FP had dissolved by this time at other points. Therefore, the slow dissolution of FP (which has already disappeared from other basins) incorporating 90Sr in the Sakhan catchment could lead to the higher k value compared with the other regions mentioned above. Overall, k is an integral parameter that can be influenced by both hydrological run-off processes and geochemical factors (controlling the leaching of 90Sr from FP and redistribution in soils) in the catchment. Further studies are needed to clarify this issue. 90Sr-Q relationship and its time-dependency. An accurate description of the response of the 90Sr con- centration to Q is another important mission in this study. The variation of 90Sr corresponded to increases in Q during the snowmelt season and subsequent decreases in summer (Fig. 2b). The recurring annual pattern in Q, which rises in the spring and falls in the summer, is clearly influenced by snow melting in spring11,25,26 and the increase in evapotranspiration in summer28. As for the 90Sr-Q relationship, studies have shown that 90Sr increases when the flow rate of the main stream of the Pripyat River increases during the snowmelt period14,15, and 90Sr concentration have also been found to increase with spring floods in small catchments under the influence of discharge levels25,26. Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 Discussion d Long-term decline of 90Sr concentration. The contamination of stream water by 90Sr continues to pose problems in the CEZ. Long-term monitoring and simple modelling are needed to ensure the safety of water usage in downstream areas and to further understand the dissolved 90Sr dynamics. For this study, we obtained data from continuous observations of dissolved-phase 90Sr concentrations and stream water discharges from 1990–2016 in the Sakhan catchment, located northwest of CNPP. We then applied a double-exponential model to the long- term 90Sr data. As shown in Fig. 2b, we were able to model the long-term decline in the 90Sr concentration. The best-fit parameter values for k1 and k2 were found to be 0.27 (1/yr) and 0.03 (1/yr), respectively. Note that our 90Sr data start from 1990, five years after the accident. In general, exponential functions for the decay of radionuclide concentrations in rivers are thought to consist of a fast “flush” of activity as a result of rapid wash-off processes, a slow decline as a result of soil fixation and redistribution processes, and a very-long-term “equilibrium” situ- ation8,9. According to Sasina et al.10, the fast flush phase of 90Sr concentrations originating from the Chernobyl accident in Belarusian rivers lasted for about 0.04 years after the accident (there is a distance of about 90 km from CNPP to the nearest point). Thus, the fast flush of activity as a result of rapid wash-off processes would already have finished by the earliest monitoring year of 1990, and so the values obtained for k1 and k2 represent the slow decline as a result of soil fixation and redistribution processes and the very-long-term equilibrium situation. For 90Sr deposited in rivers by global fallout (e.g. from atmospheric nuclear weapons testing), the slow decay after the Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ In particular, previous attempts to introduce parametric models and explain the C-Q relationship conceptually could help us to understand the time-dependency of the 90Sr-Q relationship. New parametric model of 90Sr concentration. A previous hydrochemical study19 showed that the stream water chemistry can be accurately explained by a conceptual model that builds on the interaction of the vertical profiles of lateral water fluxes and soil solution chemistry. The mathematical implementation of this conceptual model provides an analytical solution, and a physical explanation as a power function, which is empir- ically used to describe the relationship between concentration and discharge22. Here, we propose a new concep- tual hydrochemical model to explain the time-dependency of the 90Sr-Q relationship. The results of our model (Eq. (4)) are compared against measurements for 1990–2016 in Fig. 4a. Our model shows that the long-term diminishing trend of 90Sr is caused by the physical decay of 90Sr and diffusion in the environment, as well as the variation of 90Sr concentration corresponding to changes in Q. Table 1 reports the best fitting parameter with the range of credible values and the agreement between the modelled and observed 90Sr concentrations. Regardless of some uncertainty introduced by measurement errors, the model is sufficiently reliable to link 90Sr and Q for the purposes of this work (R2 = 0.66). Figure 4c shows the response of 90Sr to Q for three periods (C1: 1991–2000, C2: 2001–2010, C3: 2011–2016), and the red dashed lines show the model result for the middle of each period. Our model also adequately reproduces the time-dependency of the 90Sr-Q relationship for each of these periods (Fig. 4c). The key assumption in our model is that the distribution of the lateral flow of soil and the vertical profile of radionuclides in the soil provides a stream discharge and the radionuclide concentration in a stream. This assumption is supported by the following physical processes observed in the CEZ. During high-flow events (i.e. snowmelt, heavy rainfall), 90Sr is directly leached from the contaminated near-surface soil and/or top layer of floodplain soils in the CEZ14. The resulting high groundwater table produces lateral flow and increases the chances that more water will pass through the highly contaminated surface soil layer. This highly Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 4. Time series of observed (black) and modelled values (red) of dissolved-phase 90Sr concentrations. www.nature.com/scientificreports/ Orange dots indicate the posterior predictive credible interval (A). The relationship between observed and modelled values of dissolved-phase 90Sr concentrations. The response of dissolved phase 90Sr concentrations to discharges rate for each period are also shown in (C1-3). Red dashed lines in (C) indicate the model result for the middle of each period. Figure 4. Time series of observed (black) and modelled values (red) of dissolved-phase 90Sr concentrations. Orange dots indicate the posterior predictive credible interval (A). The relationship between observed and modelled values of dissolved-phase 90Sr concentrations. The response of dissolved phase 90Sr concentrations to discharges rate for each period are also shown in (C1-3). Red dashed lines in (C) indicate the model result for the middle of each period. Model Parameter R2 Igarashi model Symbol Best fit 2.5% 97.5% 0.66 a 0.28 0.14 1.09 b 1.37 0.74 3.05 d 2117 1032 2989 Table 1. Estimated model parameters and model agreement. Model Parameter R2 Igarashi model Symbol Best fit 2.5% 97.5% 0.66 a 0.28 0.14 1.09 b 1.37 0.74 3.05 d 2117 1032 2989 Table 1. Estimated model parameters and model agreement. Model Parameter R2 Igarashi model Symbol Best fit 2.5% 97.5% 0.66 a 0.28 0.14 1.09 b 1.37 0.74 3.05 d 2117 1032 2989 Table 1. Estimated model parameters and model agreement. Table 1. Estimated model parameters and model agreement. Table 1. Estimated model parameters and model agreement. contaminated soil water enters streams and rivers, leading to an increase in both flow rate and 90Sr concentration in watercourses. Studies conducted in the Borschi watershed of the CEZ25,26 indicate that near-channel wetland areas can act as a source of 90Sr to surface water. In this study, we assumed that the 90Sr concentration profile in the soil is represented by an exponential function with a time component. Actually, the vertical profile of the 90Sr concentration in soil changes with time according to vertical migration1,31 and transport by the water flow25,26. It has been reported that 90Sr had accumulated in deeper areas (more than 10 cm deep) and was depleted nearer the surface (~5 cm depth) of wetland sediments near the stream some 14 years after the accident26. However, we also need to focus on the source of 90Sr in the catchment. As shown by Ivanov et al.32, the peak 90Sr concentration was on the soil surface in the Sakhan catchment, even 21 years after the accident. www.nature.com/scientificreports/ This is because the source of 90Sr in Sakhan is non-oxidized FP with a slow dissolution rate2–5. Thus, these previous studies support the use of Eq. (3) to represent the temporal changes in the vertical profile of the 90Sr concentration in soil. In addition, our model structure, in which the concentration converges to zero at low flow rates (Q → 0), is justified by the actual hydrological processes. This is because the source of the river water is assumed to be deep groundwater when Q is asymptotically close to zero, and the 90Sr concentration, which originates from FP, is assumed to be low, so the 90Sr concentration eluted from the groundwater can be assumed to be close to zero.hi g This study is the first to show the long-term diminishing trend of the 90Sr concentration through the physical decay of 90Sr and diffusion in the environment, as well as variations in the 90Sr concentration corresponding to changes in the discharge rate. The mathematical implementation based on assumptions from previous studies enables us to describe the time-dependency of the 90Sr-Q relationship. If the solute concentration gradient in the soil involves hydrochemical processes in the river, our model can be applied to other radionuclides besides 90Sr. Finally, this study is the first to examine the relationship between dissolved-phase radionuclides and stream dis- charge from a hydrochemical perspective, and highlights the applicability of this scheme to other radionuclides in rivers. Scientific Reports \| (2020) 10:9818 \| https://doi.org/10.1038/s41598-020-66623-4 www.nature.com/scientificreports/ Methods Experiments were conducted at the Sakhan river in the CEZ (51.41°N, 30.00°E). This river lies within one of the sub-catchments of the Pripyat River, and has a total surface area of 186.9 km2 (Fig. 1). Annual rainfall in the CEZ is 500–600 mm/year. Monthly minimum and maximum temperatures are −6 °C and 19 °C, respectively. The catchment is largely covered by grassy meadow and wetlands. The forest area is occupied by coniferous and mixed forests33. The geological strata are composed of sandy quaternary deposits33. The topography is relatively flat. Local research institutes have been measuring the radionuclide concentrations in the local environment, including in soil and river water collected from various sites in the CEZ, over a 30-year period since soon after the CNPP accident. The 90Sr concentration and water discharge have been measured at the Sakhan river bridge mon- itoring station, located 7 km northwest of the nuclear power plant. The initial average 90Sr concentration in the soil was estimated to be 470.7 kBq/m2 (in 1986)5. The main long-term source of exchangeable and available 90Sr in the Sakhan catchment is non-oxidized FP2–5. The measurement data used in this study were obtained from two research institutes, the Ukrainian Hydrometeorological Institute (data period: 1990–1998) and the State Agency of Ukraine on Exclusion Zone Management and Chernobyl Ecocentre (data period: 1999–2016). Sampling was carried out in intervals of several weeks to several months in the initial phase. Continuous biweekly-interval sampling commenced in March, 1992. The 90Sr measurement error, available only for the period 1999–2016, was 10.9 ± 2.01% (mean ± S.D.). Water discharge data were obtained by integrating the cross-section observations of flow velocity and water depth from a concrete revetment under a bridge. To verify our estimates, flow rates were also measured directly using an electromagnetic current meter from March–May 2018, and the differences in the results were found to be insignificant. Received: 17 December 2018; Accepted: 26 May 2020; Published: xx xx xxxx Received: 17 December 2018; Accepted: 26 May 2020; Published: xx xx xxxx References Cesium-137 and strontium-90 contamination of water bodies in the are Chernobyl nuclear power plant accident: an overview. J. Environ. Radioact. 23, 103–122 (1994). 12. Vakulovsky, S. M. et al. 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Chernobyl Nuclear Power Station. Sov. Soil Sci. 23, 46–51 (1991 y , ( ) 32. Ivanov, Y. A. et al. Long-term dynamics of radionuclide vertical migration in soils of the Chernobyl nuclear power plant exclusion zone. Printed at, http://sti.srs.gov/fulltext/SRNL-STI-2009-00770.pdf (2009) y ( ) 2. Ivanov, Y. A. et al. Long-term dynamics of radionuclide vertical migration in soils of the Chernobyl nuclear power plant exclusion zone. Printed at, http://sti.srs.gov/fulltext/SRNL-STI-2009-00770.pdf (2009) 32. Ivanov, Y. A. et al. Long-term dynamics of radionuclide vertical migration in soils of the Chern zone. Printed at, http://sti.srs.gov/fulltext/SRNL-STI-2009-00770.pdf (2009) g 3. Matoshko, A. et al. Author contributions Y.I.: conception and design of the work; analysis of data. Y.O.: conception and design of the work; interpretation of data. J.S., S.O., S.K., V.D. and G.L.: provision of river data (core data of this work). H.L.: analysis of GIS and contamination map, D.B., A.K., M.Z., Y.W. and K.N.: interpretation of data. All authors reviewed the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66623-4. Correspondence and requests for materials should be addressed to Y.I. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W4245575342	https://www.frontiersin.org/articles/10.3389/fped.2018.00421/pdf	English	null	Childhood Vasculitis	Frontiers in pediatrics	2,019	cc-by	8,375	Anja Schnabel 1* and Christian M. Hedrich 1,2,3* 1 Pädiatrische Rheumatologie, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Germany, 2 Department of Women’s and Children’s Health, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom, 3 Department of Paediatric Rheumatology, Alder Hey Children’s NHS Foundation Trust Hospital, Liverpool, United Kingdom The term vasculitis covers heterogeneous disorders that share the presence of inﬂammation of blood vessel walls. Immune cell inﬁltrates can vary signiﬁcantly and involve granulocytes or mononuclear cells. Vasculitis can be a symptom of other underlying disorders or the underlying cause of organ speciﬁc or systemic disease. Classiﬁcation of childhood vasculitis is based on clinic, the size of predominantly affected vessels, and the histopathology of inﬂammatory inﬁltrates. Timely and accurate diagnosis and (where necessary) treatment initiation determine disease progression and outcomes. In light of new developments and the identiﬁcation of autoinﬂammatory conditions with vasculitis, new classiﬁcation tools may be discussed. Keywords: vasculitis, inﬂammation, systemic disease, kawasaki disease, granulomatosis with polyangitis, purpura, classiﬁcation, paediatric REVIEW published: 10 January 2019 doi: 10.3389/fped.2018.00421 Keywords: vasculitis, inﬂammation, systemic disease, kawasaki disease, granulomatosis with polyangitis, purpura, classiﬁcation, paediatric BACKGROUND Edited by: Rita Consolini, University of Pisa, Italy Vasculitis is an umbrella term for various and heterogeneous disorders sharing the presence of inﬂammation of blood vessel walls. Immune cell inﬁltrates can vary signiﬁcantly and involve granulocytes (neutrophils, eosinophils) or mononuclear cells (monocytes/macrophages, lymphocytes). Vasculitis can be a symptom of other underlying disorders (secondary vasculitis: infections, medication intake, malignancies, autoimmune/inﬂammatory conditions, etc.) or the cause of organ speciﬁc or systemic disease. In the latter case, the term “primary vasculitis” is used (1). Reviewed by: Toni Hospach, Klinikum Stuttgart, Germany Aleš Janda, Reviewed by: Toni Hospach, Klinikum Stuttgart, Germany Aleš Janda, Reviewed by: Toni Hospach, Klinikum Stuttgart, Germany Aleš Janda, University Hospital Freiburg, Germany Correspondence: Anja Schnabel anja.schnabel@uniklinikum-dresden.de Christian M. Hedrich christian.hedrich@liverpool.ac.uk It has been an ongoing challenge to classify childhood vasculitis, which is usually based on clinical phenotypes (e.g., single organ vs. systemic vasculitis), the size of predominantly aﬀected vessels (small/medium/large), and the histopathology of inﬂammatory inﬁltrates (e.g., granulomatous vs. non-granulomatous). The most commonly applied classiﬁcations are the updated Chapel Hill consensus conference criteria for systemic vasculitis (2012) (2), which have not been developed speciﬁcally for children, and the EULAR (European League against Rheumatism)/PRES (Pediatric Rheumatology European Society)/PRINTO (Pediatric Rheumatology International Trials Organization) classiﬁcation criteria for childhood vasculitis (3– 5). Both sets of criteria are largely identical for primary systemic vasculitis (Figure 1) (with the exception for giant cell arteritis), but show some diﬀerences for the classiﬁcation of secondary forms (Table 1) and vasculitis limited to single organ systems. Specialty section: This article was submitted to Pediatric Immunology, a section of the journal Frontiers in Pediatrics Received: 15 September 2018 Accepted: 19 December 2018 Published: 10 January 2019 Citation: Schnabel A and Hedrich CM (2019) Childhood Vasculitis. Front. Pediatr. 6:421. doi: 10.3389/fped.2018.00421 This manuscript provides an overview on current classiﬁcation of childhood vasculitis, focusing on primary systemic vasculitis. We deliver a brief summary on clinical signs, basic pathomechanisms involved and treatment options. More comprehensive discussion of individual forms of vasculitis, their molecular pathophysiology and treatment available is discussed in focused manuscripts of this special topic. Citation: January 2019 \| Volume 6 \| Article 421 1 Frontiers in Pediatrics \| www.frontiersin.org Schnabel and Hedrich Childhood Vasculitis FIGURE 1 \| Classiﬁcation of primary childhood vasculitis based on revised Chapel Hill criteria (2012) and the EULAR/PRES classiﬁcation (2–5). FIGURE 1 \| Classiﬁcation of primary childhood vasculitis based on revised Chapel Hill criteria (2012) and the EULAR/PRES classiﬁcation (2–5). TABLE 1 \| Selection of underlying causes in secondary vasculitis. Cause Example Infections Viral: Hepatitis A/B/C, HIV, CMV Bacterial: Neisseria, mycobacteria Fungal: Candida, aspergillus Malignancies Lymphoma, lymphoproliferative syndrome, etc. Autoimmune SLE, MCTD, JDM, IBD, etc. Medication, drugs Serum sickness, G-CSF, amphetamines, cocaine, heroin, etc. Others Multiple HIV, Human immunodeﬁciency virus; CMV, Cytomegaly virus; SLE, Systemic lupus erythematosus; MCTD, Mixed connective-tissue disease; JDM, Juvenile dermatomyositis; IBD, Inﬂammatory bowel disease; G-CSF, Granulocyte colony stimulating factor. TABLE 1 \| Selection of underlying causes in secondary vasculitis. Cause Example Infections Viral: Hepatitis A/B/C, HIV, CMV Bacterial: Neisseria, mycobacteria Fungal: Candida, aspergillus Malignancies Lymphoma, lymphoproliferative syndrome, etc. Autoimmune SLE, MCTD, JDM, IBD, etc. Medication, drugs Serum sickness, G-CSF, amphetamines, cocaine, heroin, etc. Others Multiple HIV, Human immunodeﬁciency virus; CMV, Cytomegaly virus; SLE, Systemic lupus erythematosus; MCTD, Mixed connective-tissue disease; JDM, Juvenile dermatomyositis; IBD, Inﬂammatory bowel disease; G-CSF, Granulocyte colony stimulating factor. TABLE 1 \| Selection of underlying causes in secondary vasculitis. factors, humoral, and cellular autoimmunological factors, as well as infections have been discussed as contributors. Chronic inﬂammation results in scarring and stenosis of large vessels (6, 7). Clinical presentation of TA is highly variable, ranging from mild symptoms (e.g., arterial hypertension and/or weak peripheral pulses) to cardiac and/or respiratory failure, and further organ involvement (CNS, gastrointestinal system, etc.). Disease stages prior to the development of key clinical symptoms, such as “pulselessness,” are characterized by non- speciﬁc symptoms associated with systemic inﬂammation and include fever, night sweats, malaise, arthralgia, and myalgia. As inﬂammation progresses, vascular stenosis develop and may result in intermittent claudication, vascular bruits and/or hypertension (11). Diagnosis is usually based on EULAR/PRINTO/PRES classiﬁcation criteria (Table 2) and informed by imaging (usually MRI angiography; Figure 2). Biopsies may be helpful in unclear cases (10, 12, 13). Frontiers in Pediatrics \| www.frontiersin.org LARGE VESSEL VASCULITIS Large vessel vasculitis (LVV) mainly aﬀects large arteries and includes giant cell arteritis (2). In childhood, primary LVV is limited to Takayasu arteritis (TA). Temporal giant cell arteritis, that predominantly involves the temporal artery, carotid and/or vertebral arteries, does not occur in children (1, 3, 5). Takayasu arteritis (TA) is characterized by granulomatous vasculitis of the aorta and/or its main branches, but also coronary and/or pulmonary arteries (6, 7). While TA usually manifests in the third or fourth decade, children (including infants) and adolescents can develop disease. The exact incidence and prevalence of TA in childhood is not known and variation has been reported between geographical regions for adults (UK: 0.8/million vs. Asia 2/million; likely 10–20% of who are children) (8, 9). Girls and young women are more frequently aﬀected when compared to boys (3:1) (10). The molecular pathophysiology of TA remains unclear, and genetic Treatment is guided by organ involvement and pre- existing damage and is currently not standardized (14). Pharmaceutical treatment involves corticosteroids as ﬁrst-line induction treatment (15), disease-modifying antirheumatic drugs methotrexate (MTX), azathioprine (AZA), cyclosporine A (CsA), and mycophenolate mofetil (MMF), TNF inhibitors (inﬂiximab/IFX, adalimumab/ADA, etanercept/ETA), IL- 6 inhibitors (Tocilizumab), and B cell depleting agents (Rituximab/RTX) (14, 16–19). Furthermore, Cyclophosphamide (CPM) can be discussed but should be reserved for otherwise treatment refractory or acutely life-threatening cases. If hemodynamically relevant vessel stenosis occur, interventional or surgical interventions may be necessary, but should be avoided if possible, since they are discussed to promote inﬂammation (14). January 2019 \| Volume 6 \| Article 421 Frontiers in Pediatrics \| www.frontiersin.org 2 Childhood Vasculitis Schnabel and Hedrich MEDIUM-SIZED VESSEL VASCULITIS primary childhood vasculitis (Japan 239/100,000 children under 5 years), in predominantly Caucasian populations (9/100,000 children under 5 years), Henoch Schönlein purpura (HSP) is more common (see below). The molecular pathophysiology of KD is unclear (22–24). Genetic predisposition (BLK, CASP3, CD40, FCGR2A, HLA class II, IPTKC), infectious triggers, super antigens, humoral factors and immune complexes have been suggested to contribute. In the acute phase of KD, monocytes/macrophages and T cells produce pro-inﬂammatory mediators that result in endothelial inﬂammation and the clinical picture of KD (22, 23). This group includes two forms of primary childhood vasculitis: Kawasaki disease and polyarteritis nodosa. Kawasaki disease (KD) is a systemic necrotizing vasculitis of small and medium-sized vessels (20). A relatively common and severe complication is the involvement of coronary arteries that can result in aneurysms and cardiac infarction. KD is a rare condition in adults. More than 90% of KD cases aﬀect children and infants (21). Globally, KD is the most common Most (75%) of patients develop KD before their 5th birthday. By deﬁnition, fever without focus is present in all KD patients. In children under 5 years-of-age, KD should be considered in the case of unexplained fevers over 4–5 days (22, 23, 25). Infants are less frequently but more severely (increased risk for aneurysm development) aﬀected and tend to exhibit “incomplete” clinical pictures that can be challenging to diagnose (26). The term “atypical KD” is reserved for children who fulﬁll diagnostic criteria for KD, but experience a not typical disease course, which may, amongst other symptoms include exudative pharyngitis and/or conjunctivitis, aseptic meningitis, arthritis, and/or anterior uveitis. Without timely establishment of treatment, 15– 25% of KD patients will develop coronary aneurysms, which deﬁne prognosis and long-term outcomes (27). Diagnosis is based on criteria summarized in Table 3 and Figure 3 (28, 29). TABLE 2 \| EULAR/PRINTO/PRES criteria for TA. Pathology in angiography plus one of the following Aorta or main branches, pulmonary arteries: focal-segmental occluding or dilating changes exclude: arteriosclerosis and ﬁbromuscular dysplasia (1) Absence of peripheral pulses, claudication Weak pulses over A. radialis and/or A. ulnaris; muscle pain and/or weakness after minor exercise (2) Asymmetrical BP >10 mmHg (systolic) difference between arms (3) Murmur (70%) A. subclavia, A. carotis, abdominal aorta (4) Hypertension >95. Percentile (5) Systemic inﬂammation ESR > 20 mm/h, elevated CrP BP, Blood pressure; CrP, C-reactive protein. exclude: arteriosclerosis and ﬁbromuscular dysplasia FIGURE 2 \| Takayasu arteritis in a 12-year-old girl. (A) MRI angiography showing signiﬁcant stenosis of the coeliac truncus () and lower aorta (→); artifacts are caused by stent implants in renal arteries (L) (provided by Dr. Gabriele Hahn, Pädiatrische Radiologie, Universitätsklinikum Carl Gustav Carus, TU Dresden), (B) transversal abdominal ultrasonography indicating signiﬁcant stenosis of the coeliac truncus, (C) Stenosis of coeliac truncus resulting in pathologically increased arterial blood ﬂow velocity (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). Consent of the patient was obtained to publish these images. FIGURE 2 \| Takayasu arteritis in a 12-year-old girl. (A) MRI angiography showing signiﬁcant stenosis of the coeliac truncus (*) and lower aorta (→); artifacts are caused by stent implants in renal arteries (L) (provided by Dr. Gabriele Hahn, Pädiatrische Radiologie, Universitätsklinikum Carl Gustav Carus, TU Dresden), (B) transversal abdominal ultrasonography indicating signiﬁcant stenosis of the coeliac truncus, (C) Stenosis of coeliac truncus resulting in pathologically increased arterial blood ﬂow velocity (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). Consent of the patient was obtained to publish these images. January 2019 \| Volume 6 \| Article 421 Frontiers in Pediatrics \| www.frontiersin.org 3 Schnabel and Hedrich Childhood Vasculitis Laboratory ﬁndings reﬂect systemic inﬂammation and include elevated CRP (≥30 mg/l) and ESR (>40/h), elevated liver enzymes (ALT ≥50 U/l), hypoproteinemia, elevated cholestasis parameters, thrombocytosis, leukocytosis, and/or anemia (22). Laboratory ﬁndings reﬂect systemic inﬂammation and include elevated CRP (≥30 mg/l) and ESR (>40/h), elevated liver enzymes (ALT ≥50 U/l), hypoproteinemia, elevated cholestasis parameters, thrombocytosis, leukocytosis, and/or anemia (22). usefulness of risk scores exists in Asian populations, but their value in predominantly Caucasian populations remains unclear (22, 31–33). Panarteritis nodosa (PAN) is a necrotizing systemic vasculitis of medium-sized vessels (3). TABLE 3 \| Clinical criteria for the diagnosis of “classical” KD (22). TABLE 3 \| Clinical criteria for the diagnosis of “classical” KD (22). TABLE 3 \| Clinical criteria for the diagnosis of “classical” KD (22). Fever of unknown origin for ≥5 days plus 4 of the following if not explained by another condition. The diagnosis can also be made on day 4 in the presence of ≥4 principal clinical criteria (particularly when redness and swelling of the hands and feet are present) • Bilateral Conjunctivitis (80–90%) • Changes to oropharyngeal mucous membranes, including injected and/or ﬁssured lips, strawberry tongue and enanthema (80–90%) • Palmar and/or plantar erythema and/or periungual desquamation (in convalescent phase) (80%) • Polymorphous exanthema, primarily truncal, not vesicular (>90%) • (Mostly) cervical lymphadenopathy (at least one lymph node >1.5 cm) (50%) Though generally any organ system can be involved, most patients develop vasculitis of the skin, musculoskeletal system, kidneys and/or gastrointestinal system. Less frequently, the heart and peripheral and/or central nervous system can be aﬀected. Initially, non-speciﬁc general symptoms, including fever and malaise are common (5, 34). Diagnosis can be made bases on EULAR/PRINTO/PRES criteria (Table 4) (3). The diagnosis can also be made on day 4 in the presence of ≥4 principal clinical criteria (particularly when redness and swelling of the hands and feet are present) • Changes to oropharyngeal mucous membranes, including injected and/or ﬁssured lips, strawberry tongue and enanthema (80–90%) Because of the rarity of childhood PAN, widely accepted and evidence-based treatment recommendations do not exist. Treatment is usually informed by adult rheumatology and includes corticosteroids, IVIG, DMARDS for maintenance treatment, and/or CPM for severe cases (40, 41). Recently, • Palmar and/or plantar erythema and/or periungual desquamation (in convalescent phase) (80%) FIGURE 3 \| Clinical criteria in KD. (A) Bilateral non-purulent conjunctivitis (80–90%), (B) changes to oropharyngeal mucous membranes, including injected and/or ﬁssured lips, strawberry tongue (80–90%), (C) Palmar, and/or (D) plantar erythema (E) polymorphous exanthema, primarily truncal, not vesicular (>90%), and (F,G) cervical lymphadenopathy (>1.5 cm) (50%). (G) Ultrasound of enlarged cervical lymph nodes with increased perfusion (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). (H) Periungual desquamation (in covalescent phase) (80%), (I) Beau lines; images from (22). FIGURE 3 \| Clinical criteria in KD. TABLE 2 \| EULAR/PRINTO/PRES criteria for TA. Globally, after KD and HSP, PAN is the third most common childhood primary vasculitis (34). Recently, a rare disorder caused by loss-of-function mutations in the ADA2 (or formerly CECR1) gene encoding for adenosine deaminase 2 (ADA2) has been described and accounts for a subset of particularly early-onset cases of PAN (Deﬁciency of ADA2: DADA2) (35–37). The molecular pathophysiology of remaining PAN cases is currently unclear. Interestingly, increased incidence and prevalence was recorded in individuals who experience Familial Mediterranean Fever (FMF), one of the most common autoinﬂammatory disorders in childhood (38). The result of transmural vessel inﬂammation is scarring and (name giving) nodular changes to vessel walls and aneurysm formation (24). For the diagnosis of PAN, classical angiography and (at least in some patients) angio-MRI can be applied to demonstrate vasal microaneurysms in aﬀected organs (3, 39). Early and aggressive treatment of KD aims at symptom alleviation and the prevention of vasculitis-associated damage (such as aneurysms). Standard treatment includes salicylic acid (initially during febrile period 30–50(−80) mg/kg/day, followed by 3–5 mg/kg/day for 6–8 weeks), and intravenous immunoglobulins (IVIG; usually 2 g/kg over 8–12 h). IVIG should be applied within the ﬁrst 7–10 days of fever to reduce the risk for coronary aneurysms. Concomitant treatment with prednisolone should be considered in KD patients with a high- risk for the development of coronary aneurysms or individuals who do not respond to a ﬁrst course of IVIG. In otherwise treatment refractory cases, cytokine-blocking strategies and other have been discussed (22, 23, 27, 28, 30). Evidence for the Bilateral Conjunctivitis (80 90%) • Changes to oropharyngeal mucous membranes, including injected and/or ﬁssured lips, strawberry tongue and enanthema (80–90%) • Palmar and/or plantar erythema and/or periungual desquamation (in convalescent phase) (80%) • Polymorphous exanthema, primarily truncal, not vesicular (>90%) • (Mostly) cervical lymphadenopathy (at least one lymph node >1.5 cm) (50%) • Bilateral Conjunctivitis (80–90%) SMALL VESSEL VASCULITIS Small vessel vasculitis (SVV) predominantly aﬀects parenchymal arteries, arterioles, capillaries, and venules. Medium-sized arteries and veins may also be aﬀected. This group can be subdivided into immune complex mediated and ANCA- associated vasculitis. Henoch Schönlein purpura (HSP) is a diagnosis of exclusion and no speciﬁc diagnostic tests are available. Secondary immune complex vasculitis and thrombocytopenic purpura require to be excluded. To diagnose HSP, EULAR/PRINO/PRES classiﬁcation criteria are commonly used (Table 5) (3). Clinical presentation is variable and, in addition to palpable purpura, includes relatively common symptoms of abdominal pain, arthritis or nephritis, as well as additional organ involvement and associated complications (Table 6). Laboratory ﬁndings are unspeciﬁc and include (usually) mildly elevated inﬂammatory parameters (ESR and CrP), normal or slightly reduced serum C3 and/or C4, the absence of high-titer autoantibodies (particularly ANCA and While Henoch Schönlein purpura (HSP) is the most common form of primary childhood vasculitis in Europe and North America, it is signiﬁcantly less common in adults (3–14 cases per million) (42). Exact numbers on prevalence and incidence do not exist secondary to very variable presentations and resulting variability in medical needs and providers involved. Henoch Schönlein purpura (HSP) is an immune complex vasculitis with (mainly) IgA containing immune depositions in small vessels. It can generally aﬀect all age groups (20/100,000/year), but is most common in 4–6 year-old children g y g g p (20/100,000/year), but is most common in 4–6 year-old children g y absence of high-titer autoantibodies (particularly ANCA and TABLE 4 \| EULAR/PRINTO/PRES criteria for PAN (3). Criterion Frequency (%) MAJOR CRITERIA (ONE HAS TO BE PRESENT) Histological evidence Necrotizing vasculitis of small or medium-sized vessels – Pathology on angiographic studies Aneurysm, vessel occlusion (exclude ﬁbromuscular dysplasia or arteriosclerosis) – MINOR CRITERIA (ONE HAS TO BE PRESENT) Skin involvement Livedo reticularis, painful subcutaneous nodules, skin infarction, digital gangrene 70–90 Myalgia Myalgia and/or myogelosis 40–70 Arterial hypertension >95. Percentile 20–35 Peripheral neuropathy Mononeuritis multiplex, polyneuropathy 20 Kidneys Proteinuria, hematuria, kidney failure 20 FIGURE 4 \| Pro-inﬂammatory mechanisms in immune complex vasculitis. Frontiers in Pediatrics \| www.frontiersin.org 5 January 2019 \| Volume 6 \| Article 421 TABLE 4 \| EULAR/PRINTO/PRES criteria for PAN (3). TABLE 4 \| EULAR/PRINTO/PRES criteria for PAN (3). TABLE 4 \| EULAR/PRINTO/PRES criteria for PAN (3). TABLE 3 \| Clinical criteria for the diagnosis of “classical” KD (22). (A) Bilateral non-purulent conjunctivitis (80–90%), (B) changes to oropharyngeal mucous membranes, including injected and/or ﬁssured lips, strawberry tongue (80–90%), (C) Palmar, and/or (D) plantar erythema (E) polymorphous exanthema, primarily truncal, not vesicular (>90%), and (F,G) cervical lymphadenopathy (>1.5 cm) (50%). (G) Ultrasound of enlarged cervical lymph nodes with increased perfusion (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). (H) Periungual desquamation (in covalescent phase) (80%), (I) Beau lines; images from (22). January 2019 \| Volume 6 \| Article 421 4 Frontiers in Pediatrics \| www.frontiersin.org Childhood Vasculitis Schnabel and Hedrich anti-TNF agents have been demonstrated eﬀective at least in a subset of DADA2 patients (35). anti-TNF agents have been demonstrated eﬀective at least in a subset of DADA2 patients (35). (70/100,000/year). Boys are slightly more frequently aﬀected as compared to girls (1.2–2:1) (43). While the exact pathophysiology of HSP is unknown, an association with previous infections within the past 2–4 weeks has been recorded (44). As the result of an unknown trigger mechanism, IgA containing immune complexes deposit in small vessels and cause complement activation, immune cell invasion, endothelial activation, and lastly vasculitis (Figure 4). SMALL VESSEL VASCULITIS Criterion Frequency (%) MAJOR CRITERIA (ONE HAS TO BE PRESENT) Histological evidence Necrotizing vasculitis of small or medium-sized vessels – Pathology on angiographic studies Aneurysm, vessel occlusion (exclude ﬁbromuscular dysplasia or arteriosclerosis) – MINOR CRITERIA (ONE HAS TO BE PRESENT) Skin involvement Livedo reticularis, painful subcutaneous nodules, skin infarction, digital gangrene 70–90 Myalgia Myalgia and/or myogelosis 40–70 Arterial hypertension >95. Percentile 20–35 Peripheral neuropathy Mononeuritis multiplex, polyneuropathy 20 Kidneys Proteinuria, hematuria, kidney failure 20 Necrotizing vasculitis of small or medium-sized vessels Aneurysm, vessel occlusion (exclude ﬁbromuscular dysplasia or arteriosclerosis) g Aneurysm, vessel occlusion (exclude ﬁbromuscular dysplasia or arteriosclerosis) FIGURE 4 \| Pro-inﬂammatory mechanisms in immune complex vasculitis. FIGURE 4 \| Pro-inﬂammatory mechanisms in immune complex vasculitis. January 2019 \| Volume 6 \| Article 421 Frontiers in Pediatrics \| www.frontiersin.org 5 Schnabel and Hedrich Childhood Vasculitis TABLE 5 \| EULAR/PRINTO/PRES criteria for HSP (3). Criterion Frequency (%) Palpable purpura plus one of the following - Symmetric purpura, mainly over extremities following gravity - Exclude thrombocytopenia! 100 Abdominal pain - Colic-like, postprandial - Nausea, gastrointestinal bleeding - Intussusception, infarction, perforation 50 Histopathology - Immune complex vasculitis - Proliferative glomerulonephritis (IgA deposition) – Kidney involvement - Proteinuria >0.3 g/24 h or - Albumin/creatinine ratio >30 mmol/mg - Micro-hematuria - Arterial hypertension - Nephritic or nephrotic syndrome 20–40 Joint involvement - Arthritis, mostly ankles and/or knees 70 TABLE 6 \| Non-cutaneous complications in HSP (45). Complication Frequency (%) Urogenital - Testicular bleeding - Orchitis, urethral stenosis 5–10 CNS - Coma, ataxia, intracerebral bleeding - DD: acute hypertensive crisis as a result of kidney failure 2 Lungs - Reduced gas exchange capacity, pulmonary bleeding <1 TABLE 5 \| EULAR/PRINTO/PRES criteria for HSP (3). Criterion Frequency (%) Palpable purpura plus one of the following - Symmetric purpura, mainly over extremities following gravity - Exclude thrombocytopenia! 100 Abdominal pain - Colic-like, postprandial - Nausea, gastrointestinal bleeding - Intussusception, infarction, perforation 50 Histopathology - Immune complex vasculitis - Proliferative glomerulonephritis (IgA deposition) – Kidney involvement - Proteinuria >0.3 g/24 h or - Albumin/creatinine ratio >30 mmol/mg - Micro-hematuria - Arterial hypertension - Nephritic or nephrotic syndrome 20–40 Joint involvement - Arthritis, mostly ankles and/or knees 70 TABLE 5 \| EULAR/PRINTO/PRES criteria for HSP (3). Granulomatosis with polyangitis (GPA) is rare in children. Its incidence is estimated to be around 1/1,000,000. The mean age at disease-onset is around 14 years. SMALL VESSEL VASCULITIS General symptoms, including fevers, weight loss, and fatigue are present in over 90% of all patients. Diagnosis is usually made based on EULAR/PRINTO/PRES classiﬁcation criteria (Table 8) (3), and additional symptoms can include arthritis, erythema, ulcerations, and gastrointestinal involvement (Figure 5) (51). Untreated, GPA has a mortality close to 100% within the ﬁrst year, and regardless of treatment, up to 60% of patients experience subsequent disease ﬂares. When compared to GPA in adults, children, and young people more frequently experience multi-organ involvement, kidney involvement, subglottic stenosis and nose deformities (51, 52). Eosinophilic granulomatosis with polyangitis (EGPA) is a necrotizing vasculitis characterized by eosinophilic inﬁltrates in small and medium-sized vessels (53, 54). Reliable demographic information and/or classiﬁcation criteria for the pediatric age- group do not exist, and diagnosis is informed by ACR criteria for adults with GPA (53). For classiﬁcation as EGPA, four of six criteria must be fulﬁlled: (i) Asthma, (ii) Eosinophilia (peripheral blood) >10%, (iii) Mono- or polyneuropathy, (iv) pulmonary inﬁltrates, (v) para-nasal sinus anomalies, and (vi) biopsy with extra vascular eosinophilic inﬁltrates. TABLE 6 \| Non-cutaneous complications in HSP (45). TABLE 6 \| Non-cutaneous complications in HSP (45). Complication Frequency (%) Urogenital - Testicular bleeding - Orchitis, urethral stenosis 5–10 CNS - Coma, ataxia, intracerebral bleeding - DD: acute hypertensive crisis as a result of kidney failure 2 Lungs - Reduced gas exchange capacity, pulmonary bleeding <1 The exact pathophysiology of EGPA remains unclear. Inappropriate Th2 activation and IL-4, IL-5, and Il-13 expression play a central role and reﬂect parallels to allergic disease. Progressive immune dysregulation triggers a prodromal state that can last several years and include chronic sinusitis, allergies, and severe corticosteroid-dependent asthma. Later, sinusitis, pulmonary inﬁltrations and sometimes severe gastrointestinal, cardiac, skin manifestations (Livedo, painful subcutaneous lesions, infarctions, etc.), and peripheral neuropathy can be present (54, 55). ANA) and, in 50% of all cases, elevated serum IgA and/or IgM (46). Fecal occult blood tests and urinary specimens are recommended at diagnosis. Since glomerulonephritis can manifest after the initial phase, regular urinary analysis at least for the ﬁrst 6 months after disease-onset are recommended. Microscopic polyangitis (MPA), in contrast to GPA and EGPA, is a non-granulomatous necrotizing vasculitis of small vessels (56). Immune complex deposition is limited or absent (pauci-immune vasculitis). Most patients develop pulmonary (but not upper respiratory tract) involvement with pulmonic hemorrhage and sometimes kidney involvement (27%). Frontiers in Pediatrics \| www.frontiersin.org SMALL VESSEL VASCULITIS Additional organ systems, including the gastrointestinal system, CNS, skin, musculoskeletal system, and end eyes can be aﬀected. Diagnostic or classiﬁcation criteria for childhood MPA do not exist. Thus, Microscopic polyangitis (MPA) remains a diagnosis of exclusions and is informed by adult rheumatology. The presence of ANCA can be helpful (in adult cohorts: >50% pANCA, 40% cANCA positive) (56, 57). Treatment depends on clinical presentation and organ involvement. Non-steroidal anti-inﬂammatory drugs (NSAIDs) or acetaminophen/paracetamol can be considered for analgesia in cases with arthritis or arthralgia. For gastrointestinal involvement treatment with corticosteroids (usually 1–2 mg/kg/day for 1 week, followed by taper over 2–3 weeks) can be considered. However, intestinal perforations have been seen under treatment (47, 48). Substitution of factor XIII can be discussed for gastrointestinal bleedings. Of note, studies indicate that early corticosteroids are not eﬀective in preventing HSP-associated nephritis (45, 49). Evidence for treatment is weak, and expert recommendations have been provided e.g., by the German Society for Pediatric Nephrology (GPN) (Table 7) (50). Bed rest can be considered in severe cases. Treatment of childhood ANCA-associated vasculitis is guided by disease severity. Generally, patients should receive more or less aggressive induction treatment to control inﬂammation (high-dose prednisolone or i.v. methylprednisolone pulses ± CPM) that is followed by maintenance treatment with more tolerable therapeutic agents (MTX, AZA, MMF) (58). Based on studies in adult cohorts, RTX can be discussed as alternative induction treatment in some cases and appears to be superior to CPM in GPA ﬂares after initial induction treatment with CPM (59, 60). Trimetoprim/sufamethoxazole should be considered for The group of ANCA-associated vasculitis includes granulomatosis with polyangitis (GPA, formerly Wegener’s granulomatosis), eosinophilic granulomatosis with polyangitis (EGPA, formerly Churg-Strauss syndrome), and microscopic polyangitis (MPA). All of which are characterized by destructive vasculitis of small and medium-sized arterial vessels, the presence of ANCA antibodies, and multi-organ involvement (1, 3, 4). Frontiers in Pediatrics \| www.frontiersin.org January 2019 \| Volume 6 \| Article 421 6 Schnabel and Hedrich Childhood Vasculitis apremilast, TNF inhibitors, etc.) are discussed elsewhere (1, 64). pneumocystis prophylaxis. Furthermore, ﬂare reduction has been described in patients with GPA and S. aureus colonization (61, 62). In devastating and treatment-refractory cases, autologous stem cell transplantation can be considered (63). Cogan syndrome (CS) is characterized by predominantly large vessel vasculitis, but can aﬀect any vessel size (1). SMALL VESSEL VASCULITIS CS is an extraordinarily rare multisystem inﬂammatory condition that can involve eyes (keratitis, uveitis, episcleritis) and inner ears (sensorineural deafness, vestibular dysfunction) (2, 74). Unspeciﬁc systemic symptoms occur in 50% of all patients, including arthralgia and manifestations of medium- size and small vessel vasculitis. To date, only few pediatric patients have been reported (75). Based on the rarity and lack of pathophysiological understanding of the disorder, data on eﬀective treatments are lacking. Available reports favor DMARDs (AZA, MTX) in combination with TNF inhibitors (75). VASCULITIS AFFECTING VARIABLE VESSEL SIZES Several forms of vasculitis can aﬀect vessels of variable types and diameters. Behcet’s disease (BD) is characterized by inﬂammatory lesions in vessel walls of all sizes, which may lead to endothelial damage, thrombosis, and aneurysms (64). Chronic recurrent oral and/or genital ulcers occur can be accompanied by additional cutaneous (erythema nodosum, cutaneous pustular vasculitis, etc.), ocular (posterior uveitis, retinal vasculitis), articular (non- erosive poly- or oligo-arthritis), gastrointestinal (abdominal pain, nausea, diarrhea, etc.), and/or central nervous symptoms (aseptic meningitis, vascular thrombosis) (1). Cases of BD can be seen across the globe and in all ethnicities (64). However, prevalence is highest in countries along the Silk Road, where it ranges between 77 and 100/100,000 individuals (0.1–15.9/100,000 in Western Europe) (65, 66). While most patients develop symptoms in young adulthood, 5–10% exhibit childhood-onset BD (67). The pathophysiology of BD is incompletely understood, but genetic associations are likely involved and may be inﬂuenced by environmental factors (13, 68, 69). HLA-B51/B5 allele carriers have considerably high risk for BD indicating a possible gene- dose eﬀect (70). Diagnosis can be challenging, especially since children and young people frequently do not develop the full clinical picture of BD and progress over time (64, 71). More than 15 sets of classiﬁcation or diagnostic criteria have been published (72). Based on clinical diﬀerences between age-groups, recently, pediatric classiﬁcation criteria have been suggested (1, 73). Treatment of BD can be complex and should be informed by clinical symptoms and disease severity. Topical treatment (steroids and/or sucralfate) and systemic treatments (corticosteroids, colchicine, AZA, CsA, thalidomide, TABLE 8 \| EULAR/PRINTO/PRES criteria for GPA (3). Criterion Frequency (%) 1. Histopathology - Granulomatous vasculitis of small vessels – 2. ENT - Chronic rhinitis/sinusitis - Oral and/or nasal ulceration - Nasal septum defect, saddle nose - Mastoiditis, hearing loss, recurrent epistaxis 80 3. Laryngo-tracheo- bronchial system - Subglottic, tracheal, bronchial stenosis 40 4. Lungs - Cough, dyspnea, obstruction - Pulmonary bleeding - Granuloma, cavernosis, inﬁltrates in X ray or thorax CT 80 5. Kidneys - Proteinuria > 0.3 g/24 h or - Albumin/creatinine ratio >30 mmol/mg - Hematuria - Necrotizing glomerulonephritis 75 6. ANCA antibodies - ANCA positivity (immune ﬂorescence, ELISA) 90 TABLE 8 \| EULAR/PRINTO/PRES criteria for GPA (3). TABLE 7 \| Treatment recommendations for severe HSP nephritis (50). January 2019 \| Volume 6 \| Article 421 CONCLUSIONS Vasculitis are rare conditions in children and young people that can be subdivided and classiﬁed based on clinical phenotypes (e.g., organ-speciﬁc vs. systemic) underlying causes (primary vs. secondary disease), histological patterns (granulomatous, non-granulomatous, necrotizing, etc.), and primarily aﬀected vessel sizes (Chapel Hill and EULAR/PRES classiﬁcations: small/medium/large). Timely and accurate diagnosis and (where necessary) treatment initiation are essential, provided the variable severity and outcomes of individual forms of vasculitis. In light of new developments [including the identiﬁcation of genetic causes, sometimes resulting in expansion of disease- associated phenotypes (e.g., DADA2)] and the identiﬁcation of autoinﬂammatory conditions with vasculitis as key feature [including complex genetic BD, but also monogenic disease (DADA2, SAVI, HA20)], new classiﬁcation tools may be justiﬁed in the near future. VASCULITIS AFFECTING VARIABLE VESSEL SIZES Gabriele Hahn, Pädiatrische Radiologie, Universitätsklinikum Carl Gustav Carus, TU Dresden); (B) Necrotizing skin vasculitis on legs and feet; (C) Splenomegaly, spleen with increased echogenicity, subcapsular ﬂuid accumulation, hypoechogenic necrotic areas with absence of perfusion (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden); (D) Severe enterocolitis (shown: transversal colon) with deep ulcerations as a result of necrotizing vasculitis (provided by Dr. Martin Laass, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). Consent of the patient was obtained to publish these images. SINGLE ORGAN VASCULITIS and/or vasculitis remain unclear in many cases. Thus, (with the exception of BD) vasculitis in the context of autoinﬂammatory disease is not part of currently available classiﬁcations for vasculitis, which will likely change in the years to come. Primary organ vasculitis covers a range of particularly rare disorders characterized by vasculitis of a single organ in the absence of signs indicative of systemic vasculitis (1). Various organ systems can be involved, including the CNS (primary large or small vessel CNS vasculitis) (76, 77), primary testicular vasculitis (78), cutaneous leukocytoclastic vasculitis (1), etc. Primary organ vasculitis covers a range of particularly rare disorders characterized by vasculitis of a single organ in the absence of signs indicative of systemic vasculitis (1). Various organ systems can be involved, including the CNS (primary large or small vessel CNS vasculitis) (76, 77), primary testicular vasculitis (78), cutaneous leukocytoclastic vasculitis (1), etc. VASCULITIS AFFECTING VARIABLE VESSEL SIZES 1 (mild) 2 (moderate) 3 (nephrotic syndrome) 4 (kidney failure) HSP STADIUM AND TREATMENT OPTIONS Proteinuria (g/g creatinine) <2 >2 GFR (ml/min/1.73 m2) >90 <90 Serum albumine (g/dl) >2.5 <2.5 Kidney histology – No membrane proliferation Active nephritis, membrane proliferation Therapeutic options – Ramiprile Ramiprile + Methylprednisolone potentially additional immune suppression Ramiprile + Methylprednisolone + Cyclophosphamide potentially additional immune suppression TABLE 7 \| Treatment recommendations for severe HSP nephritis (50). 3 (nephrotic syndrome) >90 January 2019 \| Volume 6 \| Article 421 Frontiers in Pediatrics \| www.frontiersin.org Schnabel and Hedrich Childhood Vasculitis FIGURE 5 \| Findings in a 16-year-old boy with GPA. (A) Abdominal MRI showing pronounced parenchymatous necrosis of the spleen with distension of the spleen capsule (provided by Dr. Gabriele Hahn, Pädiatrische Radiologie, Universitätsklinikum Carl Gustav Carus, TU Dresden); (B) Necrotizing skin vasculitis on legs and feet; (C) Splenomegaly, spleen with increased echogenicity, subcapsular ﬂuid accumulation, hypoechogenic necrotic areas with absence of perfusion (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden); (D) Severe enterocolitis (shown: transversal colon) with deep ulcerations as a result of necrotizing vasculitis (provided by Dr. Martin Laass, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). Consent of the patient was obtained to publish these images. FIGURE 5 \| Findings in a 16-year-old boy with GPA. (A) Abdominal MRI showing pronounced parenchymatous necrosis of the spleen with distension of the spleen capsule (provided by Dr. Gabriele Hahn, Pädiatrische Radiologie, Universitätsklinikum Carl Gustav Carus, TU Dresden); (B) Necrotizing skin vasculitis on legs and feet; (C) Splenomegaly, spleen with increased echogenicity, subcapsular ﬂuid accumulation, hypoechogenic necrotic areas with absence of perfusion (provided by Dr. Heike Taut, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden); (D) Severe enterocolitis (shown: transversal colon) with deep ulcerations as a result of necrotizing vasculitis (provided by Dr. Martin Laass, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden). Consent of the patient was obtained to publish these images. FIGURE 5 \| Findings in a 16-year-old boy with GPA. 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Autoinﬂammatory conditions are still relatively “new” to the ﬁeld of Rheumatology and underlying pathomechanisms of systemic inﬂammation January 2019 \| Volume 6 \| Article 421 Frontiers in Pediatrics \| www.frontiersin.org 8 Childhood Vasculitis Schnabel and Hedrich FUNDING CH’s work is supported by the Fritz-Thyssen Foundation, Novartis Pharmaceuticals (research grant), the intramural MeDDrive Program of TU Dresden, LUPUS UK, and the FAIR charity. AS and CH equally contributed to all stages of this manuscript, including conception and writing of the manuscript. AS and CH contributed to manuscript revision, read, and approved the submitted version. REFERENCES Clinical and pathological features of microscopic polyangiitis in 20 children. J Rheumatol. (2014) 41:1712–9. doi: 10.3899/jrheum.131300 38. Ozen S, Ben-Chetrit E, Bakkaloglu A, Gur H, Tinaztepe K, Calguneri M, et al. 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Pediatr Infect Dis J. (2018) 37:850–5. doi: 10.1097/INF.0000000000001923 January 2019 \| Volume 6 \| Article 421 Frontiers in Pediatrics \| www.frontiersin.org 9 Schnabel and Hedrich Childhood Vasculitis 53. Masi AT, Hunder GG, Lie JT, Michel BA, Bloch DA, Arend WP, et al. The American College of Rheumatology 1990 criteria for the classiﬁcation of Churg-Strauss syndrome (allergic granulomatosis and angiitis). Arthritis Rheum. (1990) 33:1094–100. doi: 10.1002/art.1780330806 34. Ozen S, Anton J, Arisoy N, Bakkaloglu A, Besbas N, Brogan P, et al. Juvenile polyarteritis: results of a multicenter survey of 110 children. J Pediatr. (2004) 145:517–22. doi: 10.1016/j.jpeds.2004.06.046 35. Meyts I, Aksentijevich I. Deﬁciency of Adenosine Deaminase 2 (DADA2): updates on the phenotype, genetics, pathogenesis, and treatment. J Clin Immunol. (2018) 38:569–78. doi: 10.1007/s10875-018-0525-8 54. Zwerina J, Eger G, Englbrecht M, Manger B, Schett G. 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Available online at: https://livrepository.liverpool.ac. uk/3019142/1/10067-WALSH.pdf Conﬂict of Interest Statement: 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. Copyright © 2019 Schnabel and Hedrich. 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. 77. Twilt M, Benseler SM. CNS vasculitis in children. Mult Scler Relat Disord. (2013) 2:162–71. doi: 10.1016/j.msard.2012.11.002 78. Dotan ZA, Laufer M, Heldenberg E, Langevitz P, Fridman E, Duvdevani M, et al. 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https://openalex.org/W2917587079	https://europepmc.org/articles/pmc6387974?pdf=render	English	null	Live Single-Cell Metabolomics With Matrix-Free Laser/Desorption Ionization Mass Spectrometry to Address Microalgal Physiology	Frontiers in plant science	2,019	cc-by	7,077	ORIGINAL RESEARCH published: 18 February 2019 doi: 10.3389/fpls.2019.00172 Keywords: microalgae, single-cell metabolomics, live single cell mass spectrometry, matrix-free laser desorption/ionization-mass spectrometry, multivariate statistics, diatoms Coscinodiscus (Bacillariophyceae), chlorophyceae Haematococcus pluvialis Live Single-Cell Metabolomics With Matrix-Free Laser/Desorption Ionization Mass Spectrometry to Address Microalgal Physiology Tim U. H. Baumeister1, Marine Vallet1, Filip Kaftan2, Aleš Svatoš2* and Georg Pohnert1,3* 1 Max Planck Fellow Group on Plankton Community Interaction, Max Planck Institute for Chemical Ecology, Jena, Germany, 2 Research Group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology, Jena, Germany, 3 Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics, Friedrich Schiller University Jena, Jena, Germany Unicellular phototrophic algae can form massive blooms with up to millions of individual cells per milliliter in freshwater and marine ecosystems. Despite the temporal dominance of bloom formers many algal species can co-exist and compete for nutrients and space, creating a complex and diverse community. While microscopy and single cell genomics can address the taxonomic inventory, the cellular metabolome has yet to be thoroughly explored to determine the physiological status of microalgae. This might, however, provide a key to understand the observed species diversity in the homogeneous environment. Here, we introduce an effective, rapid and versatile method to analyze living single cells from aqueous substrata with laser-desorption/ionization mass spectrometry (LDI-MS) using a simple and inexpensive matrix-free support. The cells deposited on a cultivation-medium wetted support are analyzed with minimal disturbance as they remain in their natural viable state until their disruption during LDI-MS. Metabolites desorbed from single cells are analyzed on High-Resolution Mass Spectrometry (HR-MS) using the Orbitrap FT-MS technology to ﬁngerprint cellular chemistry. This live single-cell mass spectrometry (LSC-MS) allows assessing the physiological status and strain-speciﬁcs of different microalgae, including marine diatoms and freshwater chlorophytes, at the single-cell level. We further report a reliable and robust data treatment pipeline to perform multivariate statistics on the replicated LSC-MS data. Comparing single cell MS spectra from natural phytoplankton samples and from laboratory strains allows the identiﬁcation and discrimination of inter and intra-speciﬁc metabolic variability and thereby has promising applications in addressing highly complex phytoplankton communities. Notably, the herein described matrix-free live-single-cell LDI-HR-MS approach enables monitoring dynamics of the plankton and might explain why key-players survive, thrive, avoid selective feeding or pathogenic virus and bacteria, while others are overcome and die. Edited by: Flavia Guzzo, University of Verona, Italy Reviewed by: Young Jin Lee, Iowa State University, United States Damien L. Callahan, Deakin University, Australia Andrea Anesi, Fondazione Edmund Mach, Italy *Correspondence: Aleš Svatoš svatos@ice.mpg.de Georg Pohnert georg.pohnert@uni-jena.de Specialty section: This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science Received: 08 September 2018 Accepted: 01 February 2019 Published: 18 February 2019 Citation: Baumeister TUH, Vallet M, Kaftan F, Svatoš A and Pohnert G (2019) Live Single-Cell Metabolomics With Matrix-Free Laser/Desorption Ionization Mass Spectrometry to Address Microalgal Physiology. Front. Plant Sci. 10:172. doi: 10.3389/fpls.2019.00172 February 2019 \| Volume 10 \| Article 172 1 Frontiers in Plant Science \| www.frontiersin.org LSC-MS Metabolomics of Microalgae Baumeister et al. INTRODUCTION Other non-destructive methods such as Raman microscopy enable molecular ﬁngerprinting of single cells, but Raman signals are only observed from a few functional groups (Smith et al., 2016). Single-cell mass spectrometry (SC-MS) is one of the foremost strategies to record metabolic proﬁles of single cells and the cellular metabolic activities. Several SC-MS approaches have been developed, many of which involve signiﬁcant sample preparation, including, but not limited to, treatment with an organic matrix or evacuation under vacuum. These treatments can lead to cellular degradation or other stress that can trigger wound-activated chemical transformations and alter the metabolic signature of a cell within seconds (Pohnert, 2000). Single cell analysis by ionization under ambient pressure oﬀers the lowest perturbation, granting the analysis within minutes (Comi et al., 2017; Yang et al., 2017; Sun et al., 2018; Zhang and Vertes, 2018). Materials For diatom cell wall preparation fresh diatom cultures (30 mL, see below) were centrifuged for 15 min at 3000 relative centrifugal force and clean empty bio mineralized diatom cell walls (frustules) were obtained by treating the pellet in 1 mL of hydrogen peroxide (1.5%) at 90◦C for 2 h, followed by three washing steps of the cell pellet with acetonitrile/water (1:1) (Aitken et al., 2016). The empty shells were stored in pure ethanol at 4◦C. Analytical standards Chl a, fucoxanthin, β-carotene, astaxanthin (Atx) and chemicals, if not mentioned otherwise were obtained from Merck (Darmstadt, Germany). Dimethylsulfoniopropionate (DMSP) was synthesized according to a published procedure (Chambers et al., 1987). Standards were prepared in UPLC grade pure ethanol or chloroform at 1 mg × mL−1 and 2 µL were spotted on cleaned diatom cell walls (frustules) or on humid GF/C ﬁlter stacks, air-dried and analyzed as described above. The sample preparation plays a crucial role in depicting a realistic portrait of the cellular analytes; the methods at disposal include suction of the cell content by micro capillary or extraction of the metabolites by nanomanipulation (Fujii et al., 2015; Lee et al., 2016). However, these approaches are technically challenging and often lack sensitivity. Laser desorption/ionization (LDI) strategies provide an alternative, where analytes from single cells can be ionized using a pulsed UV-laser. Cells are thereby destroyed and metabolites are simultaneously ionized. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approaches, this process is supported by an externally added matrix. However, matrix signals dominate the low molecular range of the mass spectra. Two matrix-free platforms have been optimized for LDI-MS applications. Nano post arrays (NAPA) INTRODUCTION provide nanostructured surfaces that support ionization as demonstrated, e.g., in the investigation of yeast cell metabolites (Walker et al., 2012). If cells themselves are patterned, this property can be used for a direct imaging using a pulsed laser without the support of matrix or additional structured surfaces (Jaschinski et al., 2014). Especially cells from phytoplankton, like diatoms with nanopatterned silicate cell walls fulﬁll this pre- requisite. Furthermore, phytoplankton cells inherit signiﬁcant amounts of chromophores, like chlorophyll a (Chl a) and carotenes that can be easily ionized through photoionization by a UV laser (Suzuki et al., 2009; Jaschinski et al., 2014; Mandal et al., 2019). The aforementioned techniques require evacuation of the sample to vacuum before proﬁling of the cells. With the present contribution, we overcome this limitation and introduce an ambient pressure matrix-free ionization of cells in their native state. We proceed to analyze single cells of the freshwater algal model Haematococcus pluvialis and of the marine diatom Coscinodiscus granii with LDI-HR-MS, by decrypting changes in their cellular metabolome during aging and nutrient-depletion. The present method discriminated natural and laboratory strains of diatoms Coscinodiscus granii, hence opening perspectives in taxonomic identiﬁcation at the single cell level during natural phytoplankton blooms. Cell-to-cell heterogeneity is deﬁned by diﬀerential metabolic expression resulting in diverse phenotypes in seemingly homogeneous populations (Malviya et al., 2016). The cell performance can vary tremendously within a population in response to environmental cues and other external stimuli. Moreover, cellular responses to intra- or inter-speciﬁc infochemicals released by the conspeciﬁcs or competing species can be diverse according to the physiological state of the respective cells (Zenobi, 2013). Numerous challenges are faced when addressing the concentration, structural identity and functional role of cellular metabolites (Kuhlisch and Pohnert, 2015). In particular, rapid and high throughput methods are required to cover the dynamics of metabolite expression that is causing substantial ﬂuctuations in concentrations over the course of the development of a culture or bloom (Barofsky et al., 2010; Vidoudez and Pohnert, 2012). Being able to identify and quantify metabolites in single cells, however, will support the prediction of dynamics in large populations of microalgae by assigning ﬁtness status to respective cell cohorts (Acevedo-Trejos et al., 2018). Complementary analytical tools are paramount to record the cellular heterogeneity but the inventory is rather limited. Several single cell approaches including ﬂuorescence microscopy require labeling experiments which can interfere with the cell physiology (Ettinger and Wittmann, 2014). Frontiers in Plant Science \| www.frontiersin.org Cultivation of Algae Heterogeneous phytoplankton samples were collected in German coastal waters during a bloom in August 2016. Coscinodiscus granii (isolate Helg2016) was identiﬁed and selected based on keytaxonomiccriteria(Lundholmetal.,2002;Zimmermannetal., 2011; Kesseler, 2015; Wang et al., 2015). Cultures were grown in half strength Guillard’s (F/2) enrichment medium prepared with natural sea water (ATI, Hamm, Germany). Monoclonal strains fromculturecollectionsCoscinodiscusgraniistrainSCCAP-K1834 (isolation in Denmark in 2012) and Haematococcus pluvialis strain SAG 192.80 were maintained in artiﬁcial seawater medium and freshwater Blue-Green (BG11) medium (Stanier et al., 1971), February 2019 \| Volume 10 \| Article 172 Frontiers in Plant Science \| www.frontiersin.org 2 LSC-MS Metabolomics of Microalgae Baumeister et al. scaled. Only signals occurring in more than half of the samples of the group (nutrients status, age or strain, respectively) were selected and processed. The MS spectra were recorded in positive polarity. For the quantile-quantile plots, TIC normalized and Pareto scaled peak intensities of Chl a (m/z 892.5345), β-carotene (m/z 536.4371), and fucoxanthin (m/z 658.4214) of the Helg2016 and SCCAP-K1834 (Day 1) data were selected. Quantile-quantile plots were created using the R stats package to visualize if the data were normally distributed. Normality test was assessed with the Shapiro-Wilk test (R Core Team, 2018). Unsupervised principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to highlight metabolic variations between LSC-MS proﬁles, using the MetaboanalystR package (Gromski et al., 2015; Xia et al., 2015). After PLS-DA calculation, permutation test and cross validation (CV) were performed to verify the signiﬁcance of the test and validate the selected model, respectively. Signiﬁcant features were determined by calculating the sum of squares of the PLS loadings for each component, giving a variable of importance in projection (VIP score), including 10 top features without taking into account an absolute threshold. To assess the signiﬁcance of class discrimination, a permutation test on 1000 simulations based on prediction accuracy was performed. Further validation was inferred with the Random Forest algorithm to construct classiﬁcation trees which were grown by random sample selection from a bootstrap sample at each branch. The random forest provided an OOB (out-of-bag) error to obtain an unbiased estimate of the classiﬁcation error. respectively. To study the inﬂuence of aging and nutrient depletion on the cellular metabolome, the algae under study were cultivated for 15 days under daylight ﬂuorescent lamps (irradiance 100 mE × m−2 × s−1) with a 14 h photoperiod coupled to a thermo-regulated cycle (16◦C/12◦C day/night). Cultivation of Algae The growth was monitored by counting with a Sedgewick-Rafter chamber (Pyser Optics, Kent, United Kingdom) every second day. For Haematococcus, cultures were left incubating for 15 days until observing cells in “red” phenotype. Pictures were taken with an inverted microscope Axiovert2plus coupled to AxioCam MRc 5 camera (Carl Zeiss AG, Oberkochen, Germany). One culture per species or strain was used to randomly recover a minimum of 20 cells for single cell proﬁling. Proﬁling of Living Single Cells by LDI-HR-MS Prior to single cell analysis GF/C glass ﬁber ﬁlters (Whatman, Maidstone, United Kingdom) were cut in rectangles of 15 × 12 mm and washed thrice with methanol (LiChrosolv hypergrade for LC-MS, Merck, Germany) and n-hexane (Rotipuran ≥99% p.a., Roth, Germany). Algae cells were manually collected from cultures with a Pasteur pipette and promptly applied to a three layered stack of washed GF/C ﬁlters wetted beforehand by adding 50 µL of F2 medium homogeneously over the ﬁlter. Filters with cells were placed on a clean microscopic glass slide (15 × 12 mm) that was ﬁxed on a holder. All samples were analyzed via an AP-SMALDI (AP-SMALDI10, TransMIT, Gießen, Germany) ion source equipped with an UV (337 nm) nitrogen laser (LTB MNL-106, LTB, Germany) with a spot size of 10 µm. The AP-SMALDI ion source was coupled to the mass spectrometer Q-Exactive Plus (Thermo Fisher Scientiﬁc, Bremen, Germany), which provided high resolution mass spectra. Data were collected using an Xcalibur software version 2.8 Build 1824 (Thermo Fisher Scientiﬁc, Bremen, Germany). Samples were analyzed in both polarities with the number of laser shots per spot set to 30 (approximately 1.5 µJ × shot−1) within the laser frequency of 60 Hz. Mass spectra were recorded in the mass range from m/z 100–1000 Da with the peak resolution of 70,000. The analysis of one single cell yielded a live single-cell mass spectrum (LSC-MS) and 20 individual cells were analyzed in each sample. Raw data was converted into the netCDF format using the Thermo File Converter. Spectra of media blanks were obtained before each experiment. Spectra from diﬀerent cells (N > 20) per treatment (species, strain, age) were collected from single cells that were selected visually. Datasets and the script employed are available upon request. Mass Spectrometry Proﬁling of Living Single Cells With LDI-MS g We developed a rapid and eﬀective method to obtain the chemical proﬁle of living single cells without prior preparation or application of matrix. As described in Figure 1, unicellular microalgae were sampled from their culture by pipetting cells with their culture medium on a GF/C ﬁlter. These were then directly analyzed with ambient pressure LDI-HR-MS in both polarities. Single cells in size above 10 µm were easily visualized on GF/C ﬁlter by a mounted AP-SMALDI camera. This enables visual targeting the laser directly at a single cell. We did not evaluate the minimum cell size, for the detection of MS-spectra which will be below this value. The laser irradiation completely disrupted the cell within a minute and desorbed/ionized molecules formed cell-speciﬁc chemical patterns (Figures 2, 3). After that process, no visually recognizable cellular material was left on the GF/C ﬁlter. The analysis in positive polarity yielded higher number of mass peaks than in negative mode (Figure 2). Raw spectra were processed with an in-house script based on the MALDIquant R package, reducing the background noise signal, removing the signals from the culture medium, and including a total ion current (TIC) normalization as recommended when working with MALDI-MS (Gibb and Strimmer, 2012; Emara et al., 2017). Since application of internal standard is not feasible Signiﬁcant Features Analysis Converted raw data was pre-processed in R (R Core Team, 2018), using the packages MALDIquant, and MALDIquantForeign (Gibb and Strimmer, 2012). Noise was estimated via median intensity and signals below a signal-to-noise ratio of 5 were removed from further processing. The peaks were aligned and those detected in the blank medium were excluded from the peak matrix. Peak intensities were TIC normalized and Pareto February 2019 \| Volume 10 \| Article 172 Frontiers in Plant Science \| www.frontiersin.org 3 LSC-MS Metabolomics of Microalgae Baumeister et al. FIGURE 1 \| Scheme for acquisition and data treatment of single cell MS proﬁling of unicellular algae. FIGURE 1 \| Scheme for acquisition and data treatment of single cell MS proﬁling of unicellular algae. FIGURE 1 \| Scheme for acquisition and data treatment of single cell MS proﬁling of unicellular algae. FIGURE 1 \| Scheme for acquisition and data treatment of single cell MS proﬁling of unicellular algae. FIGURE 2 \| LDI-MS proﬁling of a single cell of diatom C. granii acquired in positive (A) and (B) negative mode. as it would induce stress in the living cell, the relative quantiﬁcation based on TIC normalization was selected. To test the reproducibility of our MS proﬁling, the chemical signatures of single algal cells recovered from one culture of C. granii isolate Helg2016 and one culture of a diﬀerent strain of the same species (SCCAP-K1834) were acquired (Figure 3 shows Frontiers in Plant Science \| www.frontiersin.org 4 February 2019 \| Volume 10 \| Article 172 FIGURE 2 \| LDI-MS proﬁling of a single cell of diatom C. granii acquired in positive (A) and (B) negative mode. FIGURE 2 \| LDI-MS proﬁling of a single cell of diatom C. granii acquired in positive (A) and (B) negative mode. of single algal cells recovered from one culture of C. granii isolate Helg2016 and one culture of a diﬀerent strain of the same species (SCCAP-K1834) were acquired (Figure 3 shows of single algal cells recovered from one culture of C. granii isolate Helg2016 and one culture of a diﬀerent strain of the same species (SCCAP-K1834) were acquired (Figure 3 shows as it would induce stress in the living cell, the relative quantiﬁcation based on TIC normalization was selected. Signiﬁcant Features Analysis To test the reproducibility of our MS proﬁling, the chemical signatures February 2019 \| Volume 10 \| Article 172 Frontiers in Plant Science \| www.frontiersin.org LSC-MS Metabolomics of Microalgae Baumeister et al. FIGURE 3 \| Blank subtracted LSC-MS spectra of nine different consecutively analyzed cells of the diatom Coscinodiscus granii isolate Helg2016 recovered randomly from one culture at the same incubation time. FIGURE 3 \| Blank subtracted LSC-MS spectra of nine different consecutively analyzed cells of the diatom Coscinodiscus granii isolate Helg2016 recovered randomly from one culture at the same incubation time. exemplary processed LSC-MS spectra of nine cells). A total of 326 and 400 peaks were recovered, respectively, from the LSC- MS spectra. Data from C. granii isolate Helg2016 and strain SCCAP-K1834 were compared by a PCA and did not display any obvious pattern (Supplementary Figure S1). Furthermore, three prominent signals (Chl a, fucoxanthin, and β-carotene) were chosen from the dataset and tested if the respective intensities followed a normal distribution using quantile-quantile plots and the Shapiro-Wilk test (Figure 4 and Supplementary Table S1). A deviation from the normal distribution would indicate heterogeneity in the tested cell population. The signal intensities of the chosen photosynthetic pigments obtained from isolate Helg2016 were normally distributed, but not from strain SCCAP-K1834. Cells in cultures of the strain SCCAP-K1834 were very heterogeneous in size (Supplementary Figure S2), which may explain the observed variability of photosynthetic pigments. Indeed, the size of the cells substantially inﬂuences the pigment content in Coscinodiscus granii (Yan et al., 2018). This result indicates that our LSC-MS approach may be also able to address the single cell heterogeneity within a population. exemplary processed LSC-MS spectra of nine cells). A total of 326 and 400 peaks were recovered, respectively, from the LSC- MS spectra. Data from C. granii isolate Helg2016 and strain SCCAP-K1834 were compared by a PCA and did not display any obvious pattern (Supplementary Figure S1). Furthermore, three prominent signals (Chl a, fucoxanthin, and β-carotene) were chosen from the dataset and tested if the respective intensities followed a normal distribution using quantile-quantile plots and the Shapiro-Wilk test (Figure 4 and Supplementary Table S1). A deviation from the normal distribution would indicate heterogeneity in the tested cell population. The signal intensities of the chosen photosynthetic pigments obtained from isolate Helg2016 were normally distributed, but not from strain SCCAP-K1834. Frontiers in Plant Science \| www.frontiersin.org Signiﬁcant Features Analysis and its identity was further veriﬁed in cells extract of C. granii with LC-MS as described in Spielmeyer and Pohnert (2010) (Supplementary Figure S3). Interestingly, fragments of Chl a were observed (m/z 614, 452, 467, 481), suggesting in-source fragmentation. This in-source fragmentation could not be avoided as no signal could be detected anymore when reducing the laser energy. In particular photosynthetic algal pigments that are commonly used for algal taxonomic characterization could be identiﬁed (Table 1). It is hence assumed that signals in the m/z 200–500 range may arise from fragments or diﬀerent adducts. Common techniques employ liquid- or gas-chromatography and Fourier-transform infrared spectroscopy proﬁling of pigments (Biller and Ross, 2014; Meng et al., 2014; Agirbas et al., 2015), but our LSC-MS approach oﬀers direct analysis and higher resolution of the pigment composition at the single cell level. The multifunctional zwitterionic metabolite DMSP (m/z 135) was also detected Signiﬁcant Features Analysis Cells in cultures of the strain SCCAP-K1834 were very heterogeneous in size (Supplementary Figure S2), which may explain the observed variability of photosynthetic pigments. Indeed, the size of the cells substantially inﬂuences the pigment content in Coscinodiscus granii (Yan et al., 2018). This result indicates that our LSC-MS approach may be also able to address the single cell heterogeneity within a population. HR-MS allows assignment of certain heteroatoms, e.g., N, O, S, and halogens due to characteristic mass defects and the characteristic relative intensity of isotope peaks. Only at high molecular weight isotopic deconvolution is not straightforward, as illustrated in the [M+H]+ of Chl a (m/z 893.5371) which comprises the isotopolog containing 25Mg 13C, 15N. To simplify readability of this text, nominal mass-charge ratios will be used from here on (compare tables for accurate mass-charge ratios). MS2 analysis could not be achieved from signals from single cells due to insuﬃcient ion intensities. However, the MS2 fragmentation of commercially available analytes that were pipetted on empty frustules of C. granii could be achieved (Jaschinski et al., 2014) to conﬁrm the identity of cellular and commercial metabolites. Furthermore, analysis of extracts in LC- MS and LC-MS-MS can help in the identiﬁcation of detected metabolites. With this approach, main constituents of the algal cells within a broad mass range were identiﬁed. The LSC-MS signals in positive polarity for photosynthetic pigments, including Chl a, fucoxanthin and β-carotene, dominate the chemical proﬁle of the unicellular algal cells. Frontiers in Plant Science \| www.frontiersin.org February 2019 \| Volume 10 \| Article 172 Frontiers in Plant Science \| www.frontiersin.org 5 LSC-MS Metabolomics of Microalgae Baumeister et al. FIGURE 4 \| Quantile-Quantile plots to assess if observed peak intensities from signals obtained from several analyzed microalgal cells follow a normal distribution. Selected signals were chlorophyll a (m/z 892), fucoxanthin (m/z 658), and β-carotene (m/z 536). Diatom cells from the same culture at day 1 early growth of Coscinodiscus granii (A) isolate Helg2016 (N = 20) and (B) strain SCCAP-K1834 (N = 25). FIGURE 4 \| Quantile-Quantile plots to assess if observed peak intensities from signals obtained from several analyzed microalgal cells follow a normal distribution. Selected signals were chlorophyll a (m/z 892), fucoxanthin (m/z 658), and β-carotene (m/z 536). Diatom cells from the same culture at day 1 early growth of Coscinodiscus granii (A) isolate Helg2016 (N = 20) and (B) strain SCCAP-K1834 (N = 25). Frontiers in Plant Science \| www.frontiersin.org Strain- and Age-Speciﬁc Proﬁling of Diatoms C. granii Unraveled by Matrix-Free LDI-MS (B) PLS-DA score plot of LSC-MS proﬁles of H. pluvialis cells in early and late growth stage. The ellipses represented the 95% conﬁdence region estimated by the statistical calculation. (C) Comparison of pigments content (represented as TIC normalized intensity) in selected group of LSC-MS spectra of red, green and overlap cells. FIGURE 5 \| Statistical comparison of LSC-MS proﬁles of Coscinodiscus granii strain SCCAP-K1834 and isolate Helg2016 recovered from early (Day 1) and late (Day 15) growth stages. (A) PCA score plot (B) PLS-DA score plots displaying dissimilarities between LSC-MS proﬁles of young (day 1 early exponential growth phase) and old (day 15 early stationary growth phase) cells of C. granii. The LSC-MS proﬁles of both strains are included in the analysis. The ellipses represented the 95% conﬁdence region estimated by the statistical calculation. FIGURE 5 \| Statistical comparison of LSC-MS proﬁles of Coscinodiscus granii strain SCCAP-K1834 and isolate Helg2016 recovered from early (Day 1) and late (Day 15) growth stages. (A) PCA score plot (B) PLS-DA score plots displaying dissimilarities between LSC-MS proﬁles of young (day 1 early exponential growth phase) and old (day 15 early stationary growth phase) cells of C. granii. The LSC-MS proﬁles of both strains are included in the analysis. The ellipses represented the 95% conﬁdence region estimated by the statistical calculation. FIGURE 6 \| (A) Microscopy observations of a palmella cell (green stage), immature and mature haematocyst (red stage). Scale bar 10 µm. (B) PLS-DA score plot of LSC-MS proﬁles of H. pluvialis cells in early and late growth stage. The ellipses represented the 95% conﬁdence region estimated by the statistical calculation. (C) Comparison of pigments content (represented as TIC normalized intensity) in selected group of LSC-MS spectra of red, green and overlap cells. FIGURE 6 \| (A) Microscopy observations of a palmella cell (green stage), immature and mature haematocyst (red stage). Scale bar 10 µm. (B) PLS-DA score plot of LSC-MS proﬁles of H. pluvialis cells in early and late growth stage. The ellipses represented the 95% conﬁdence region estimated by the statistical calculation. (C) Comparison of pigments content (represented as TIC normalized intensity) in selected group of LSC-MS spectra of red, green and overlap cells. fucoxanthin (m/z 658) and Chl a (m/z 892) were absent from LSC-MS proﬁling of old Coscinodiscus granii in stationary phase, but carotene (m/z 536) and alloxanthin (m/z 566) were still detected. Frontiers in Plant Science \| www.frontiersin.org Strain- and Age-Speciﬁc Proﬁling of Diatoms C. granii Unraveled by Matrix-Free LDI-MS Monoclonal C. granii (strain SCCAP-K1834) and the non-clonal isolate (Helg2016) sampled during a bloom in August 2016 in German coastal waters were grown in triplicate in their respective medium over 15 days. C. granii isolate (Helg2016) reached a cell count of 200 cells × mL−1 in six days while three times higher cell density was obtained for the strain SCCAP- K1834 (Supplementary Figure S2). Single cells were isolated and proﬁled with LSC-MS to determine metabolic diﬀerences between strains and to compare cells in logarithmic (day 1) and stationary (day 15) growth phases. The peak matrix contained 595 peaks which were found at least in 50% of the samples after background subtraction. An unbiased PCA was performed on processed LSC-MS proﬁles and explained 48.2% of total variance (Figure 5A). The strain clearly explained most of the variance observed (PC1 35%) whereas the cell age (PC2) accounted for 13.2% of variance. To further assess our ability to distinguish cell age with the LSC-MS method, a PLS-DA was carried out on the LSC-MS proﬁles of young and old C. granii cells sampled at early exponential and early stationary growth phases (Figure 5B). The cumulative variance was 45.3%, while most of the variance was explained by component 1 (31.5%). The model ﬁtness was TABLE 1 \| Summary of measured compounds and detected signals with LDI-MS. Analytes Monoisotopic ion molecular mass (u) Measured ion (m/z) MS1 (positive polarity) Measured ion (m/z) MS2 (positive polarity) Precursor Fragment Chlorophyll a 892.5342 892.5352 892.5357 614.2366, 481.1866 Fucoxanthin 658.4227 658.4226 658.4201 515.3516, 473.3516 β-carotene 536.4377 536.4370 Astaxanthin 596.3860 596.3856 597.1625 582.1386 DMSP 135.0474 135.0475 TABLE 1 \| Summary of measured compounds and detected signals with LDI-MS. February 2019 \| Volume 10 \| Article 172 6 LSC-MS Metabolomics of Microalgae Baumeister et al. FIGURE 5 \| Statistical comparison of LSC-MS proﬁles of Coscinodiscus granii strain SCCAP-K1834 and isolate Helg2016 recovered from early (Day 1) and late (Day 15) growth stages. (A) PCA score plot (B) PLS-DA score plots displaying dissimilarities between LSC-MS proﬁles of young (day 1 early exponential growth phase) and old (day 15 early stationary growth phase) cells of C. granii. The LSC-MS proﬁles of both strains are included in the analysis. The ellipses represented the 95% conﬁdence region estimated by the statistical calculation. FIGURE 6 \| (A) Microscopy observations of a palmella cell (green stage), immature and mature haematocyst (red stage). Scale bar 10 µm. LSC-MS Proﬁling of H. pluvialis Response to Nutrient Depletion the taxonomic diversity and health status of phytoplankton communities in aquatic ecosystems. The commercially important chlorophyceae Haematoccocus pluvialis strain SAG 192.80 was selected as model to study the eﬀect of nutrient stress on the metabolome at the single-cell level. The alga evolves from a motile, small, green cell to a large, deep red colored cell upon nutrient depletion (Shah et al., 2016). The palmella cells (A) belong to the green vegetative phase. Later red hematocysts are formed (Figure 6A). Cultures of H. pluvialis were grown until nutrient exhaustion for 15 days and random cells in early and late cultures (N > 20) were proﬁled with LSC-MS. Partial Least Square Discriminant Analysis (PLS- DA) was carried out to compare the LSC-MS proﬁles of early and late stage cultures (Figure 6B). A total explained variance of 57.4% was observed and the features in the component 1 (phenotype early and late) explained 43.3% of the variance. Algal populations of H. pluvialis are usually asynchronous with cells at various encystment stages, and hence several phenotypes can be simultaneously observed in one culture. This contributes to the non-complete separation of the groups in the PLS- DA (Figure 6B). The model ﬁtness was assessed by the 1000 permutation test (p-value <0.001). Signiﬁcant features were associated with either phenotype “early” or “late.” The early palmella cells are characterized by the presence of Chl a (m/z 892) and other masses (m/z 184, 305, 335, 526, 555, 568, 481, 559, 690, 423) for which the associated metabolites were not further identiﬁed. The late cultures are dominated by hematocyst cells and were distinguished by their xanthophyll content (m/z 550, 573) and two metabolites tentatively assigned to the class of lipids (m/z 603, 459). H. pluvialis is known to produce the antioxidant carotenoid astaxanthin through conversion of carotene in multiple steps, generating intermediates such as pheonicaxanthin, cryptoxanthin or adonixanthin (Lemoine and Schoefs, 2010; Shah et al., 2016). In accordance with the observed pigmentation astaxanthin (m/z 596) is up-regulated in the “red” stage (Figure 6C). The content Astaxanthin and Chl a in red and green cells were found signiﬁcantly diﬀerent by a Kruskal-Wallis one-way ANOVA (Atx: H = 20.545, Chl a: H = 22.516, p-value <0.001). It is remarkable that only the analysis of 20 cells is suﬃcient for a statistically sound physiological characterization. CONCLUSION We are now able to proﬁle the metabolome of microalgae (diatoms and chlorophyceae) and to discriminate the physiological status at the single cell level. This cellular proﬁling of unicellular bloom-forming algae was achieved by single-cell metabolomics using a matrix-free live-single cell LDI-SC-HR-MS approach. Sample preparation ensured that analyzed cells were in native state just until the data collection. The statistical pipeline on MS data permits to discriminate cells in diﬀerent degree of nutrient-depletion, age and taxonomy. Our method is a straightforward approach to analyze live single cells using ambient ionization mass spectrometry conditions and opens up new avenues of research. We are now able to proﬁle the metabolome of microalgae (diatoms and chlorophyceae) and to discriminate the physiological status at the single cell level. This cellular proﬁling of unicellular bloom-forming algae was achieved by single-cell metabolomics using a matrix-free live-single cell LDI-SC-HR-MS approach. Sample preparation ensured that analyzed cells were in native state just until the data collection. The statistical pipeline on MS data permits to discriminate cells in diﬀerent degree of nutrient-depletion, age and taxonomy. Our method is a straightforward approach to analyze live single cells using ambient ionization mass spectrometry conditions and opens up new avenues of research. ACKNOWLEDGMENTS We acknowledge Prof. Marco Thines (Biodiversity and Climate Research Centre, Frankfurt am Main) for sampling and identifying the wild isolate of Coscinodiscus granii Helg2016. FUNDING This work was supported by an MPG Fellowship awarded to GP and the DFG CRC 1127 “ChemBioSys”. Financial support from the Max Planck Society is highly acknowledged. AUTHOR CONTRIBUTIONS FK developed here presented LSC-MS method including sample support and cell handling before the LDI-HR-MS analysis. MV maintained the algae collection and designed the biological experiments. TB developed the data analysis pipeline. MV and TB analyzed the data and wrote the manuscript with the help of GP and AS. All authors conceived this study and approved the manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00172/ full#supplementary-material Together with an automatized single cell sorting, this presented LSC-MS method is a powerful tool to address LSC-MS Proﬁling of H. pluvialis Response to Nutrient Depletion The loadings plot for the PLS-DA reveals Chl a (m/z 892 and related fragments m/z 467, 614, 481) as discriminating the early phase (Supplementary Figure S5). This was conﬁrmed by paired t-test (Supplementary Figure S6). The ability to identify high astaxanthin producing single H. pluvialis cells might in the future enable an analytics-supported breeding of commercially relevant pigment producers and reduce the necessity for genetic engineering (Steinbrenner and Sandmann, 2006). Strain- and Age-Speciﬁc Proﬁling of Diatoms C. granii Unraveled by Matrix-Free LDI-MS Pigment reduction has been previously observed in diatoms under nitrogen deﬁciency (Thomas and Dodson, 1972; Alipanah et al., 2015) consistent with the phenotype observed here in C. granii when reaching the stationary phase. It is also known that some diatoms can increase their production of defensive polyunsaturated aldehydes in aged and nutrient-depleted cultures (Alipanah et al., 2015; Sayanova et al., 2017). These metabolites are produced from lipids that are initially lysed to release free fatty acids for further processing. In agreement, some metabolites were putatively assigned to a neutral glycosphingolipid (m/z 732.5613) and glycerophosphoserines (m/z 826.5358, 791.4664). conﬁrmed by a 1000 permutation test (p-value <0.001) and the predictive ability to assign a “young” or “old” age status was assessed and visualized by cross-validation test (Supplementary Figure S4). An in silico classiﬁcation test was performed (random forest) to conﬁrm the reliability of assigning one data set from a single cell to the right class with out-of-bag errors (data not shown). These tests demonstrated that there was a very low percentage of overall error of 4.5% in assigning a group (old, young, strain) to a cell by its sole LSC-MS proﬁle. To identify metabolites related to cell aging or strain-speciﬁc markers, the variables of importance in projection (VIP score) from the PLS-DA analysis were calculated, leading to features that were tentatively assigned by matching HR-MS spectra. 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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. Meng, Y., Yao, C., Xue, S., and Yang, H. (2014). Application of fourier transform infrared (FT-IR) spectroscopy in determination of microalgal compositions. Bioresour. Technol. 151, 347–354. doi: 10.1016/j.biortech.2013.10.064 Pohnert, G. (2000). Wound-activated chemical defense in unicellular planktonic algae. Angew. Chem. Int. Ed. 39, 4352–4354. doi: 10.1002/1521-3773(20001201) 39:23<4352::AID-ANIE4352>3.0.CO;2-U February 2019 \| Volume 10 \| Article 172 Frontiers in Plant Science \| www.frontiersin.org 9
https://openalex.org/W2179235000	https://zenodo.org/records/899577/files/CheckList_article_17709.pdf	English	null	Reptilia, Squamata, Teiidae, Tupinambis longilineus: distribution extension	Check list	2,008	cc-by	1,123	Check List 4(3): 240–243, 2008. ISSN: 1809-127X Check List 4(3): 240–243, 2008. ISSN: 1809-127X Reptilia, Squamata, Teiidae, Tupinambis longilineus: Distribution extension. Ana Caroline de Lima Flávio Eduardo Pimenta Universidade Federal do Rio de Janeiro, Museu Nacional, Departamento de Vertebrados. Quinta da Boa Vista s/n. CEP 20940-040. Rio de Janeiro, RJ, Brazil. E-mail: analima02@hotmail.com of ventrals across midbody (20 in T. longilineus, 30-40 in T. merianae, 46-50 in T. rufescens, and 46 in T. duseni). In all these characteristics, T. longilineus is closer to T. teguixin. These two species differ from T. merianae in having distinctly large supra-temporals, lower number of pores and gular region spotless or with small, black or grey spots. Tupinambis longilineus differs from T. teguixin by its slender body and limbs, with body compressed and rectangular in cross section (cylindrical in T. teguixin), temporal scales comparatively larger, higher number of femoral pores; slightly lower numbers of dorsal scales around midbody and ventrals across midbody, and lower number of subdigital lamellae, especially under fourth finger (Ávila-Pires 1995). The genus Tupinambis Daudin, 1803 occurs in the most part of South America, from east of Andes to Uruguay and northern Argentina (Ávila-Pires 1995). It contains seven species: T. teguixin (Linnaeus, 1758), T. merianae (Duméril & Bibron, 1839), T. rufescens (Günther, 1871), T. duseni Lönnberg, 1896, T. longilineus Ávila- Pires, 1995, T. quadrilineatus Manzani & Abe, 1997, and T. palustris Manzani and Abe, 2002 (SBH 2007). Tupinambis longilineus was described by Ávila-Pires (1995), at his catalogue of lizards from the Brazilian Amazonia, based on a specimen from Alvorada d'Oeste, state of Rondônia (11°25' S, 62°22' W). According to Pianka and Vitt (2003) this species also occurs in Rio Ituxi, near Lábrea, in the state of Amazonas (7º18' S, 64º51' W). We present a new locality of occurrence to Tupinambis longilineus, at the municipality of Juruti (2º30' S, 56º10' W), state of Pará, Brazil (Figure 1). This represent the first record for the state of Pará and an extension of 1,100 airline km from the nearest locality with record of the species (Rio Ituxi; Pianka and Vitt 2003), and 1,200 airline km from the type locality. Tupinambis longilineus differs from T. merianae, T. rufescens, and from T. duseni by the presence of only one loreal scale (two loreals in the latter three species); supraciliaries in direct contact with supraoculars (second to fourth separated from the supraciliaries by a row of scales); presence of a supernumerary antegular fold; and smaller number Figure 1. Distribution of Tupinambis longilineus in Brazilian Amazonia. Figure 1. Reptilia, Squamata, Teiidae, Tupinambis longilineus: Distribution extension. Distribution of Tupinambis longilineus in Brazilian Amazonia. Figure 1. Distribution of Tupinambis longilineus in Brazilian Amazonia. 240 Check List 4(3): 240–243, 2008. ISSN: 1809-127X NOTES ON GEOGRAPHIC DISTRIBUTION e 2. Male adult of Tupinambis longilineus collected at Juruti, Pará; A, head; B, body. Photo by F. E. Pim Figure 2. Male adult of Tupinambis longilineus collected at Juruti, Pará; A, head; B, body. Photo by F. E. Pimenta. 241 Check List 4(3): 240–243, 2008. ISSN: 1809-127X NOTES ON GEOGRAPHIC DISTRIBUTION A male and a female of T. longilineus were collected and deposited in the herpetological collection of the Museu Paraense Emílio Goeldi, Pará, Brazil. The female was collected on 31 October 2006 and the male (Figure 2) on 19 May 2007, during a program of faunal rescue as consequence of deforestation activities in the future industrial area of an ALCOA mining project (Figure 3). The area is characterized by regenerated vegetation in medium to advanced stages and by forest with signs of exploration. The present record is an indication that the species occurs throughout the area between Tapajós and Purus rivers. The lack of more records of this lizard, that reaches at least 20 cm snout-vent length, shows how scarce is the knowledge on the herpetofauna of this large area. With such patchy data, nothing can be said concerning the conser- vation status of populations of this species. We hope that examples like this one concerning T. longilineus motivate further faunal surveys in this region, in order to propitiate more efficient conservation actions. The present record is an indication that the species occurs throughout the area between Tapajós and Purus rivers. The lack of more records of this lizard, that reaches at least 20 cm snout-vent length, shows how scarce is the knowledge on the herpetofauna of this large area. With such patchy data, nothing can be said concerning the conser- vation status of populations of this species. We hope that examples like this one concerning T. longilineus motivate further faunal surveys in this region, in order to propitiate more efficient conservation actions. Figure 3. Study area showing one deforestation site to mining project at Juruti, Pará. Photo by Christian Knepper. Figure 3. Study area showing one deforestation site to mining project at Juruti, Pará. Photo by Christian Knepper. Acknowledgements We are grateful to ALCOA Aluminum and CNEC Engenharia for financial support, to Teresa Cristina de Ávila Pires and Marinus S. Hoogmoed (both MPEG) for the species identity confirmation, to Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA) for the license (034-2006), and to Ana Lúcia da Costa Prudente, Paulo Sérgio Bernarde, Teresa Cristina de Ávila Pires, and an anonymous referee by kindly reviewing this article. Acknowledgements g We are grateful to ALCOA Aluminum and CNEC Engenharia for financial support, to Teresa Cristina de Ávila Pires and Marinus S. Hoogmoed (both MPEG) for the species identity confirmation, to Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA) for the license (034-2006), and to Ana Lúcia da Costa Prudente, Paulo Sérgio Bernarde, Teresa Cristina de Ávila Pires, and an anonymous referee by kindly reviewing this article. 242 Check List 4(3): 240–243, 2008. ISSN: 1809-127X SBH, 2007. Lista de espécies de répteis do Brasil: an online reference. Electronic Database accessible at: http://www2.sbherpetologia.org.br/checklist/repteis.h tm. Sociedade Brasileira de Herpetologia, Brazil. Captured on 04 May 2007. Literature cited Á Pianka, E. R. and L. J. Vitt. 2003. Lizards: Windows to the evolution of diversity. Berkeley: University of California Press. 333 p. Ávila-Pires, T. C. 1995. Lizards of Brazilian Amazonia (Reptilia: Squamata). Zoologische Verhandelingen Leiden 299: 1-706. SBH, 2007. Lista de espécies de répteis do Brasil: an online reference. Electronic Database accessible at: http://www2.sbherpetologia.org.br/checklist/repteis.h tm. Sociedade Brasileira de Herpetologia, Brazil. Captured on 04 May 2007. Manzani, P. R. and A. S. Abe. 1997. A new species of Tupinambis Daudin, 1802 (Squamata, Teiidae) from central Brazil. Boletim do Museu Nacional, Série Zoologia, 382: 1-10. Manzani, P. R. and A. S. Abe. 2002. A new species of Tupinambis (Daudin, 1803) from southeastern Brazil (Squamata, Teiidae). Arquivos do Museu Nacional 60(4): 295-302. Received September 2007 Accepted July 2008 Published online August 2008 Received September 2007 Accepted July 2008 Published online August 2008 243
https://openalex.org/W3027355984	https://www.research-collection.ethz.ch/bitstream/20.500.11850/492418/2/chemistry-02-00030.pdf	English	null	On the Importance of the Thiazole Nitrogen in Epothilones: Semisynthesis and Microtubule-Binding Affinity of Deaza-Epothilone C	Chemistry	2,020	cc-by	11,439	ETH Library Author(s): ( ) Edenharter, Adriana; Ryckewaert, Lucie; Cintulová, Daniela; Estévez-Gallego, Juan; Díaz, José F.; Altmann, Karl-Heinz Received: 18 April 2020; Accepted: 21 May 2020; Published: 23 May 2020 p p y y Ab t t D hil C hi h i hi h Abstract: Deaza-epothilone C, which incorporates a thiophene moiety in place of the thiazole heterocycle in the natural epothilone side chain, has been prepared by semisynthesis from epothilone p , p p y p heterocycle in the natural epothilone side chain, has been prepared by semisynthesis from epothilone A, in order to assess the contribution of the thiazole nitrogen to microtubule binding. y p p p y y p A, in order to assess the contribution of the thiazole nitrogen to microtubule binding. The synthesis was based on the esteriﬁcation of a known epothilone A-derived carboxylic acid fragment and a fully synthetic alcohol building block incorporating the modiﬁed side chain segment and subsequent ring-closure by ring-closing oleﬁn metathesis. The latter proceeded with unfavorable selectivity and in low yield. Distinct diﬀerences in chemical behavior were unveiled between the thiophene-derived advanced intermediates and what has been reported for the corresponding thiazole-based congeners. Compared to natural epothilone C, the free energy of binding of deaza-epothilone C to microtubules was reduced by ca. 1 kcal/mol or less, thus indicating a distinct but non-decisive role of the thiazole nitrogen in the interaction of epothilones with the tubulin/microtubule system. In contrast to natural epothilone C, deaza-epothilone C was devoid of antiproliferative activity in vitro up to a concentration of 10 µM, presumably due to an insuﬃcient stability in the cell culture medium. p , g g The synthesis was based on the esterification of a known epothilone A-derived carboxylic acid fragment and a fully synthetic alcohol building block incorporating the modified side chain segment and subsequent ring-closure by ring-closing olefin metathesis. The latter proceeded with unfavorable selectivity and in low yield. Distinct differences in chemical behavior were unveiled between the thiophene-derived advanced intermediates and what has been reported for the corresponding thiazole-based congeners. Compared to natural epothilone C, the free energy of binding of deaza-epothilone C to microtubules was reduced by ca. 1 kcal/mol or less, thus indicating a distinct but non-decisive role of the thiazole nitrogen in the interaction of epothilones with the tubulin/microtubule system. In contrast to natural epothilone C, deaza-epothilone C was devoid of antiproliferative activity in vitro up to a concentration of 10 μM, presumably due to an insufficient stability in the cell culture medium. Keywords: binding aﬃnity; epothilones; deaza-epothilone; microtubules; structure-activity relationship; thiophene Keywords: binding affinity; epothilones; deaza-epothilone; microtubules; structure-activity relationship; thiophene. Article On the Importance of the Thiazole Nitrogen in Epothilones: Semisynthesis and Microtubule-Binding Affinity of Deaza-Epothilone C On the Importance of the Thiazole Nitrogen in Epothilones: Semisynthesis and Microtubule- Binding Affinity of Deaza-Epothilone C Adriana Edenharter 1, Lucie Ryckewaert 1, Daniela Cintulová 1, Juan Estévez-Gallego 2, José Fernando Díaz 2 and Karl-Heinz Altmann 1,* Adriana Edenharter , Lucie Ryckewaert , Daniela Cintulová , Juan Estévez Gallego , José Fernando Díaz 2 and Karl-Heinz Altmann 1,* 1 ETH Zü i h D t t f Ch i t d A li d Bi i I tit t f Ph ti l S i Adriana Edenharter 1, Lucie Ryckewaert 1, Daniela Cintulová 1, Juan Estévez-Gallego 2, José Fernando Díaz 2 and Karl-Heinz Altmann 1,* A ia a E e a e , ucie yc e ae , a ie a i u o á , Jua Es é e Ga ego , José Fernando Díaz 2 and Karl-Heinz Altmann 1,* 1 ETH Zü i h D f Ch i d A li d Bi i I i f Ph i l S i 1 ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, CH-8093 Zürich, Switzerland; adriana.edenharter@gmx.com (A.E.); lucie.ryckewaert@yahoo.com (L.R.); daniela.cintulova@tuwien.ac.at (D.C.) 1 ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, CH 8093 Zürich, Switzerland; adriana.edenharter@gmx.com (A.E.); lucie.ryckewaert@yahoo.com (L.R.); daniela.cintulova@tuwien.ac.at (D.C.) 2 Centro de Investigaciones Biológicas Margarita Salas Consejo Superior de Investigaciones Científicas 1 ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, CH-8093 Zürich, Switzerland; adriana.edenharter@gmx.com (A.E.); lucie.ryckewaert@yahoo.com (L.R.); daniela.cintulova@tuwien.ac.at (D.C.) ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, CH 8093 Zürich, Switzerland; adriana.edenharter@gmx.com (A.E.); lucie.ryckewaert@yahoo.com (L.R.); daniela.cintulova@tuwien.ac.at (D.C.) 2 Centro de Investigaciones Biológicas Margarita Salas Consejo Superior de Investigaciones Científicas 1 ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, CH-8093 Zürich, Switzerland; adriana.edenharter@gmx.com (A.E.); lucie.ryckewaert@yahoo.com (L.R daniela.cintulova@tuwien.ac.at (D.C.) ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, C 8093 Zürich, Switzerland; adriana.edenharter@gmx.com (A.E.); lucie.ryckewaert@yahoo.com (L.R.); daniela.cintulova@tuwien.ac.at (D.C.) 2 Centro de Investigaciones Biológicas Margarita Salas Consejo Superior de Investigaciones Científicas ( ) 2 Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Cientíﬁcas, 28040 Madrid, Spain; jeg@cib.csic.es (J.E.-G.); fer@cib.csic.es (J.F.D.) 2 Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; jeg@cib.csic.es (J.E.-G.); fer@cib.csic.es (J.F.D.) * Correspondence: karl heinz altmann@pharma ethz ch 2 Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Cientíﬁcas, 28040 Madrid, Spain; jeg@cib.csic.es (J.E.-G.); fer@cib.csic.es (J.F.D.) Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; jeg@cib.csic.es (J.E.-G.); fer@cib.csic.es (J.F.D.) * Correspondence: karl heinz altmann@pharma ethz ch * Correspondence: karl-heinz.altmann@pharma.ethz.ch * Correspondence: karl-heinz.altmann@pharma.ethz.ch Originally published in: This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Article Article Chemistry 2020, 2, 30; doi:10.3390/chemistry2020030 1. Introduction 1. Introduction Interest in epothilones then surged in 1995, when Bollag et al. demonstrated that Epo A and B were new microtubule-stabilizing agents and, thus, inhibited cell proliferation by a taxol- like mechanism [5]. At the time of discovery of their mode of action, Epo A and B were the only non- taxane compound class known to stabilize microtubules, but in contrast to taxol they retained almost full activity (i.e., IC50 values in the nM range) against multidrug-resistant cancer cells expressing the P-glycoprotein efflux pump or harboring tubulin mutations [6,7]. Numerous total syntheses of natural epothilones have been reported in the literature, and these Numerous total syntheses of natural epothilones have been reported in the literature, and these eﬀorts have been reviewed extensively [8–13]. Based on the chemistry developed in the context of the total synthesis work, hundreds of synthetic analogues of epothilones have been prepared for structure-activity relationship (SAR) studies and with the objective to deliver compounds with an improved overall pharmacological proﬁle [14,15]. In addition, semisynthetic approaches have been pursued to explore the epothilone-like structural space, in particular by Höﬂe and co-workers. (This work is summarized in ref. [16]). The most important semisynthetic epothilone derivative is the Epo B lactam ixabepilone, which is approved by the FDA (under the tradename Ixempra®) for the treatment of metastatic or locally advanced breast cancer [17]. At least eight additional epothilone-type agents have been advanced to clinical trials in oncology, including the natural product Epo A [14,15], and Epo D has also been investigated in Phase I clinical trials for Alzheimer’s disease [18]. The development of most of these compounds has been discontinued (including the development of Epo D for Alzheimer’s disease), but an analog termed utidelone (or UTD1) is currently being studied in Phase III clinical trials against breast cancer (in combination with capecitabine) [19]. y p p efforts have been reviewed extensively [8–13]. Based on the chemistry developed in the context of the total synthesis work, hundreds of synthetic analogues of epothilones have been prepared for structure-activity relationship (SAR) studies and with the objective to deliver compounds with an improved overall pharmacological profile [14,15]. In addition, semisynthetic approaches have been pursued to explore the epothilone-like structural space, in particular by Höfle and co-workers. (This work is summarized in ref. [16]). 1. Introduction 1. Introduction Epothilones A and B (Epo A and B) (Figure 1) are 16-membered, polyketide-derived macrolides that were discovered in 1987 by Reichenbach et al. in the context of a screening for new antifungal agents from the soil-dwelling myxobacterium Sorangium cellulosum Soce 90 [1,2]. A number of closely related, but less prevalent polyketides, like epothilones C (Epo C) and D (Epo D) were later identiﬁed in larger scale fermentations of S. cellulosum [3]. Epothilones A and B (Epo A and B) (Figure 1) are 16-membered, polyketide-derived macrolides that were discovered in 1987 by Reichenbach et al. in the context of a screening for new antifungal agents from the soil-dwelling myxobacterium Sorangium cellulosum Soce 90 [1,2]. A number of closely related, but less prevalent polyketides, like epothilones C (Epo C) and D (Epo D) were later identified in larger scale fermentations of S. cellulosum [3]. rstpage Lastpage; doi: www mdpi com Figure 1. Molecular structures of natural epothilones A–D (Epo A–D). Figure 1. Molecular structures of natural epothilones A–D (Epo A–D). L t d i d i Figure 1. Molecular structures of natural epothilones A–D (Epo A–D). Figure 1. Molecular structures of natural epothilones A–D (Epo A–D). Chemistry 2020, 2, 30; doi:10.3390/chemistry2020030 www.mdpi.com/journal/chemistry Chemistry 2020, 2, 30 Chemistry 2020, 2, x Chemistry 2020, 2, 30 Chemistry 2020, 2, x 500 of 5092 500 of 5092 500 of 5092 Epo A and B were subsequently found to be highly cytotoxic in the 60-cell line panel of the National Cancer Institute, although the mechanistic underpinnings of this eﬀect remained unknown at the time [4]. Interest in epothilones then surged in 1995, when Bollag et al. demonstrated that Epo A and B were new microtubule-stabilizing agents and, thus, inhibited cell proliferation by a taxol-like mechanism [5]. At the time of discovery of their mode of action, Epo A and B were the only non-taxane compound class known to stabilize microtubules, but in contrast to taxol they retained almost full activity (i.e., IC50 values in the nM range) against multidrug-resistant cancer cells expressing the P-glycoprotein eﬄux pump or harboring tubulin mutations [6,7]. Epo A a d we e subseque t y ou d to be ig y cytoto ic i t e 60 ce i e pa e o t e National Cancer Institute, although the mechanistic underpinnings of this effect remained unknown at the time [4]. 1. Introduction 1. Introduction The most important semisynthetic epothilone derivative is the Epo B lactam ixabepilone, which is approved by the FDA (under the tradename Ixempra®) for the treatment of metastatic or locally advanced breast cancer [17]. At least eight additional epothilone- type agents have been advanced to clinical trials in oncology, including the natural product Epo A [14,15], and Epo D has also been investigated in Phase I clinical trials for Alzheimer’s disease [18]. The development of most of these compounds has been discontinued (including the development of Epo D for Alzheimer’s disease), but an analog termed utidelone (or UTD1) is currently being studied in Phase III clinical trials against breast cancer (in combination with capecitabine) [19]. While numerous side chain-modified epothilone analogs have been investigated as part of While numerous side chain-modiﬁed epothilone analogs have been investigated as part of comprehensive SAR studies [15,16], a speciﬁc question that has been addressed only indirectly relates to the importance of the N-atom in the thiazole ring for microtubule binding and cellular potency. Thus, Nicolaou and co-workers have shown that among the three possible pyridyl-Epo B variants (Figure 2), the isomer with the N-atom in the position ortho to the vinyl linker between the pyridine ring and the macrolactone core (i.e., o-Pyr-Epo B) is the most potent with regard to both the promotion of tubulin polymerization and the inhibition of cancer cell growth in vitro [20]. At the same time, the meta and para isomers (m-Pyr-Epo B and p-Pyr-Epo B, respectively) still retained a signiﬁcant tubulin-polymerizing capacity, which reﬂects the ability of a compound to stabilize microtubules. p g g p comprehensive SAR studies [15,16], a specific question that has been addressed only indirectly relates to the importance of the N-atom in the thiazole ring for microtubule binding and cellular potency. Thus, Nicolaou and co-workers have shown that among the three possible pyridyl-Epo B variants (Figure 2), the isomer with the N-atom in the position ortho to the vinyl linker between the pyridine ring and the macrolactone core (i.e., o-Pyr-Epo B) is the most potent with regard to both the promotion of tubulin polymerization and the inhibition of cancer cell growth in vitro [20]. At the same time, the meta and para isomers (m-Pyr-Epo B and p-Pyr-Epo B, respectively) still retained a significant tubulin-polymerizing capacity, which reflects the ability of a compound to stabilize microtubules. Figure 2. 1. Introduction 1. Introduction In our own work, we have demonstrated that the 501 of 509 501 of 509 501 of 509 Chemistry 2020, 2, 30 microtubule-binding aﬃnities of quinoline-based epothilone analogs (Figure 3) [22] that incorporate the side chain N-atom in the “natural” position (i.e., m-Quin-Epo B and m-Quin-Epo D) are ca. one order of magnitude higher than those of the corresponding regioisomers p-Quin-Epo B and p-Quin-Epo D, respectively (Kb’s of 92 × 107 and 88 × 107 for m-Quin-Epo B and m-Quin-Epo D, respectively, vs. 6.9 × 107 and 6.1 × 107 for p-Quin-Epo B and p-Quin-Epo D). Quite intriguingly, and for reasons not understood, the diﬀerence in the microtubule-binding aﬃnity between m-Quin-Epo D and p-Quin-Epo D translates into a corresponding diﬀerence in the cellular activity, while the epoxides m-Quin-Epo B and p-Quin-Epo B are virtually equipotent (and highly active). 3) [22] that incorporate the side chain N-atom in the “natural” position (i.e., m-Quin-Epo B and m- Quin-Epo D) are ca. one order of magnitude higher than those of the corresponding regioisomers p- Quin-Epo B and p-Quin-Epo D, respectively (Kb’s of 92 × 107 and 88 × 107 for m-Quin-Epo B and m- Quin-Epo D, respectively, vs. 6.9 × 107 and 6.1 × 107 for p-Quin-Epo B and p-Quin-Epo D). Quite intriguingly, and for reasons not understood, the difference in the microtubule-binding affinity between m-Quin-Epo D and p-Quin-Epo D translates into a corresponding difference in the cellular activity, while the epoxides m-Quin-Epo B and p-Quin-Epo B are virtually equipotent (and highly active). Figure 3. Molecular structures of quinoline-derived epothilone analogs. Figure 3. Molecular structures of quinoline-derived epothilone analogs. Figure 3. Molecular structures of quinoline-derived epothilone analogs. Figure 3. Molecular structures of quinoline-derived epothilone analogs. While our data for quinoline-based epothilone analogs seem to re-enforce the conclusions derived from the earlier studies by Nicolaou and Danishefsky on pyridyl- and phenyl-based epothilone analogs, respectively, they need to be interpreted with some care, in light of the significant overall modification of the side chain vs. natural epothilones. Finally, the recent X-ray crystal structure of a tubulin-Epo A complex [23] invokes a hydrogen bond between the thiazole nitrogen and the side chain hydroxy group of βThr297. Overall, the available experimental data suggest that the presence of a properly positioned heterocyclic N-atom in side chain-modified epothilone analogs is required, in order to maximize interactions with the tubulin/microtubule system. 1. Introduction 1. Introduction Molecular structures of the three possible pyridyl-Epo B variants and of phenyl-Epo D. Figure 2. Molecular structures of the three possible pyridyl-Epo B variants and of phenyl-Epo D. Figure 2. Molecular structures of the three possible pyridyl-Epo B variants and of phenyl-Epo D. Figure 2. Molecular structures of the three possible pyridyl-Epo B variants and of phenyl-Epo D. Nicolaou’s data were in line with the results of an earlier study by Danishefsky and co-workers who had reported the phenyl-based Epo D analog Ph-Epo D (Figure 2) to be a potent inducer of tubulin polymerization, albeit to a lower extent than the natural product Epo D itself [21]. These data suggested that the presence of a heterocyclic N-atom next to the vinyl linker moiety in epothilone analogs is required for maximum induction of tubulin assembly and, consequently, for the inhibition of cancer cell proliferation. However, neither of the above studies included a quantitative assessment of the microtubule-binding affinity of the analogs investigated. In this context, it needs to be recognized that the assessment of tubulin polymerization induction is useful for the unequivocal identification of compounds with poor tubulin assembly properties, but is less suited for the high- resolution quantitative differentiation between potent assembly inducers. In our own work, we have d d h h i b l bi di ffi i i f i li b d hil l (Fi Nicolaou’s data were in line with the results of an earlier study by Danishefsky and co-workers who had reported the phenyl-based Epo D analog Ph-Epo D (Figure 2) to be a potent inducer of tubulin polymerization, albeit to a lower extent than the natural product Epo D itself [21]. These data suggested that the presence of a heterocyclic N-atom next to the vinyl linker moiety in epothilone analogs is required for maximum induction of tubulin assembly and, consequently, for the inhibition of cancer cell proliferation. However, neither of the above studies included a quantitative assessment of the microtubule-binding aﬃnity of the analogs investigated. In this context, it needs to be recognized that the assessment of tubulin polymerization induction is useful for the unequivocal identiﬁcation of compounds with poor tubulin assembly properties, but is less suited for the high-resolution quantitative diﬀerentiation between potent assembly inducers. 1. Introduction 1. Introduction At the same time, the magnitude of the effect associated with the simple removal of this nitrogen from the natural side chain, quite surprisingly, has never been assessed. We have thus been interested for some time in the synthesis of a thiophene-containing analog of a natural epothilone and the determination of its microtubule-binding affinity in comparison with the natural parent structure. In this paper, we describe the results of these efforts. While our data for quinoline-based epothilone analogs seem to re-enforce the conclusions derived from the earlier studies by Nicolaou and Danishefsky on pyridyl- and phenyl-based epothilone analogs, respectively, they need to be interpreted with some care, in light of the signiﬁcant overall modiﬁcation of the side chain vs. natural epothilones. Finally, the recent X-ray crystal structure of a tubulin-Epo A complex [23] invokes a hydrogen bond between the thiazole nitrogen and the side chain hydroxy group of βThr297. Overall, the available experimental data suggest that the presence of a properly positioned heterocyclic N-atom in side chain-modiﬁed epothilone analogs is required, in order to maximize interactions with the tubulin/microtubule system. At the same time, the magnitude of the eﬀect associated with the simple removal of this nitrogen from the natural side chain, quite surprisingly, has never been assessed. We have thus been interested for some time in the synthesis of a thiophene-containing analog of a natural epothilone and the determination of its microtubule-binding aﬃnity in comparison with the natural parent structure. In this paper, we describe the results of these eﬀorts. 2. Materials and Methods 2. Materials and Methods Detailed protocols for the synthesis of new compounds and the associated analytical data can be found in the Supplementary Material. Detailed protocols for the synthesis of new compounds and the associated analytical data can be found in the Supplementary Materials. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Reagents and conditions: a) nBuLi, −78 °C to RT, 0%. Scheme 1. Reagents and conditions: a) nBuLi, −78 ◦C to RT, 0%. efin (Scheme 1). Scheme 1. Reagents and conditions: a) nBuLi, −78 °C to RT, 0%. Scheme 1. Reagents and conditions: a) nBuLi, −78 °C to RT, 0%. Scheme 1. Reagents and conditions: a) nBuLi, −78 ◦C to RT, 0%. Scheme 1 Reagents and conditions: a) nBuLi −78 °C to RT 0% In light of a previous report by Höfle and co-workers, who had been unable of convert 2 into phenyl-Epo A or to reconstruct Epo A from 2 by a variety of olefination methods [24], we did not further pursue the direct elaboration of 2 into 1. Instead, we turned our attention to a different aspect of the chemistry of Epo A that had been unveiled by Höfle’s work on semisynthetic epothilone derivatives and that involves the targeted removal of the C13–C15 segment of the macrocycle to generate acid 8 [28] (Scheme 2). The latter was then to be esterified with alcohol 7, and the resulting diene would be cyclized by ring-closing olefin metathesis (RCM) in analogy to previous work on the total synthesis of Epo A [29–31]. Deprotection would then yield deaza-Epo C (5). In light of a previous report by Höﬂe and co-workers, who had been unable of convert 2 into phenyl-Epo A or to reconstruct Epo A from 2 by a variety of oleﬁnation methods [24], we did not further pursue the direct elaboration of 2 into 1. Instead, we turned our attention to a diﬀerent aspect of the chemistry of Epo A that had been unveiled by Höﬂe’s work on semisynthetic epothilone derivatives and that involves the targeted removal of the C13–C15 segment of the macrocycle to generate acid 8 [28] (Scheme 2). The latter was then to be esteriﬁed with alcohol 7, and the resulting diene would be cyclized by ring-closing oleﬁn metathesis (RCM) in analogy to previous work on the total synthesis of Epo A [29–31]. Deprotection would then yield deaza-Epo C (5). In light of a previous report by Höfle and co-workers, who had been unable of convert 2 into phenyl-Epo A or to reconstruct Epo A from 2 by a variety of olefination methods [24], we did not further pursue the direct elaboration of 2 into 1. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Instead, we turned our attention to a different aspect of the chemistry of Epo A that had been unveiled by Höfle’s work on semisynthetic epothilone derivatives and that involves the targeted removal of the C13–C15 segment of the macrocycle to generate acid 8 [28] (Scheme 2). The latter was then to be esterified with alcohol 7, and the resulting diene would be cyclized by ring-closing olefin metathesis (RCM) in analogy to previous work on the total synthesis of Epo A [29 31] Deprotection would then yield deaza Epo C (5) Scheme 2. Retrosynthesis of deaza-Epo C (5). Scheme 2. Retrosynthesis of deaza-Epo C (5). Scheme 2. Retrosynthesis of deaza-Epo C (5). Scheme 2. Retrosynthesis of deaza-Epo C (5). Scheme 2. Retrosynthesis of deaza-Epo C (5). Scheme 2. Retrosynthesis of deaza-Epo C (5). When elaborating this strategy, we were cognizant of the fact that the conversion of the Epo C analog 5 into 1 might be impaired by the competing epoxidation of the C16–C17 double bond, which is more nucleophilic than in Epo C, and/or oxidation at sulfur [32]. At the same time, we also felt that our question about the effects of the removal of the thiazole nitrogen could be addressed by comparing the microtubule-binding affinity of 5 with that of Epo C, as the latter had also been reported to be a potent inducer of tubulin polymerization [33] (although no microtubule binding data for the compound exist in the literature). In the forward direction the synthesis of acid 8 commenced with the deoxygenation of Epo A When elaborating this strategy, we were cognizant of the fact that the conversion of the Epo C analog 5 into 1 might be impaired by the competing epoxidation of the C16–C17 double bond, which is more nucleophilic than in Epo C, and/or oxidation at sulfur [32]. At the same time, we also felt that our question about the effects of the removal of the thiazole nitrogen could be addressed by comparing the microtubule-binding affinity of 5 with that of Epo C, as the latter had also been reported to be a potent inducer of tubulin polymerization [33] (although no microtubule binding data for the compound exist in the literature). 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) The initial synthetic target of our work was deaza-Epo A (1), which we planned to access from ketone 2 by means of the Wittig or Horner–Woodsworth–Emmons (HWE) reaction (Scheme 1). Ketone 2 can be accessed from Epo A by TMS-protection and ozonolysis [24]; and its conversion into TMS-protected 3 via the transformation of the ketone moiety into the corresponding vinyl boronic acid, followed by iodination and Stille coupling, had been described earlier by Höfle and co-workers The initial synthetic target of our work was deaza-Epo A (1), which we planned to access from ketone 2 by means of the Wittig or Horner–Woodsworth–Emmons (HWE) reaction (Scheme 1). Ketone 2 can be accessed from Epo A by TMS-protection and ozonolysis [24]; and its conversion into TMS-protected 3 via the transformation of the ketone moiety into the corresponding vinyl boronic acid, followed by iodination and Stille coupling, had been described earlier by Höﬂe and co-workers [25]. 502 of 509 502 of 509 502 of 509 Chemistry 2020, 2, 30 However, no experimental protocols for this reaction sequence are provided in ref. [25], and no yields are reported for the iodination and Stille coupling steps. On the other hand, we had been successful ourselves in the elaboration of ketone 2 into epothilone A analogs bearing modiﬁed thiazole, pyrimidine or pyridine moieties in place of the natural thiazole heterocycle by means of HWE chemistry (Hauenstein & Altmann, unpublished experiments; see also refs. [26,27]). Unfortunately, the attempted HWE coupling of 2 with phosphonate 4 did not yield any of the desired oleﬁn (Scheme 1). Chemistry 2020, 2, x 4 successful ourselves in the elaboration of ketone 2 into epothilone A analogs bearing modified thiazole, pyrimidine or pyridine moieties in place of the natural thiazole heterocycle by means of HWE chemistry (Hauenstein & Altmann, unpublished experiments; see also refs. [26] and [27]). Unfortunately, the attempted HWE coupling of 2 with phosphonate 4 did not yield any of the desired olefin (Scheme 1). Chemistry 2020, 2, x 4 successful ourselves in the elaboration of ketone 2 into epothilone A analogs bearing modified thiazole, pyrimidine or pyridine moieties in place of the natural thiazole heterocycle by means of HWE chemistry (Hauenstein & Altmann, unpublished experiments; see also refs. [26] and [27]). Unfortunately, the attempted HWE coupling of 2 with phosphonate 4 did not yield any of the desired Scheme 1. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Reagents and conditions: a) 10, TFA, DCM, 6 h, RT, 43%; b) Hoveyda–Grubbs-II cat., DCM, 20 h, RT, 61%; c) i) 2,6-lutidine, DCM, 10 min, RT, ii) TBSOTf, 5 min, 110 °C, microwave; d) LiOH, H2O, iPrOH, 5 min, 150 °C, microwave, 61% over two steps. Scheme 3. Reagents and conditions: a) 10, TFA, DCM, 6 h, RT, 43%; b) Hoveyda–Grubbs-II cat., DCM, 20 h, RT, 61%; c) i) 2,6-lutidine, DCM, 10 min, RT, ii) TBSOTf, 5 min, 110 ◦C, microwave; d) LiOH, H2O, iPrOH, 5 min, 150 ◦C, microwave, 61% over two steps. The synthesis of alcohol 7 started from 3-thienylmethanol (11), which was TBS-protected; deprotonation of TBS-ether 12 with nBuLi (1.1 equiv) and reaction with a two-fold excess of MeI then provided TBS-ether 13 in 62% yield (Scheme 4). The deprotection of 13 with TBAF, followed by oxidation of the ensuing free alcohol under Swern conditions, afforded the desired aldehyde 15 in 71% overall yield. The Wittig reaction of 15 with 16 then delivered homologated aldehyde 17 (71% yield). The deoxygenation reaction gave Epo C in highly variable yields, due to incomplete conversion and the formation of an unknown side product that was diﬃcult to remove. It was found eventually, however, that the subsequent ring-opening reaction proceeded equally well without the prior removal of this impurity, thus obviating the need for the tedious puriﬁcation of the intermediate Epo C. The ring-opening of the macrocycle with ethylene in the presence of the Grubbs–Hoveyda II catalyst furnished diene 9 (as described [28]), which was converted into acid 8 by sequential treatment with TBSOTf and LiOH at elevated temperature under microwave conditions in 61% overall yield. Scheme 3. Reagents and conditions: a) 10, TFA, DCM, 6 h, RT, 43%; b) Hoveyda–Grubbs-II cat., DCM, 20 h, RT, 61%; c) i) 2,6-lutidine, DCM, 10 min, RT, ii) TBSOTf, 5 min, 110 °C, microwave; d) LiOH, H2O, iPrOH, 5 min, 150 °C, microwave, 61% over two steps. Th th i f l h l 7 t t d f 3 thi l th l (11) hi h TBS t t d The synthesis of alcohol 7 started from 3-thienylmethanol (11), which was TBS-protected; deprotonation of TBS-ether 12 with nBuLi (1.1 equiv) and reaction with a two-fold excess of MeI then provided TBS-ether 13 in 62% yield (Scheme 4). 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) The deprotection of 13 with TBAF, followed by oxidation of the ensuing free alcohol under Swern conditions, aﬀorded the desired aldehyde 15 in 71% overall yield. The Wittig reaction of 15 with 16 then delivered homologated aldehyde 17 (71% yield). The synthesis of alcohol 7 started from 3-thienylmethanol (11), which was TBS-protected; deprotonation of TBS-ether 12 with nBuLi (1.1 equiv) and reaction with a two-fold excess of MeI then provided TBS-ether 13 in 62% yield (Scheme 4). The deprotection of 13 with TBAF, followed by oxidation of the ensuing free alcohol under Swern conditions, afforded the desired aldehyde 15 in 71% overall yield. The Wittig reaction of 15 with 16 then delivered homologated aldehyde 17 (71% yield) Scheme 4. Reagents and conditions: a) TBSCl, imidazole, DMF, 5 h, 45 °C, quant.; b) nBuLi, MeI, THF, 18 h, −35 °C to RT, 62%; c) TBAF, THF, 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 °C to RT, 71% (2 steps); e) 16, benzene, 23 h, 103 °C, 71%; f) 18, dibutylboron triflate, DIPEA, DCM, 4 h, −78 °C to 0 °C, 51%; g) TBSCl, imidazole, DMF, 4.5 h, 45 °C, 79%; h) DIBAL-H, DCM, 2 h, −78 °C, 80%; i) 22, NaHMDS, THF, 2 h, −78 °C, 88%; j) TBAF, THF, 5 h, 0 °C to RT, 94%. The reaction of 17 with the dibutylboron enolate of acetylsultam 18, obtained by the successi atment of the latter with dibutylboron triflate and DIPEA [22,35], proceeded with only modera ectivity, to afford a ca. 2:1 mixture of aldol products, in favor of the desired isomer 19 [3 eparing the dibutylboron triflate in situ from triethylborane and trifluoromethanesulfonic acid d t lead to an improved dr. Although tedious, isomer separation was possible by colum romatography, and 19 was finally obtained as a single isomer in 51% yield; the latter was th otected as its TBS-ether 20. The reductive cleavage of the auxiliary with DIBAL-H, followed Scheme 4. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) When elaborating this strategy, we were cognizant of the fact that the conversion of the Epo C analog 5 into 1 might be impaired by the competing epoxidation of the C16–C17 double bond, which is more nucleophilic than in Epo C, and/or oxidation at sulfur [32]. At the same time, we also felt that our question about the eﬀects of the removal of the thiazole nitrogen could be addressed by comparing the microtubule-binding aﬃnity of 5 with that of Epo C, as the latter had also been reported to be a potent inducer of tubulin polymerization [33] (although no microtubule binding data for the compound exist in the literature). In the forward direction, the synthesis of acid 8 commenced with the deoxygenation of Epo A with 3-methylbenzo[d]thiazole-2(3H)-selenone (10), prepared according to Calo and co-workers by refluxing methylbenzothiazolium iodide and elemental selenium in pyridine [34] (Scheme 3). The deoxygenation reaction gave Epo C in highly variable yields, due to incomplete conversion p In the forward direction, the synthesis of acid 8 commenced with the deoxygenation of Epo A with 3-methylbenzo[d]thiazole-2(3H)-selenone (10), prepared according to Calo and co-workers by refluxing methylbenzothiazolium iodide and elemental selenium in pyridine [34] (Scheme 3). In the forward direction, the synthesis of acid 8 commenced with the deoxygenation of Epo A with 3-methylbenzo[d]thiazole-2(3H)-selenone (10), prepared according to Calo and co-workers by reﬂuxing methylbenzothiazolium iodide and elemental selenium in pyridine [34] (Scheme 3). 503 of 509 reatment Chemistry 2020, 2, 30 catalyst furnished Scheme 3. Reagents and conditions: a) 10, TFA, DCM, 6 h, RT, 43%; b) Hoveyda–Grubbs-II cat., DCM, 20 h, RT, 61%; c) i) 2,6-lutidine, DCM, 10 min, RT, ii) TBSOTf, 5 min, 110 °C, microwave; d) LiOH, H2O, iPrOH, 5 min, 150 °C, microwave, 61% over two steps. Scheme 3. Reagents and conditions: a) 10, TFA, DCM, 6 h, RT, 43%; b) Hoveyda–Grubbs-II cat., DCM, 20 h, RT, 61%; c) i) 2,6-lutidine, DCM, 10 min, RT, ii) TBSOTf, 5 min, 110 ◦C, microwave; d) LiOH, H2O, iPrOH, 5 min, 150 ◦C, microwave, 61% over two steps. hemistry 2020, 2, x atalyst furnished diene 9 (as described [28]), which was converted into acid 8 by sequential treatm ith TBSOTf and LiOH at elevated temperature under microwave conditions in 61% overall yield istry 2020, 2, x lyst furnished diene 9 (as descr h TBSOTf and LiOH at elevate Scheme 3. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Reagents and conditions: a) TBSCl, imidazole, DMF, 5 h, 45 °C, quant.; b) nBuLi, MeI, THF, 18 h, −35 °C to RT, 62%; c) TBAF, THF, 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 °C to RT, 71% (2 steps); e) 16, benzene, 23 h, 103 °C, 71%; f) 18, dibutylboron triflate, DIPEA, DCM, 4 h, −78 °C to 0 °C, 51%; g) TBSCl, imidazole, DMF, 4.5 h, 45 °C, 79%; h) DIBAL-H, DCM, 2 h, −78 °C, 80%; i) 22, NaHMDS, THF, 2 h, −78 °C, 88%; j) TBAF, THF, 5 h, 0 °C to RT, 94%. Scheme 4. Reagents and conditions: a) TBSCl, imidazole, DMF, 5 h, 45 ◦C, quant.; b) nBuLi, MeI, THF, 18 h, −35 ◦C to RT, 62%; c) TBAF, THF, 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 ◦C to RT, 71% (2 steps); e) 16, benzene, 23 h, 103 ◦C, 71%; f) 18, dibutylboron triﬂate, DIPEA, DCM, 4 h, −78 ◦C to 0 ◦C, 51%; g) TBSCl, imidazole, DMF, 4.5 h, 45 ◦C, 79%; h) DIBAL-H, DCM, 2 h, −78 ◦C, 80%; i) 22, NaHMDS, THF, 2 h, −78 ◦C, 88%; j) TBAF, THF, 5 h, 0 ◦C to RT, 94%. TBSCl, imidazole, DMF, 5 h, 45 °C, quant.; b) nBuLi, MeI, T 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 °C 103 °C, 71%; f) 18, dibutylboron triflate, DIPEA, DCM, 4 h, DMF, 4.5 h, 45 °C, 79%; h) DIBAL-H, DCM, 2 h, −78 °C, 80% TBAF, THF, 5 h, 0 °C to RT, 94%. boron enolate of acetylsultam 18, obtained by the su TBSCl, imidazole, DMF, 5 h, 45 °C, quant.; b) nBuLi, MeI, T 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 °C 103 °C, 71%; f) 18, dibutylboron triflate, DIPEA, DCM, 4 h, MF, 4.5 h, 45 °C, 79%; h) DIBAL-H, DCM, 2 h, −78 °C, 80% TBAF, THF, 5 h, 0 °C to RT, 94%. boron enolate of acetylsultam 18, obtained by the su me 4. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Preparing the dibutylboron triﬂate in situ from triethylborane and triﬂuoromethanesulfonic acid did not lead to an improved dr. Although tedious, isomer separation was possible by column chromatography, and 19 was ﬁnally obtained as a single isomer in 51% yield; the latter was then protected as its TBS-ether 20. The reductive cleavage of the auxiliary with DIBAL-H, followed by Julia−Kocienski oleﬁnation of the ensuing aldehyde 21 with sulfone 22 and subsequent TBS-deprotection, gave homoallylic alcohol 7 in ca. 8.5% overall yield for the 10-step sequence from 3-thienylmethanol (11). The attempted Wittig oleﬁnation of aldehyde 21 with methyltriphenylphosphonium bromide in combination with various bases had been found previously to induce the elimination of TBSOH (Cintulová & Altmann, unpublished). Chemistry 2020, 2, x 6 Julia−Kocienski olefination of the ensuing aldehyde 21 with sulfone 22 and subsequent TBS- deprotection, gave homoallylic alcohol 7 in ca. 8.5% overall yield for the 10-step sequence from 3- thienylmethanol (11). The attempted Wittig olefination of aldehyde 21 with methyltriphenylphosphonium bromide in combination with various bases had been found previously to induce the elimination of TBSOH (Cintulová & Altmann unpublished) The esteriﬁcation of alcohol 7 with acid 8 under Yamaguchi conditions [37] at RT furnished diene 6 in moderate but acceptable yields of 44%−65% (Scheme 5). previously to induce the elimination of TBSOH (Cintulová & Altmann, unpublished). The esterification of alcohol 7 with acid 8 under Yamaguchi conditions [37] at RT furnished diene 6 in moderate but acceptable yields of 44%−65% (Scheme 5). Scheme 5. a) Reagents and conditions: NEt3, 2,4,6-trichlorobenzoyl chloride (TCBC), DMAP, 3 h, RT, 44%–65%; b) Grubbs II cat., toluene, 40 °C, 17%. Scheme 5. a) Reagents and conditions: NEt3, 2,4,6-trichlorobenzoyl chloride (TCBC), DMAP, 3 h, RT, 44%–65%; b) Grubbs II cat., toluene, 40 ◦C, 17%. Scheme 5. a) Reagents and conditions: NEt3, 2,4,6-trichlorobenzoyl chloride (TCBC), DMAP, 3 h, RT, 44%–65%; b) Grubbs II cat., toluene, 40 °C, 17%. Scheme 5. a) Reagents and conditions: NEt3, 2,4,6-trichlorobenzoyl chloride (TCBC), DMAP, 3 h, RT, 44%–65%; b) Grubbs II cat., toluene, 40 ◦C, 17%. A series of screening experiments was then performed on an analytical scale to identify the best conditions for the crucial ring-closure reaction, including a range of solvents (DCM, benzene, toluene, THF) and different metathesis catalysts (Grubbs I and II, Hoveyda−Grubbs II, Grubbs III) [38]. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Reagents and conditions: a −35 °C to RT, 62%; c) TBAF, THF 1% (2 steps); e) 16, benzene, 23 h 0 °C, 51%; g) TBSCl, imidazole, aHMDS, THF, 2 h, −78 °C, 88%; j eaction of 17 with the dibuty f 17 w tment of the latter with dibutylboron triflate and DIPEA [22,35], proceeded with only moder ctivity, to afford a ca. 2:1 mixture of aldol products, in favor of the desired isomer 19 [3 paring the dibutylboron triflate in situ from triethylborane and trifluoromethanesulfonic acid d lead to an improved dr. Although tedious, isomer separation was possible by colum omatography, and 19 was finally obtained as a single isomer in 51% yield; the latter was th d h h d l f h l h I A f ll d Scheme 4. Reagents and conditions: a) TBSCl, imidazole, DMF, 5 h, 45 °C, quant.; b) nBuLi, MeI, THF, 18 h, −35 °C to RT, 62%; c) TBAF, THF, 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 °C to RT, 71% (2 steps); e) 16, benzene, 23 h, 103 °C, 71%; f) 18, dibutylboron triflate, DIPEA, DCM, 4 h, −78 °C to 0 °C, 51%; g) TBSCl, imidazole, DMF, 4.5 h, 45 °C, 79%; h) DIBAL-H, DCM, 2 h, −78 °C, 80%; i) 22, NaHMDS, THF, 2 h, −78 °C, 88%; j) TBAF, THF, 5 h, 0 °C to RT, 94%. Scheme 4. Reagents and conditions: a) TBSCl, imidazole, DMF, 5 h, 45 ◦C, quant.; b) nBuLi, MeI, THF, 18 h, −35 ◦C to RT, 62%; c) TBAF, THF, 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 ◦C to RT, 71% (2 steps); e) 16, benzene, 23 h, 103 ◦C, 71%; f) 18, dibutylboron triﬂate, DIPEA, DCM, 4 h, −78 ◦C to 0 ◦C, 51%; g) TBSCl, imidazole, DMF, 4.5 h, 45 ◦C, 79%; h) DIBAL-H, DCM, 2 h, −78 ◦C, 80%; i) 22, NaHMDS, THF, 2 h, −78 ◦C, 88%; j) TBAF, THF, 5 h, 0 ◦C to RT, 94%. Chemistry 2020, 2, 30 504 of 509 504 of 509 The reaction of 17 with the dibutylboron enolate of acetylsultam 18, obtained by the successive treatment of the latter with dibutylboron triﬂate and DIPEA [22,35], proceeded with only moderate selectivity, to aﬀord a ca. 2:1 mixture of aldol products, in favor of the desired isomer 19 [36]. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Not unexpectedly, the reaction under all conditions investigated suffered from low selectivity; low selectivity has also been reported for the RCM of bis-TBS-protected Epo C with the Grubbs I catalyst [29–31]). However, in contrast to the latter, which delivered the E/Z isomeric mixture of macrocycles in high yield, diene 6 appeared to be of limited stability under the reaction conditions and/or to be consumed by alternative reaction pathways. Overall, the screening experiments, unfortunately, did not provide consistent guidance on how to maximize the yield of the desired macrocycle 23. Ultimately, the reaction was conducted with 0.1 equiv. of Grubbs II catalyst in toluene at 40 °C and quenched before complete conversion of the starting material. On a 20 mg scale, these conditions provided 3.5 mg of slightly impure 23 (17%) together with 6.9 mg (33%) of the corresponding 12,13- E isomer. When the reaction was carried out for 3 h at reflux, the E isomer of 23 was obtained in 51% isolated yield. A series of screening experiments was then performed on an analytical scale to identify the best conditions for the crucial ring-closure reaction, including a range of solvents (DCM, benzene, toluene, THF) and diﬀerent metathesis catalysts (Grubbs I and II, Hoveyda−Grubbs II, Grubbs III) [38]. Not unexpectedly, the reaction under all conditions investigated suﬀered from low selectivity; low selectivity has also been reported for the RCM of bis-TBS-protected Epo C with the Grubbs I catalyst [29–31]). However, in contrast to the latter, which delivered the E/Z isomeric mixture of macrocycles in high yield, diene 6 appeared to be of limited stability under the reaction conditions and/or to be consumed by alternative reaction pathways. Overall, the screening experiments, unfortunately, did not provide consistent guidance on how to maximize the yield of the desired macrocycle 23. Ultimately, the reaction was conducted with 0.1 equiv. of Grubbs II catalyst in toluene at 40 ◦C and quenched before complete conversion of the starting material. On a 20 mg scale, these conditions provided 3.5 mg of slightly impure 23 (17%) together with 6.9 mg (33%) of the corresponding 12,13-E isomer. When the reaction was carried out for 3 h at reﬂux, the E isomer of 23 was obtained in 51% isolated yield. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) y The difficulties with the RCM of diene 6 were then further aggravated by the fact that no conditions could be identified that would have allowed for the clean deprotection of 23, including conditions that have been employed successfully in the deprotection of bis-TBS-protected Epo C [29– 31]. These findings, which point to a distinct instability of 23 (or 5) compared to (protected) Epo C, called for a change to a protecting group more easily removable than TBS. An obvious candidate that would meet this requirement was the TMS group and we felt that the corresponding acid 8a (Figure 4) could be readily available from 9 in analogy to the preparation of 8, by simply substituting TMSOTf The diﬃculties with the RCM of diene 6 were then further aggravated by the fact that no conditions could be identiﬁed that would have allowed for the clean deprotection of 23, including conditions that have been employed successfully in the deprotection of bis-TBS-protected Epo C [29–31]. These ﬁndings, which point to a distinct instability of 23 (or 5) compared to (protected) Epo C, called for a change to a protecting group more easily removable than TBS. An obvious candidate that would meet this requirement was the TMS group and we felt that the corresponding acid 8a (Figure 4) could be readily available from 9 in analogy to the preparation of 8, by simply substituting TMSOTf for TBSOTf. 505 of 509 ( g g TMSOTf Chemistry 2020, 2, 30 4) could be readily f O f Chemistry 2020, 2, 30 4) could be readily f SO f Figure 4. Molecular structures of acid 8a. Figure 4. Molecular structures of acid 8a. Figure 4. Molecular structures of acid 8a. Figure 4. Molecular structures of acid 8a. Unfortunately, the treatment of 9 with TMSOTf under a variety of conditions did not lead to complete conversion to the bis-TMS ether 8a, even if a large excess of TMSOTf was employed (up to 75-fold); in addition, the retro aldol cleavage of the C3−C4 bond was frequently observed as a side reaction [39]. The puriﬁcation of acid 8a by silica gel chromatography was possible to some extent, but was complicated by the limited stability of the compound under the chromatographic conditions. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) Reagents and conditions: a) TMSOTf, 2,6-lutidine, DCM, 1.5 h, 0 °C to RT, 53%; b) 7, TCBC, NEt3, THF, DMAP, 2 h, 0 °C to RT, 39% for the 2:1 mixture (not separated) of 26 (26%) and 27 (13%); c) Grubbs I cat., toluene, RT, 18 h, 13% (28) and 19% (29); d) PPTS, EtOH, 0 °C, 90 min, 67%. Scheme 6. Reagents and conditions: a) TMSOTf, 2,6-lutidine, DCM, 1.5 h, 0 ◦C to RT, 53%; b) 7, TCBC, NEt3, THF, DMAP, 2 h, 0 ◦C to RT, 39% for the 2:1 mixture (not separated) of 26 (26%) and 27 (13%); c) Grubbs I cat., toluene, RT, 18 h, 13% (28) and 19% (29); d) PPTS, EtOH, 0 ◦C, 90 min, 67%. Thus, the reaction of 26 with 7 under Yamaguchi conditions gave the desired ester 26 and the dimeric structure 27 [40] in a ca. 2:1 ratio and with an overall yield of 39%. The separation of 26 and 27 was possible, if tedious, but was better performed after the RCM step. Intriguingly, two attempts at the Yamaguchi esterification of doubly TMS-protected acid 8a only led to slow decomposition, with alcohol 7 being recovered in almost quantitative yields. Thus, the reaction of 26 with 7 under Yamaguchi conditions gave the desired ester 26 and the dimeric structure 27 [40] in a ca. 2:1 ratio and with an overall yield of 39%. The separation of 26 and 27 was possible, if tedious, but was better performed after the RCM step. Intriguingly, two attempts at the Yamaguchi esteriﬁcation of doubly TMS-protected acid 8a only led to slow decomposition, with alcohol 7 being recovered in almost quantitative yields. RCM with 60 mg of the 2:1 mixture of 26 and 27 with Grubbs I catalyst gave 4.5 mg (13%) of Z- product 28 and 6.6 mg (19%) of E-isomer 29 after two preparative RP-HPLC runs. Furthermore, 6.9 mg (19%) of diene 26 were recovered; no other products were characterized. The configurational assignment of the C12–C13 double bond in 28 and 29 was based on J coupling constants obtained via NMR decoupling experiments. For Z-product 28, a coupling constant of 10.9 Hz was determined, while E-isomer 29 showed a coupling constant of 15.3 Hz (see the Supporting Material). 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) In contrast to 8a, mono-TMS-ether 26 (Scheme 6) could be obtained in 53% yield after column chromatography, when TMSOTf and 2,6-lutidine were premixed in DCM before the addition of 9 to the reaction mixture (Scheme 6). While the use of 26 presented its own problems in the subsequent esteriﬁcation step (vide infra), suﬃcient quantities of this material could be produced to complete the synthesis of the target structure. Unfortunately, the treatment of 9 with TMSOTf under a variety of conditions did not lead to complete conversion to the bis-TMS ether 8a, even if a large excess of TMSOTf was employed (up to 75-fold); in addition, the retro aldol cleavage of the C3−C4 bond was frequently observed as a side reaction [39]. The purification of acid 8a by silica gel chromatography was possible to some extent, but was complicated by the limited stability of the compound under the chromatographic conditions. In contrast to 8a, mono-TMS-ether 26 (Scheme 6) could be obtained in 53% yield after column chromatography, when TMSOTf and 2,6-lutidine were premixed in DCM before the addition of 9 to the reaction mixture (Scheme 6). While the use of 26 presented its own problems in the subsequent esterification step (vide infra), sufficient quantities of this material could be produced to complete the synthesis of the target structure. Scheme 6. Reagents and conditions: a) TMSOTf, 2,6-lutidine, DCM, 1.5 h, 0 °C to RT, 53%; b) 7, TCBC, NEt3, THF, DMAP, 2 h, 0 °C to RT, 39% for the 2:1 mixture (not separated) of 26 (26%) and 27 (13%); c) Grubbs I cat., toluene, RT, 18 h, 13% (28) and 19% (29); d) PPTS, EtOH, 0 °C, 90 min, 67%. Scheme 6. Reagents and conditions: a) TMSOTf, 2,6-lutidine, DCM, 1.5 h, 0 ◦C to RT, 53%; b) 7, TCBC, NEt3, THF, DMAP, 2 h, 0 ◦C to RT, 39% for the 2:1 mixture (not separated) of 26 (26%) and 27 (13%); c) Grubbs I cat., toluene, RT, 18 h, 13% (28) and 19% (29); d) PPTS, EtOH, 0 ◦C, 90 min, 67%. Scheme 6. 3.2. Biochemical and Cellular Assessment With deaza-Epo C (5) in hand, we assessed the binding of the compound to cross-linked microtubules at diﬀerent temperatures in comparison with natural Epo C. As can be seen from the data presented in Table 1, for temperatures up to 35 ◦C, the binding constant of deaza-Epo C (5) for microtubules is ca. 4-fold to 6-fold lower than that of Epo C, corresponding to a free energy diﬀerence ∆∆G of ca. 0.7 kcal/mol to 1 kcal/mol. Thus, the loss in binding free energy incurred by the replacement of the thiazole nitrogen in Epo C by a CH group is rather modest and appears to be comparable with the loss in binding energy observed upon removal of the epoxide oxygen from Epo A (to form Epo C) (Table 1) or the Epo B→Epo D transformation [41]. The diﬀerence appears somewhat less pronounced than for the quinoline-based Epo D analogs o-Quin-Epo D and p-Quin Epo D (Figure 3), where the diﬀerence in binding constants is >10-fold; however, the diﬀerences are small and should not be overinterpreted. At the same time, the data for the quinoline-based epothilones depicted in Figure 3 suggest that the diﬀerence in binding free energy observed here between Epo C and deaza-Epo C (5) can most likely be extrapolated to epothilones A, B and D and their corresponding deaza analogs. For temperatures above 35 ◦C, a marked drop in the microtubule-binding aﬃnity of deaza-Epo C (5) was observed (Kb of 5 × 105 at 42 ◦C); the eﬀect is signiﬁcantly more pronounced than for Epo C (Kb of 88 × 105 at 42 ◦C). The cause for this behavior is unclear at this point, but may be related to the limited chemical stability of the compound 5 at higher temperatures (vide infra). Table 1. Binding constants of deaza-Epo C (5), Epo C and Epo A for stabilized microtubules. 1 Compound Kb [107 M−1] 26 ◦C 30 ◦C 35 ◦C 5 0.39 ± 0.03 0.33 ± 0.02 0.37 ± 0.05 Epo C 1.46 ± 0.33 1.19 ± 0.15 1.93 ± 0.27 Epo A 2 7.48 ± 1.00 5.81 ± 1.08 3.63 ± 0.51 1 Association constant Kb with glutaraldehyde-stabilized microtubules at diﬀerent temperatures, determined as described in ref. [42]. Numbers are average values from three independent experiments ± standard deviation. 2 From ref. [41]. Table 1. Binding constants of deaza-Epo C (5), Epo C and Epo A for stabilized microtubules. 3.1. Synthesis of Deaza-Epo C (5) 3.1. Synthesis of Deaza-Epo C (5) With the protected macrocycle in hand, removal of the TMS moiety was then attempted with citric acid RCM with 60 mg of the 2:1 mixture of 26 and 27 with Grubbs I catalyst gave 4.5 mg (13%) of Z-product 28 and 6.6 mg (19%) of E-isomer 29 after two preparative RP-HPLC runs. Furthermore, 6.9 mg (19%) of diene 26 were recovered; no other products were characterized. The conﬁgurational assignment of the C12–C13 double bond in 28 and 29 was based on J coupling constants obtained via NMR decoupling experiments. For Z-product 28, a coupling constant of 10.9 Hz was determined, while E-isomer 29 showed a coupling constant of 15.3 Hz (see the Supporting Material). With the protected macrocycle in hand, removal of the TMS moiety was then attempted with citric acid (MeOH, RT, 12 h), Chemistry 2020, 2, 30 506 of 509 506 of 509 as this had proven successful for other TMS-protected epothilone analogs [27]; however, the mass of 5 was not detected in the reaction mixture, and RP-HPLC indicated the formation of multiple products. In contrast, the ﬁnal deprotection of 28 with PPTS in EtOH gratifyingly aﬀorded deaza-Epo C (5) in 67% yield after HPLC puriﬁcation (Scheme 6). 3.2. Biochemical and Cellular Assessment 1 Epo A 7.48 ± 1.00 5.81 ± 1.08 3.63 ± 0.51 1 Association constant Kb with glutaraldehyde-stabilized microtubules at diﬀerent temperatures, determined as described in ref. [42]. Numbers are average values from three independent experiments ± standard deviation. 2 From ref. [41]. Epothilone analogs with similar Kb’s as deaza-Epo C (5) have been reported to exhibit sub-µM antiproliferative activity [26], and it was, therefore, surprising that 5 showed no growth inhibition of the human cancer cell lines MCF7 (breast) or A549 (lung) up to a concentration of 10 µM. In contrast, and as expected from the literature, Epo C inhibited the growth of MCF7 and A549 cells with IC50 values of 9 nM and 103 nM, respectively [33]. These ﬁndings triggered experiments on the stability of 5 in cell culture medium, which revealed that the compound was degraded with a half-life of less than 2 h; this is signiﬁcantly lower than the half-life of Epo C under identical conditions (see the Supporting Material). Due to limitations in the sensitivity of the analytical system, the experiments had to be carried out at a 100 µM concentration, which is 10-fold higher than the highest concentration tested in the growth inhibition experiments. While our stability data, thus, are largely qualitative in nature, they do indicate that the limited stability of deaza-Epo C (5) in cell culture medium may contribute to the lack of cellular potency. 4. Conclusions Deaza-epothilone C (5), which incorporates a thiophene heterocycle in place of the thiazole moiety in natural epothilones, has been prepared by semisynthesis from epothilone A. The synthesis Chemistry 2020, 2, 30 507 of 509 507 of 509 of building blocks 8 (from epothilone A) and 7 (from 3-thienylmethanol) was accomplished with reasonable eﬃciency; the synthesis of the alternatively protected acids 8a and 23 proved to be more diﬃcult. Likewise, the elaboration of these building blocks into deaza-epothilone C was hampered by signiﬁcant diﬃculties, which (partly) reﬂect distinct diﬀerences in the chemical behavior between the thiophene-containing intermediates and their thiazole-derived congeners. As a consequence, deaza-epothilone C (5) was obtained from 7 in only 2.3% overall yield (three steps). Nevertheless, suﬃcient material could be procured to assess the binding of 5 to microtubules and its cellular activity. While the replacement of the thiazole nitrogen by a simple CH group causes a drop in the the free energy of binding of 5 relative to epothilone C, in agreement with structural data for the tubulin/Epo A complex and also previous (binding) data for quinoline-based epothilone analogs, the magnitude of the decrease (between 0.7 and 1 kcal/mol) is rather modest. Thus, the data indicate that, in principle, thiophene-derived analogs of epothilone A or B (or macrocycle-modiﬁed variants thereof) should be high-aﬃnity microtubule binders. However, high-aﬃnity microtubule-binding of such analogs may not translate into potent cellular activity, as indicated by the reduced stability (compared to epothilone C) and lack of cellular activity observed for deaza-epothilone C (5). At the same time, stability may be enhanced by appropriate modiﬁcation of the thiophene ring (e.g., by the replacement of the methyl group by a quasi-isosteric electron-withdrawing chlorine atom). These questions will have to be clariﬁed in future experiments. Supplementary Materials: The following are available online at http://www.mdpi.com/2624-8549/2/2/30/s1, synthesis protocols and analytical data for all new compounds, Figure S1: Decoupling experiments with 28, Figure S2: Decoupling experiments with 29, Figure S3: Stability of deaza-Epo C (5) in Dulbecco’s Modiﬁed Eagle Medium (DMEM) over a 2 h time period, Figure S4: Stability of deaza-Epo C (5) in DMEM over a 24 h time period, Figure S5: Stability of Epo C in DMEM over a 2 h time period, Figure S6: Stability of Epo C in DMEM over a 24 h time period. 4. Conclusions Author Contributions: Conceptualization, K.-H.A.; methodology, K.-H.A., A.E., J.F.D.; formal analysis, A.E., K.-H.A., J.F.D.; investigation, A.E., L.R., D.C., J.E.-G.; data curation, A.E., J.E.-G.; writing, K.-H.A.; supervision, K.-H.A.; funding acquisition, J.F.D. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by institutional funding from ETH Zürich (A.E., K.-H.A., L.R., D.C.) and by Ministerio de Economia y Competitividad grants BFU2016-75319-R to J.F.D. (both AEI/FEDER, UE) and H2020-MSCA-ITN-2019 860070 TUBINTRAIN to J.F.D. and a donation from Club deportivo Escuela Hungaresa de Pontevedra. No funds were received to cover the costs for publication in open access. Acknowledgments: The authors acknowledge networking contributions by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”. We are indebted to Bernhard Pfeiﬀer (ETHZ) for NMR support, and to Xiangyang Zhang, Louis Bertschi, Rolf Häﬂiger and Oswald Greter (ETHZ) for HRMS spectra. Kurt Hauenstein is acknowledged for excellent technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2485554478	https://research-information.bris.ac.uk/files/145380589/Full_text_PDF_final_published_version_.pdf	English	null	Is there a role for <scp>IGF</scp>‐1 in the development of second primary cancers?	Cancer medicine	2,016	cc-by	12,300	Shanmugalingam, T., Bosco, C., Ridley, A. J., & Van Hemelrijck, M. (2016). Is there a role for IGF-1 in the development of second primary cancers? Cancer Medicine, 5(11), 3353-3367. https://doi.org/10.1002/cam4.871 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1002/cam4.871 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Wiley at https://doi.org/10.1002/cam4.871 . Please refer to any applicable terms of use of the publisher. Shanmugalingam, T., Bosco, C., Ridley, A. J., & Van Hemelrijck, M. (2016). Is there a role for IGF-1 in the development of second primary cancers? Cancer Medicine, 5(11), 3353-3367. https://doi.org/10.1002/cam4.871 Shanmugalingam, T., Bosco, C., Ridley, A. J., & Van Hemelrijck, M. (2016). Is there a role for IGF-1 in the development of second primary cancers? Cancer Medicine, 5(11), 3353-3367. https://doi.org/10.1002/cam4.871 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 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. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Cancer Medicine 2016; 5(11):3353–3367 doi: 10.1002/cam4.871 doi: 10.1002/cam4.871 doi: 10.1002/cam4.871 Correspondence Thurkaa Shanmugalingam, Division of Cancer Studies, Cancer Epidemiology Group, Research Oncology, King’s College London, 3rd Floor, Bermondsey Wing, Guy’s Hospital, London SE1 9RT, United Kingdom. Tel: +44 0 20 7188 7904; Fax: +44 0 20 7188 9986; E mail: thurkaa t shanmugalingam@kcl ac uk Thurkaa Shanmugalingam, Division of Cancer Studies, Cancer Epidemiology Group, Research Oncology, King’s College London, 3rd Floor, Bermondsey Wing, Guy’s Hospital, London SE1 9RT, United Kingdom. Tel: +44 0 20 7188 7904; Fax: +44 0 20 7188 9986; Fax: +44 0 20 7188 9986; E-mail: thurkaa.t.shanmugalingam@kcl.ac.uk E-mail: thurkaa.t.shanmugalingam@kcl.ac.uk Abstract Breast cancer, colorectal cancer, IGF-1, lung cancer, prostate cancer, second primary cancer Cancer survival rates are increasing, and as a result, more cancer survivors are exposed to the risk of developing a second primary cancer (SPC). It has been hypothesized that one of the underlying mechanisms for this risk could be mediated by variations in insulin-like growth factor-1 (IGF-1). This review sum- marizes the current epidemiological evidence to identify whether IGF-1 plays a role in the development of SPCs. IGF-1 is known to promote cancer develop- ment by inhibiting apoptosis and stimulating cell proliferation. Epidemiological studies have reported a positive association between circulating IGF-1 levels and various primary cancers, such as breast, colorectal, and prostate cancer. The role of IGF-1 in increasing SPC risk has been explored less. Nonetheless, several experimental studies have observed a deregulation of the IGF-1 pathway, which may explain the association between IGF-1 and SPCs. Thus, measuring serum IGF-1 may serve as a useful marker in assessing the risk of SPCs, and therefore, more translational experimental and epidemiological studies are needed to further disentangle the role of IGF-1 in the development of specific SPCs. Funding Information This research was supported by the Experimental Cancer Medicine Centre at King’s College London, Cancer Research UK (AJR), and also by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. Received: 9 October 2015; Revised: 8 June 2016; Accepted: 24 June 2016 Cancer Medicine 2016; 5(11):3353–3367 Cancer Medicine Open Access IGF-1 in First Primary Cancers This section provides an overview of evidence for the emerging role of IGF-1 in the development of first pri- mary cancers, with a focus on epidemiological studies (Table 1) as well as experimental studies investigating the underlying biological mechanisms. y g g IGF-1 is a single-chain polypeptide growth factor [28–30] that is related to insulin and IGF-2 [31]. IGF-1 stimulates cell growth, proliferation, and differentiation, and is essen- tial for normal organismal growth and development [32, 33]. IGF-1 binds to the insulin-like growth factor 1 recep- tor (IGF-1R), which is a tyrosine kinase receptor [34]. IGF-1 has a higher binding affinity than IGF-2 for IGF-1R. IGF-1R initiates a cascade of downstream signal transduc- tion pathways known to be involved in cell growth, pro- liferation, and cancer, including Ras/Raf/ERK and PI3K/ Akt/mTOR [35]. The majority of IGF-1 found in the circulation is produced by the liver, functioning as an endocrine hormone. IGF-1 is also produced in other organs where autocrine or paracrine mechanisms have a role [36]. Ample evidence indicates that IGF-1 and IGF-1R are important for growth and survival of cancer cells [37, 38] (Fig. 1). The expression of the IGF-1 gene is primarily regulated by growth hormone (GH), and to a smaller extent by various other hormones [35]. By contrast, IGF-1 that is synthesized locally in an autocrine or paracrine manner may stimulate growth of some cancers [36]. The circulating levels of IGF-1 change markedly with age, peaking at puberty, and slowly declining with increasing age; this fluctuation is regulated by GH, which itself has mitogenic and proliferative properties [35]. However, in other cell types, for example, cartilage cells, the growth- stimulating effects of IGF-1 are GH-independent [39]. Furthermore, GH deficiency is the most common disorder seen in survivors of childhood cancer, and there are con- cerns regarding its use in treating cancer survivors as it might increase the risk of SPCs [40]. Although IGF-1 possesses antiapoptotic, cell survival, and transforming activities, it is not classed as an oncogene. Recently, several studies have identified IGF-1 to be associated with an increased risk of developing a number of common cancers, including lung [21], breast [22], colorectal [23], and prostate [24]. Circulating levels of IGF-1 have been linked to the development of SPCs in men with head and neck squamous cell carcinoma [20]. Introduction cancers (SPCs). Therefore, identifying which cancer sur- vivors have a high risk of developing SPCs is crucial. It is well known that smoking [4, 5], obesity [6, 7], and insulin resistance [8, 9] are risk factors for the development of first primary cancers. However, the role of these risk factors in the development of SPCs in cancer survivors is less clear. There is some evidence that SPCs may be the result of genetic and hormonal risk factors [10–12], of late effects of chemo- and radio- therapy [2, 13], smoking and alcohol effects [14, 15], as well as nonmodifiable variables such as age and gen- der. For instance, a study based on the Swedish Family- Cancer Database concluded that, compared with the cancers (SPCs). Therefore, identifying which cancer sur- vivors have a high risk of developing SPCs is crucial. Within the last 40 years, cancer survival rates have doubled in the UK [1], mainly due to advances in treatment, and the increased detection of cancer at an early stage [2]. In England and Wales, approximately 50% of adult cancer patients diagnosed in 2010 to 2011 are predicted to survive 10 years or more [1]. In the US, the 5-year relative sur- vival rate for all cancers diagnosed in 2004–2010 was 68%, an increase from 49% in 1975–1977 [3]. Side-effects of cancer treatment and possible underlying etiological mechanisms, such as IGF-1 metabolism, are thought to be implicated in the development of second primary It is well known that smoking [4, 5], obesity [6, 7], and insulin resistance [8, 9] are risk factors for the development of first primary cancers. However, the role of these risk factors in the development of SPCs in cancer survivors is less clear. There is some evidence that SPCs may be the result of genetic and hormonal risk factors [10–12], of late effects of chemo- and radio- therapy [2, 13], smoking and alcohol effects [14, 15], as well as nonmodifiable variables such as age and gen- der. For instance, a study based on the Swedish Family- Cancer Database concluded that, compared with the 3353 IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. “prostate cancer”, “colorectal cancer”, and “meta analysis”, combined with the keywords “second primary cancer” and “IGF 1”. All references of the selected articles were checked using hand searches. Introduction general population, males and females diagnosed with an initial primary cancer were 1.3–1.6 times more likely to develop a second cancer, respectively [16]. Epidemiological evidence suggests that survivors of cer- tain types of cancers have a higher risk of developing SPCs. For example, contralateral breast cancer is the most common SPC that develops in patients diagnosed with a first breast cancer, accounting for approximately 50% of all SPCs [17]. Furthermore, breast cancer has emerged as the most common solid cancer among female survivors of Hodgkin’s lymphoma (diagnosed in childhood), which is largely due to the high-dose chest irradiation for Hodgkin’s lymphoma [2, 18]. In addition, urological can- cers (bladder, kidney, testes, and penile cancers) are con- sistently more prevalent among men with prostate cancer [19]. Indeed, it has been hypothesized that variations in the insulin-like growth factor (IGF) pathway, specifically IGF-1 and its binding protein 3 (IGFBP-3), could account for the increased risk of SPCs [20]. IGF-1 in First Primary Cancers However, so far a role of IGF-1 in development of SPCs following diagnosis of prostate cancer, breast cancer, colo- rectal cancer, or lung cancer has not been analyzed. In addition, a disorder known as Laron syndrome which is associated with low circulating levels of IGF-1 and IGFBP-3 [25] are protected from developing cancer, but instead can develop diabetes and cardiovascular disease [26]. With the increase in number of cancer survivors, the long-term health outcomes of this population need to be carefully examined. Approximately, one in five cancers is diagnosed in those with a previous diagnosis of cancer, and hence, these “second primary cancers” are a leading cause of morbidity and mortality among cancer survivors [27]. It is therefore of interest to investigate the role of IGF-1 in the development of various SPCs as it can help us understand the potential underlying mechanism for carcinogenesis. This review therefore aims to identify whether IGF-1 plays a role in the development of SPCs, by assessing epidemiological evidence available to date. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Breast cancer We used a computerized literature search database (PubMed and EMBASE) to identify full text and abstract studies of English language, using human subjects and published between the years 1999 and 2015. Searches were performed with and without the Medical Subject Heading (MeSH) terms for “cancer”, “breast cancer”, “lung cancer”, Findings to date on the role of IGF-1 in breast cancer development vary depending on the study. An early case– control study conducted in 1993 demonstrated that cir- culating levels of IGF-1 were higher in women with breast cancer compared to women without breast cancer [41]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3354 IGF-1 in the development of first and second primary cancers IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. Table 1. Studies of cancer risk related to IGF-1 level. Author (Year) Control (n) Cases (n) Cancer risk related to IGF-1 level Reference Breast cancer Peyrat (1993) 92 44 Median concentrations: 26 ng/mL (BCa) versus 20 ng/mL (controls) [38] Endogenous Hormones and Breast Cancer Collaborative Group (2010) 1839 1032 OR for BCa in the highest versus lowest fifth of IGF1 concentration was 1.28 (95% CI: 1.14–1.44; P < 0.0001) [39] Rinaldi (2006) 312 202 Highest versus lowest quintile OR 1.38 (95% CI: 1.02–1.86; P = 0.01) for women who develop breast cancer after 50 years of age [40] Kaaks (2014) 259 193 OR=1.41 (95% CI: 1.01–1.98; P = 0.01 for the highest versus lowest quartile, for ER+ breast tumors overall (pre- and postmenopausal women combined) [41] Baglietto (2007) 42961 119 HR for BCa comparing the fourth with the first quartiles was 1.20 (95% CI: 0.87–1.65). [42] 19542 versus 736 68 versus 9 HR for BCa in older women comparing the fourth with the first quartiles (+60 years) was 1.61 (95% CI: 1.04–2.51) versus 0.60 (95% CI: 0.25–1.45) for younger women (<50 years) Renehan (2004) Meta-analysis of 4 studies Meta-analysis of 4 studies High concentrations of IGF-1 were associated with an increased risk of premenopausal BCa (OR comparing 75th with 25th percentile 1.65, 95% CI: 1.26–2.08; P < 0.001) [44] Shi (2004) 1306 779 Premenopausal women: Nearly 40% increase in BCa risk among those who had higher IGF-1 in the circulation (overall OR 1.39, 95% CI: 1.16–1.66). [45] 1552 911 No association in postmenopausal women (overall OR 0.93, 95% CI: 0.80–1.10). (Continued) Breast cancer Sugumar (2004) 1471 764 Subjects with higher circulating levels of IGF-1 had increased risk of premenopau- sal BCa with an OR of 1.74 (95% CI: 0.97–3.13; P = 0.06) [46] Schernhammer (2006) 158 79 RR for top versus bottom quartile of IGF-1 was 0.98 (95% CI: 0.69–1.39; P = 0.77) [47] Hankinson (1998) 92 46 Postmenopausal women: No association between IGF-1 concentrations and BCa risk (top vs. bottom quintile of IGF-1, RR = 0.85 [95% CI: 0.53–1.39]). [22] 35 35 RR of BCa among premenopausal women by IGF-1 concentration (top vs. bottom tertile) was 2.33 (95% CI: 1.06–5.16; P = 0.08) Lung cancer Ahn (2006) 101 38 OR for LCa risk by IGF-1 concentrations (highest vs. lowest quartile) was 0.69 (95% CI: 0.41–1.15); P = 0.26 [51] London (2002) 159 51 OR for LCa risk by IGF-1 concentrations (highest vs. lowest quartile) was 0.73 (95% CI: 0.43–1.24); P = 0.80 [53] Lukanova (2001) 47 23 OR for LCa risk by IGF-1 concentrations [54] Table 1. Studies of cancer risk related to IGF-1 level. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3355 IGF-1 in the development of first and second primary cancers IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. Author (Year) Control (n) Cases (n) Cancer risk related to IGF-1 level Reference Morris (2006) 11,072 843 Meta-analysis: OR for LCa risk by IGF-1 concentrations (highest vs. lowest quartile) was 1.02 (95% CI: 0.80–1.31); P = 0.64 [55] Yu (1999) 54 74 High plasma levels of IGF-1 were associated with an increased risk of LCa (OR = 2.06; 95% CI: 1.19–3.56; P = 0.01) [21] Chen (2009) Meta-analysis of 6 studies Meta-analysis of 6 studies Pooled OR for LCa risk by IGF-1 concentra- tions (highest vs. lowest quartile) was 0.87 (95% CI: 0.60–1.13) [56] Cao (2012) Meta-analysis of 6 studies Meta-analysis of 6 studies OR for LCa risk by IGF-1 concentrations (highest vs. lowest quartile) was 1.05 (95% CI: 0.80–1.37); P = 0.74 [57] Prostate cancer Mantzoros (1997) 52 51 Increment of 60 ng mL corresponded to an OR of 1.91 (95% CI: 1.00–3.73; P = 0.05) [62] Colorectal cancer Nomura (2003) 282 177 (colon cancer) 105 (rectal cancer) Weakly positive association of IGF-I with colon cancer. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Breast cancer Colon cancer cases in third (IGF-1 of 137–174 ng/mL) and fourth quartile (IGF-1 > 174 ng/mL) had increased risk compared with controls (OR of 2.2 and 1.8, respectively) No association of IGFI with rectal cancer [68] Palmqvist (2002) 336 110 (colon cancer) 58 (rectal cancer Increase in colon cancer risk with increasing levels of IGF-1 (OR of 2.30 and 2.66 for third and fourth quartile, respectively) Rectal cancer risk was inversely related to levels of IGF-1 (OR of 0.33 and 0.33 for third and fourth quartile, respectively) [69] Tripkovic (2007) 52 52 Increase in IGF-1 level was followed by a 3.15-fold increased risk for developing colon cancer with levels of IGF-1 > 310 ng/mL, whereas twice as many controls exhibited levels of IGF-1 < 107 ng/mL [70] Ma (1999) 318 193 Men in the highest quintile for IGF-I had an increased risk of colorectal cancer compared with men in the lowest quintile (RR = 2.51; 95% CI: 1.15–5.46; P = 0.02) [71] Kaaks (2000) 200 102 Colorectal cancer risk showed a modest but statistically nonsignificant positive association with levels of IGF-I [72] BCa, breast cancer; OR, odds ratio; CI, confidence intervals; HR, hazard ratios; RR, relative risk; LCa, lung cancer. 1B t d l l t d f th 2284 ith IGF I d Table 1. Studies of cancer risk related to IGF-1 level. (Continued) Table 1. Studies of cancer risk related to IGF-1 level. (Continued) Control (n) Cases (n) Cancer risk related to IGF-1 level Reference 11,072 843 Meta-analysis: OR for LCa risk by IGF-1 concentrations (highest vs. lowest quartile) was 1.02 (95% CI: 0.80–1.31); P = 0.64 [55] 54 74 High plasma levels of IGF-1 were associated with an increased risk of LCa (OR = 2.06; 95% CI: 1.19–3.56; P = 0.01) [21] Meta-analysis of 6 studies Meta-analysis of 6 studies Pooled OR for LCa risk by IGF-1 concentra- tions (highest vs. lowest quartile) was 0.87 (95% CI: 0.60–1.13) [56] Meta-analysis of 6 studies Meta-analysis of 6 studies OR for LCa risk by IGF-1 concentrations (highest vs. lowest quartile) was 1.05 (95% CI: 0.80–1.37); P = 0.74 [57] 52 51 Increment of 60 ng mL corresponded to an OR of 1.91 (95% CI: 1.00–3.73; P = 0.05) [62] 282 177 (colon cancer) 105 (rectal cancer) Weakly positive association of IGF-I with colon cancer. Breast cancer Colon cancer cases in third (IGF-1 of 137–174 ng/mL) and fourth quartile (IGF-1 > 174 ng/mL) had increased risk compared with controls (OR of 2.2 and 1.8, respectively) No association of IGFI with rectal cancer [68] 336 110 (colon cancer) 58 (rectal cancer Increase in colon cancer risk with increasing levels of IGF-1 (OR of 2.30 and 2.66 for third and fourth quartile, respectively) Rectal cancer risk was inversely related to levels of IGF-1 (OR of 0.33 and 0.33 for third and fourth quartile, respectively) [69] 52 52 Increase in IGF-1 level was followed by a 3.15-fold increased risk for developing colon cancer with levels of IGF-1 > 310 ng/mL, whereas twice as many controls exhibited levels of IGF-1 < 107 ng/mL [70] 318 193 Men in the highest quintile for IGF-I had an increased risk of colorectal cancer compared with men in the lowest quintile (RR = 2.51; 95% CI: 1.15–5.46; P = 0.02) [71] 200 102 Colorectal cancer risk showed a modest but statistically nonsignificant positive association with levels of IGF-I [72] nfidence intervals; HR, hazard ratios; RR, relative risk; LCa, lung cancer. culated from the 2284 women with IGF-I measured. GF-1 level. (Continued) BCa, breast cancer; OR, odds ratio; CI, confidence intervals; HR, hazard ratios; RR, relative risk; LCa, lung cancer. 1Breast cancer cases and person-years calculated from the 2284 women with IGF-I measured. 2Breast cancer cases and person-years. Since then, several epidemiological studies have reported that higher circulating levels of IGF-1 are associated with an increased risk of breast cancer [22, 42–45]. This may arise as higher levels of IGF-1 are associated with accel- eration of early carcinogenesis [36]. More recently, three meta-analyses demonstrated positive associations between IGF-1 and risk of breast cancer among premenopausal, but not postmenopausal women [46–48]. A study by Schernhammer et al. aims to explain these observations by showing that premenopausal women with high IGF-1 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3356 IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. Figure 1. The effects of IGF-1 and IGF-1R on normal and cancerous cells. Cancer cell Normal cell IGF-1 IGF-1 IGF-1R IGF-1R Binds to Binds to Proliferation Proliferation Apoptosis Apoptosis Stimulate, Inhibit, Increased levels Transformation Binds to Binds to IGF-1R Proliferation Apoptosis Proliferation Apoptosis Cancer cell Transformation Increased levels Stimulate, Figure 1. Breast cancer The effects of IGF-1 and IGF-1R on normal and cancerous cells. particularly in premenopausal women. Moreover, experi- mental evidence suggests that a link between IGF-1 and estrogen may explain this positive association, but perhaps only in breast cancers that express the estrogen receptor. levels were at risk of higher IGF-1R activation in mam- mary epithelial cells, which is suggested to increase survival of these cells with accumulating DNA damage, thereby facilitating stepwise carcinogenesis [49]. These results may indicate the importance of IGF-1 levels in younger women in early life or its possible interaction with other hormones such as estrodial [36] and growth hormone [39]. In con- trast to these studies, a large prospective study pooling two Swedish cohorts found no association between cir- culating IGF-1 and risk of breast cancer, regardless of menopausal status [50]. It is unclear why there is such a discrepancy in study findings. However, differences in findings may be due to timing of blood sampling, the patient cohort or the subset of breast cancer. Prospective studies are advantageous over retrospective studies since blood samples to measure IGF-1 levels are collected before the clinical diagnosis of cancer and hence reverse causa- tion (i.e., effects of an undiagnosed cancer on levels of IGF-1) is less likely to play a role [50]. levels were at risk of higher IGF-1R activation in mam- mary epithelial cells, which is suggested to increase survival of these cells with accumulating DNA damage, thereby facilitating stepwise carcinogenesis [49]. These results may indicate the importance of IGF-1 levels in younger women in early life or its possible interaction with other hormones such as estrodial [36] and growth hormone [39]. In con- trast to these studies, a large prospective study pooling two Swedish cohorts found no association between cir- culating IGF-1 and risk of breast cancer, regardless of menopausal status [50]. It is unclear why there is such a discrepancy in study findings. However, differences in findings may be due to timing of blood sampling, the patient cohort or the subset of breast cancer. Prospective studies are advantageous over retrospective studies since blood samples to measure IGF-1 levels are collected before the clinical diagnosis of cancer and hence reverse causa- tion (i.e., effects of an undiagnosed cancer on levels of IGF-1) is less likely to play a role [50]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Lung cancer Studies to date have investigated the association between IGF-1 and lung cancer. Several studies have shown that circulating IGF-1 levels were not associated with an increased risk of lung cancer [53–57]. One case–control study found a positive association between IGF-1 and risk of lung cancer (OR: 2.06; 95% CI: 1.19–3.56) [21]. Furthermore, this study identified that the levels of IGF-1 and IGF-2 in plasma were not influenced by cigarette smoking [21]. IGFBP-3 is the main IGF-1-binding protein in blood. IGFBP-3 is generally considered to act as a tumor sup- pressor gene by reducing the ability of IGF-1 to promote cell survival and proliferation [58]. Although epidemio- logical studies overall found no association for IGF-1, a reduced risk of lung cancer is reported with higher cir- culating levels of IGFBP-3, when comparing the highest quartile versus lowest quartile of IGFBP-3 in a Chinese prospective study (OR: 0.50, 95% CI: 0.25–1.02) [55]. Moreover, several meta-analyses have also reported an inverse association between IGFBP-3 and risk of lung cancer [58, 59]. Estrogen plays an important role in the etiology of breast cancer, and there are experimental studies reporting cross-talk between IGF-1 and the estrogen receptor (ER) in mammary cells, possibly through synergistic effects that contribute to breast carcinogenesis [44, 51]. Stewart et al. showed that estrogen increases IGF-1 binding and IGF-1R mRNA levels in the estrogen-sensitive MCF-7 cell line by 7- and 6.5-fold, respectively [52]. This suggests that one potential mechanism by which estrogen stimulates breast cancer cell proliferation may involve sensitization of IGF-1 [52]. It is possible that both IGF-1 and IGFBP-3 contribute to the development of lung cancer. Cell culture studies have found that lung cancer cell lines, regardless of their Thus far, epidemiological evidence overall suggests a positive association between IGF-1 and breast cancer risk, 3357 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. histological subtypes, have the capacity to express IGF-1 and its binding protein, IGFBP-3, both in tumors and blood [60, 61]. vivo studies have shown significantly reduced proliferation rates in PC-3 prostate cancer cell lines in IGF-1-deficient hosts, compared to control hosts [66]. Exogenous IGF-1 increased the invasive potential of the DU145 prostate cancer cell line, which was dependent on IGF-1R, the ERK MAPK pathway, and the PI3K pathway [67]. Prostate cancer Associations between prostate cancer and IGF-1 have been studied extensively, and consistently show a positive asso- ciation. Since 1993, it has been investigated whether higher circulating IGF-1 levels are associated with an increased risk of prostate cancer [62, 63]. Early studies failed to demonstrate an association between IGF-1 and prostate cancer risk. The first significant positive association between IGF-1 and prostate cancer was examined in a case–control study by Mantzoros et al. By comparing men with prostate cancer to healthy controls, the odds ratio per 60 ng/mL increment in circulating levels of IGF-1 was 1.91 (95% CI: 1.00–3.73) [64]. Furthermore, the authors also men- tioned that this association is further reinforced by the lack of association between IGF-1 and benign prostatic hyperplasia. Lung cancer Furthermore, prostate cancer epithelial cells can stimulate their own growth by synthesizing and responding to IGF-1 in an autocrine manner (Fig. 2), as opposed to paracrine signaling [68]. Thus, until now, there is little evidence for a link between IGF-1 and lung cancer risk, but an inverse asso- ciation between IGFBP-3 and lung cancer risk has been observed. These epidemiological observations are consistent with experimental data, which demonstrates that IGFBP-3 block the mitogenic and antiapoptotic effects of IGF-1 on lung cancer cells [21, 55]. Overall, epidemiological and experimental evidence to date suggest a positive association between circulating IGF-1 and prostate cancer risk. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Colorectal cancer Overall, studies have provided data showing that colorectal cancer is positively associated with IGF-1 levels [23, 69–72]. Early studies in the late 1990s, suggested that high cir- culating IGF-1 concentrations are associated with an increasing risk of colorectal cancer [23, 73]. Nomura et al. evaluated the association between IGF-1 and colon and rectal cancer separately [69]. They showed a higher risk of colon cancer for those with IGF-1 levels in the third (137–174 ng/mL) and fourth quartiles (IGF-1 > 174 ng/ mL) as compared to the controls (OR of 2.2 and 1.8, respectively). There was no association between IGF-1 and rectal cancer. However, another study found a decreased risk of rectal cancer with high levels of circulating IGF-1 [70]. The authors commented that this may be due to rectal cancer presenting at an earlier stage than colon cancer, which may have masked the association with IGF-1 IGF-1 is known to stimulate the growth of prostate cancer cells by inducing cell proliferation and inhibiting apoptosis [65]. The effect of IGF-1 on prostate cancer cell lines has been extensively explored. For example, in Figure 2. The autocrine and paracrine actions of IGF-1. Stromal cell IGF-1R IGF-1 Paracrine action Cancer cell Figure 2. The autocrine and paracrine actions of IGF-1. Stromal cell IGF-1R IGF-1 Paracrine action Cancer cell 3358 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Figure 2. The autocrine and paracrine actions of IGF-1. IGF-1R Cancer cell IGF-1 Autocrine action Stromal cell IGF-1R IGF-1 Paracrine action Cancer cell IGF-1R Cancer cell IGF-1 Autocrine action Stromal cell Cancer cell IGF-1 Cancer cell Figure 2. The autocrine and paracrine actions of IGF-1. Figure 2. The autocrine and paracrine actions of IGF-1. 3358 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. levels [70]. On the other hand, data from two nested case–control study showed no statistically significant asso- ciation between IGF-1 and colorectal cancer risk [74, 75]. lymphoma during radiation treatment, or the duration and dose of treatment [88, 89]. Even in the absence of treatment effects, in general, younger women are at a greater risk of developing a second primary breast cancer than older women [90, 91]. For instance, a cohort study by Hancock et al. Colorectal cancer Therefore, further research is required to investigate the role of obe- sity, ethnicity, and dietary habits possibly as confounders to IGF-1 and colorectal cancer risk. Breast cancer The risk of developing a second primary breast cancer in a patient diagnosed with a first breast cancer has been studied extensively. It has been shown that in women with breast cancer, the risk of developing a new primary breast cancer in the contralateral breast is much higher than for healthy women developing a first breast cancer [84, 85]. Only a small portion of this large risk is attrib- utable to effects of treatment: lifestyle and genetic factors also need to be taken into account [84], and possibly the role of IGF-1 in increasing breast cancer risk. IGF-1 in Second Primary Cancers This section summarizes studies on the emerging role of IGF-1 in the development of SPCs with a focus on the definition of SPC as well as their potential link with IGF-1 in the case of patients with primary breast, lung, prostate, and colorectal cancer. SPCs are defined as malignant tumors diagnosed at the same time as the primary tumor or later, which are in a different organ [82] and are not a metastasis or recur- rence of the original primary cancer [83]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Colorectal cancer which reviewed women treated for Hodgkin’s disease between 1961 and 1990 (mean follow- up, 10 years) concluded that the greatest risk was seen in young women treated before the age of 15 years (RR: 136, 95% CI: 34–371), with a significant decline in rela- tive risk with advancing age (above 30 years of age, RR: 0.7, 95% CI: 0.2–1.8) [92]. The epidemiological data on IGF-1 and colorectal cancer is supported by laboratory evidence. The IGF-1R is expressed in both normal colonic mucosa and human colorectal cancers [70]. IGF-1 has been speculated to cause proliferation of colorectal cancer cells and promote over- expression of the IGF-1R in several studies, with no uni- form consensus [76–79]. Furthermore, in acromegaly, a condition that is associated with high IGF-1 levels, studies have shown that there is increased proliferation of normal colonic epithelium with an increased risk of developing colorectal adenomas and cancers in acromegaly patients [80, 81]. From a biological point of view, the pubertal growth of the mammary gland is mediated predominantly by the actions of IGF-1 and GH via estrogen [93]. It is postu- lated that this mechanism may make younger women more prone to developing a secondary breast cancer due to the increased levels of both IGF-1 and estrogen during puberty, and the promoting effects of IGF-1 [41, 94]. According to the evidence from in situ hybridization, in breast cancer, IGF-1 is predominantly expressed in the stromal cells (mainly fibroblasts) and very rarely in the breast epithelium [28, 95]. This supports the concept of a paracrine role of IGF-1 in breast cancer (Fig. 2). It is possible that there is also an endocrine role, given that circulating IGF-1 in the bloodstream is implicated in the malignant transformation of breast tissue [28, 43, 96]. IGF-1 functions to protect breast cancer cells from apop- tosis and induces survival [28], suggesting that locally synthesized IGF-1 influences the growth of human breast cancer cells. It can therefore be hypothesized that IGF-1 produced by the stromal cells is increased in breast cancer (Fig. 2). This may then promote growth of a second primary breast cancer by entering the blood stream and acting in an endocrine fashion [94, 97]. In colorectal cancer, the circulating levels of IGF-1 is particularly influenced by nutritional status. Lung cancer The lungs are often regarded as one of the most common organs to develop a SPC [11, 98, 99]. Common causes of a secondary lung cancer include a resected primary lung cancer, treatment-related complications in breast cancer and head and neck cancers [99–101], as well as continued smoking [102, 103]. In a US study, Johnson reported a 2–14% risk of developing a second lung cancer per person per year, with the risk increasing from twofold to sevenfold after 10 years of initial lung cancer diagnosis [104]. Breast cancer is also a common SPC in itself. For instance, it is the most common SPC that develops in young women treated for Hodgkin’s lymphoma with supradiaphragmatic irradiation [86]. The estimates of cumulative risks of developing breast cancer ranged from 35% by 40 years of age [87] to 48% by 40 years of age [88] after treatment. Much of this variation may be due to the differences in the age of diagnosis of Hodgkin’s Head and neck cancer patients are at an increased risk of developing lung cancer with a standardized incidence ratio (SIR) of 3.75 (95% CI: 3.01–4.62) [98]. In addition, according to data from the Surveillance, Epidemiology, and End Results (SEER) registries, approximately 5% of 3359 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. cancers [111, 112]; this seems to be influenced by pelvic radiation therapy for prostate cancer [108]. Moreover, diagnostic bias is thought to play a role due to anatomy. However, Chrouser et al. did not observe an increased risk of bladder cancer after radiotherapy for prostate cancer [113], and there are some uncertainties in relation to the possible mechanism for the lack of association observed in this study. It is possible that there may have been an increased risk in this study that was not detected due to a short mean follow-up period of 7.1 years or likelihood of underreporting SPCs. Based on the current evidence, it seems that the risk of developing a SPC after prostate cancer is higher, particularly for other urological cancers. breast cancer survivors are diagnosed with a second pri- mary lung cancer [11]. Additionally, treatment of breast cancer patients with radiation postmastectomy has been shown to approximately double the risk of second primary lung cancer, especially in the ipsilateral lung [99]. Prostate cancer The risk of all SPCs following a diagnosis of a first pri- mary prostate cancer has been studied with variable results. In 1999, a Swiss study based on data collected from the Cancer Registries of the Swiss Cantons of Vaud and Neuchâtel investigated the risk of SPCs in prostate cancer survivors between 1974 and 1994. They found a signifi- cantly reduced incidence rate of all cancers in men diag- nosed with prostate cancer, compared with the general population (SIR: 0.7, 95% CI: 0.6–0.8) [107]. More recently, in 2014, a cohort study from the Swiss Canton of Zurich investigated the risk of SPCs in prostate cancer survivors between 1980 and 2010 [19]. They found an increased risk of SPCs among men with prostate cancer, compared to the general population (SIR: 1.11, 95% CI: 1.06–1.17). The inconsistency between these two studies may be due to the diagnosis of prostate cancer at an advanced stage with shorter survival in the earlier years of study [19]. Therefore, the chance of developing a SPC was lower than what it is currently, when prostate cancer is gener- ally diagnosed at a less advanced stage. In contrast to the biological mechanisms of breast and lung cancer, prostate cancer epithelial cells can stimulate their own growth by synthesizing and responding to IGF-1 [65, 68]. Furthermore, there is evidence that IGF-1 enhances the adhesion of prostate cancer cells and this promotes prostate cancer metastasis, possibly through the actions of IL-17 [118]. The potential data does not suggest a direct causative role for IGF-1 signaling in the progression and invasiveness of prostate cancer. The IGF-1 pathway activates a number of downstream signaling pathways, including the phosphatidylinositol-3 kinase (PI3-K) path- way, the protein kinase C pathway, the CREB pathway, and the mitogen-activated protein kinase (MAPK) pathway. These pathways contribute to prostate cancer through deregulation and constitutive activation of the pathway [67]. While the etiology of IGF-1 in second primary pros- tate cancer is unknown, it is plausible that those who develop prostate cancer may possess a common genetic, hormonal, or environmental factor that protects them from developing a SPC [119]. Prostate cancer survivors have a lower risk of developing cancers of the stomach, lung, and pancreas [108, 120], raising the question of whether these patients are “protected” against these malig- nancies, or whether it is simply that they are above the When looking at specific cancer types, Davies et al. Lung cancer The rates of second primary lung cancer among women diag- nosed with breast cancer before the age of 50 years is rising significantly, with the increase being as early as 1 year after breast cancer diagnosis [105]. Radiotherapy treatment for breast cancer seems unlikely as the sole cause of this rise (see methodological section) because a long-term latency period (5–10 years) is usually associated with radiotherapy treatment [105]; proteins or hormones such as IGF-1 should also be considered as predisposing factors. Prostate cancer is also commonly observed as a SPC in itself. Kok et al. concluded that in the first year fol- lowing a first cancer diagnosis, male cancer survivors have a 30% increased risk of developing prostate cancer as a SPC, partly due to increased diagnostic activity of the urological organs or incidental finding following health check-ups [114]. Other studies have also shown an excess risk of developing prostate cancer as a SPC after a diag- nosis of a bladder cancer as a first cancer [115, 116]. In addition, cancer survivors diagnosed with a first primary urological cancer may request for prostate-specific antigen (PSA) testing as a consequence of anxiety or persisting urological symptoms [114]. Furthermore, survivors of melanoma are also at increased risk of developing prostate cancer [117]. When considering the biological effects of IGF-1 in second primary lung cancer, it has been shown that lung mesenchymal cells locally synthesize IGF-1 which acts on the bronchial epithelium in a paracrine manner [56]. A possible mechanism explaining the role of IGF-1 in second primary lung cancer is that higher IGF-1 levels detected in lung cancer are probably regulated by the levels of tissue-derived IGF-1, but not circulating IGF-1 [106]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Colorectal cancer With the exposure to radiotherapy, one hypothesis suggests that in individuals with higher IGF bioactivity, there is enhanced survival of partially transformed cells which leads to a larger pool of targets for subsequent “hits” initiating colo- rectal carcinogenesis via the process of stepwise carcino- genesis and malignant transformation. A second hypothesis suggests that the time needed for the progression of a fully transformed cell to fully developed cancer is inversely associated with IGF bioactivity [69]. Secondly, when evaluating the effect of IGF-1 on SPCs, one has to consider sources of errors that cause misclas- sification of this biomarker. Nondifferential misclassifica- tion of IGF-1 may occur due to laboratory errors (e.g., specimen collection, processing, and storage) or changes in IGF-1 assays [126]. In addition, a single measurement of IGF-1 may not reflect the actual underlying levels. Repeated measurements would reflect long-term exposure and may be useful in the context of carcinogenesis [127]. Aside from misclassification of data for IGF-1, it is also possible to have misclassification related to the SPCs because it is not always possible from a pathological point of view to make a distinction between local recurrences, metastases, or a true SPC. In normal colonic tissue, IGF-1 binds with high affinity to the IGF-1R and activates specific insulin receptor sub- strates, which can modulate several downstream pathways involved in gene transcription, cell proliferation, and apoptosis [124]. Although the etiology of IGF-1 in second primary colorectal cancer is unknown, based on findings from normal colonic tissues, we can speculate about the potential complexity of this carcinogenic mechanism. With the exposure to radiotherapy, one hypothesis suggests that in individuals with higher IGF bioactivity, there is enhanced survival of partially transformed cells which leads to a larger pool of targets for subsequent “hits” initiating colo- rectal carcinogenesis via the process of stepwise carcino- genesis and malignant transformation. A second hypothesis suggests that the time needed for the progression of a fully transformed cell to fully developed cancer is inversely associated with IGF bioactivity [69]. Thirdly, when studying the association between IGF-1 and SPCs, one has to be aware of potential confounders such as smoking or treatment. In the case of lung cancer, the effect of current and past smoking needs to be removed to maximize the efficiency of the study [56] as some studies have shown that smoking decreases the levels of IGF-1 [128], while others have found no relationship [129]. Colorectal cancer It is therefore possible that cigarette-related car- cinogen exposure may overshadow the more subtle effects of IGF-1 on cancer development, which could explain the general lack of an association between IGF-1 and risk of lung cancer [54]. As a result, smoking may have an effect on IGF-1 levels as well as the risk of developing a SPC, but it is unlikely to be an intermediate in the pathway between IGF-1 and SPCs [130]. Furthermore, treatment received for the primary cancer may confound the association between IGF-1 and SPCs [114, 130]. Similarly to smoking, treatment may also alter the effect of IGF-1 as well as the risk of developing a SPC, but it is again unlikely to be an intermediate in the pathway between IGF-1 and SPCs [130]. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. IGF-1 in the development of first and second primary cancers age at which the risk of these tumors typical peak, which is at an earlier age [119]. Even though several studies suggest a link between IGF-1 and development of SPCs, several methodological issues need to be considered when assessing these epidemiologi- cal findings. Firstly, diagnostic bias may occur when the SPC is the main outcome of interest, as it may be detected following a diagnostic intervention related to the first primary tumor [115]. Aside from diagnostic activity, treat- ment related to this first primary tumor may also increase the risk of developing a second primary tumor (e.g., chemotherapy and radiation therapy) [115, 125]. Methodological considerations for epidemiological studies investigating the link between IGF-1 and risk of second primary cancers In the clinical setting, it may be problematic to absolutely define whether the second tumor is in fact a SPC or a recurrence or a metastasis, and a definitive diagnosis may only be possible histologically. Whether the results showed in this review were strictly according to the standard are unclear, so therefore we need to consider the results with caution. Prostate cancer reported that survivors of prostate cancer had a 40% lower risk of developing a SPC compared to the general male US population; the risk was lower for leukemias and cancers of the oral cavity, stomach, colon, liver, lung, and pancreas [108]. However, they observed a higher risk of developing bladder [109, 110], renal, and endocrine © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3360 Colorectal cancer Several studies have demonstrated an increased risk of developing secondary colorectal cancer following radio- therapy exposure, in particular, rectal cancer following radiation for prostate cancer and colorectal cancer fol- lowing abdominopelvic radiation for cervical cancer. Brenner et al. investigated the risk in prostate cancer patients who underwent radiotherapy or surgery and reported a significantly increased risk of rectal cancer in the radiotherapy group, particularly for long-term survi- vors, when comparing with the surgery group [121]. Furthermore, Baxter et al. observed a significant increase in the development of rectal cancer postradiation for prostate cancer [122]. However, radiation did not promote development of cancer in the remainder of the colon, suggesting that the effect of radiation is limited directly to irradiated tissue. In addition to prostate cancer patients, cervical cancer patients also seem to be at risk of devel- oping colon cancer, as observed by Chatruvedi et al. [123]. Several studies have demonstrated an increased risk of developing secondary colorectal cancer following radio- therapy exposure, in particular, rectal cancer following radiation for prostate cancer and colorectal cancer fol- lowing abdominopelvic radiation for cervical cancer. Brenner et al. investigated the risk in prostate cancer patients who underwent radiotherapy or surgery and reported a significantly increased risk of rectal cancer in the radiotherapy group, particularly for long-term survi- vors, when comparing with the surgery group [121]. Furthermore, Baxter et al. observed a significant increase in the development of rectal cancer postradiation for prostate cancer [122]. However, radiation did not promote development of cancer in the remainder of the colon, suggesting that the effect of radiation is limited directly to irradiated tissue. In addition to prostate cancer patients, cervical cancer patients also seem to be at risk of devel- oping colon cancer, as observed by Chatruvedi et al. [123]. In normal colonic tissue, IGF-1 binds with high affinity to the IGF-1R and activates specific insulin receptor sub- strates, which can modulate several downstream pathways involved in gene transcription, cell proliferation, and apoptosis [124]. Although the etiology of IGF-1 in second primary colorectal cancer is unknown, based on findings from normal colonic tissues, we can speculate about the potential complexity of this carcinogenic mechanism. Conflict of Interest None declared. 14. McLaughlin, V. H., A. Trentham-Dietz, J. M. Hampton, P. A. Newcomb, and B. L. Sprague. 2014. Lifestyle factors and the risk of a second breast cancer after ductal carcinoma in situ. Cancer Epidemiol. Biomark. Prev. 23:450–460. Acknowledgments 11. Mariotto, A. B., J. H. Rowland, L. A. Ries, S. Scoppa, and E. J. Feuer. 2007. Multiple cancer prevalence: a growing challenge in long-term survivorship. Cancer Epidemiol. Biomark. Prev. 16:566–571. This research was supported by the Experimental Cancer Medicine Centre at King’s College London, Cancer Research UK (AJR) and also by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. 12. Travis, L. B., W. Demark Wahnefried, J. M. Allan, M. E. Wood, and A. K. Ng. 2013. Aetiology, genetics and prevention of secondary neoplasms in adult cancer survivors. Nature reviews. Clin. Oncol. (R. Coll. Radiol.) 10:289–301. 13. Rheingold, S. R., A. I. Neugut, and A. T. Meadows. 2003. Therapy-related secondary cancers. 6th ed. BC Decker. Medicine H-FC, editor. Conclusion In spite of a consistent positive observation between IGF-1 and risk of first primary cancers (especially breast, prostate, and colorectal), the evidence for the role of IGF-1 in the development of SPCs is less clear. Some of the evidence we gathered came from targeting the IGF system in cell 3361 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. study of obesity and cancer risk (Sweden). Cancer Causes Control 12:13–21. culture studies, and therefore, we need to see the results with caution on whether it can be compared to clinical situations. However, the relevant influences of these path- ways in SPCs are unknown. This lack of an association may be partly explained by methodological issues. 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https://openalex.org/W4294267611	https://www.nature.com/articles/s41598-022-17607-z.pdf	English	null	Phase-based fast 3D high-resolution quantitative T2 MRI in 7 T human brain imaging	Scientific reports	2,022	cc-by	12,233	OPEN Amir Seginer1 & Rita Schmidt 2,3* Magnetic resonance imaging (MRI) is a powerful and versatile technique that offers a range of physiological, diagnostic, structural, and functional measurements. One of the most widely used basic contrasts in MRI diagnostics is transverse relaxation time (T2)-weighted imaging, but it provides only qualitative information. Realizing quantitative high-resolution T2 mapping is imperative for the development of personalized medicine, as it can enable the characterization of diseases progression. While ultra-high-field (≥ 7 T) MRI offers the means to gain new insights by increasing the spatial resolution, implementing fast quantitative T2 mapping cannot be achieved without overcoming the increased power deposition and radio frequency (RF) field inhomogeneity at ultra-high-fields. A recent study has demonstrated a new phase-based T2 mapping approach based on fast steady- state acquisitions. We extend this new approach to ultra-high field MRI, achieving quantitative high-resolution 3D T2 mapping at 7 T while addressing RF field inhomogeneity and utilizing low flip angle pulses; overcoming two main ultra-high field challenges. The method is based on controlling the coherent transverse magnetization in a steady-state gradient echo acquisition; achieved by utilizing low flip angles, a specific phase increment for the RF pulses, and short repetition times. This approach simultaneously extracts both T2 and RF field maps from the phase of the signal. Prior to in vivo experiments, the method was assessed using a 3D head-shaped phantom that was designed to model the RF field distribution in the brain. Our approach delivers fast 3D whole brain images with submillimeter resolution without requiring special hardware, such as multi-channel transmit coil, thus promoting high usability of the ultra-high field MRI in clinical practice. Non-invasive biomedical imaging provides high-impact medical diagnostics and offers an ideal means of pro- moting preventative medicine. This is indeed the case when it comes to ultra-high field (≥ 7 T) Magnetic Reso- nance Imaging (MRI)1–3. One high-value diagnostic MRI method is based on estimating the T2 relaxation time of tissues—either T2-weighted4 imaging or quantitative-T2 mapping5–7. T2-weighted MRI of the brain is one of the most widely employed routine diagnostic methods in cancer and neurodegenerative diseases. It is essential for the detection of hyperintense lesions pronounced in demyelinating diseases, such as multiple sclerosis8–10, and in the monitoring of disease progression9. In multiple sclerosis, improved precision at early stages of lesion formation would allow their clear categorization and aid in developing new tools to delay or eliminate the relapse. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2022) 12:14088 1Siemens Healthcare Ltd, Rosh Ha’ayin, Israel. 2Department of Brain Sciences, Weizmann Institute of Science, Rehovot, Israel. 3The Azrieli National Institute for Human Brain Imaging and Research, Weizmann Institute of Science, Rehovot, Israel. email: rita.schmidt@weizmann.ac.il OPEN Left—a design of a steady-state configuration based on Bloch simulations that provides θ(T2,α) for specific φinc, which was thereafter utilized to generate T2 and α in the 2D space (θ1, θ2). The new space allows to extract T2 and α from θ1 and θ2. Center—the estimation algorithm was assessed via simulations and brain-like phantom measurements. In these measurements, a realistic signal S was acquired, providing \|S\| and ∠S , from which the T2 and α (or B1 distribution) were estimated. Right—human imaging at 7 T MRI provided high-resolution whole-brain T2 maps, while coping with the B1 distribution. which are simultaneous multi-parametric acquisitions18,19 as well as a design for T1 and T2 weighted images in highly inhomogeneous static magnetic fields20. These include DESPOT221 and phase-cycled balanced steady- state free precession22–24 (bSSFP). A method called TESS19,25 shows promising results for T2 mapping without RF field dependence, however, currently it was demonstrated only as a 2D implementation for brain imaging26. Finally, a method analyzing the complex signal of a set of unbalanced GRE scans at 7 T gave T1 and T2 maps18, but included a long total scan duration (16:36 min) and used parallel transmission to mitigate the transmit field inhomogeneity. g y Recently, a new method was introduced based on a steady-state spoiled gradient-echo (SGRE) acquisition that utilizes low flip angles and short repetition times (TRs) to obtain T2 maps at 3 T MRI27, assuming a uniform and a priori known flip angle. While most of the GRE-based studies have focused on magnitude images20,21,28, in this study, phase information was highlighted, which offers a new and attractive method for T2 mapping. Building on this work we elucidate the dependence of the phase-based method on the (unknown) excitation flip angle in addition to the RF pulse phase, with an eye to design an approach suited for T2 mapping of the brain at 7 T. This new extension to the steady state method includes both T2 and RF field estimation and is designed to cover the relevant flip angle range arising in the brain due to the RF field inhomogeneity at 7 T MRI (see Fig. 1). The advantage of this approach is its ability to simplify the signal dependencies and reduce the confounding variables. This includes the removal of the static magnetic field (B0) dependence and a reduced dependence on the longitudinal relaxation time (T1). OPEN Recent studies at 7 T MRI have shown that we can detect smaller lesions than previously possible and so better monitor disease progression8. However, the robust characterization of disease progression with MRI requires quantitative T2 mapping, the use of which in clinics is impeded by its long scan duration. Novel fast methods11–13 encounter extra challenges in ultra-high field MRI among which are the severe RF field inhomogeneity14, which reduces the accuracy of the quantification, and the increased power deposition that results in prolonged scan duration. Common T2 methods are especially prone to the above drawbacks since they are spin-echo-based, requiring refocusing pulses that are high in Specific Absorption Rate (SAR)15 and whose effectiveness is sensitive to RF field inhomogeneity.h i g y Recent studies have proposed another solution—called Magnetic Resonance Fingerprinting (MRF)16,17. This method allows parametric MR mapping (including T1 and T2 maps), thus eliminating the dependence on the specific scan parameter or scanner. However, MRF is not easily translated into ultra-high field MRI, since over- coming the RF field inhomogeneity further complicates the acquired dataset17. Designs based on the steady-state gradient echo (GRE) pulse sequences offer a plethora of pathways toward multi-contrast fast acquisitions, among 1Siemens Healthcare Ltd, Rosh Ha’ayin, Israel. 2Department of Brain Sciences, Weizmann Institute of Science, Rehovot, Israel. 3The Azrieli National Institute for Human Brain Imaging and Research, Weizmann Institute of Science, Rehovot, Israel. email: rita.schmidt@weizmann.ac.il \| https://doi.org/10.1038/s41598-022-17607-z www.nature.com/scientificreports/ Figure 1. Schematics of the steady state method for T2 and RF field estimation—its design and verification. Starting from simulations, through assessment of the estimation algorithm, via a 3D head-shaped brain-like phantom, to human imaging. Left—a design of a steady-state configuration based on Bloch simulations that provides θ(T2,α) for specific φinc, which was thereafter utilized to generate T2 and α in the 2D space (θ1, θ2). The new space allows to extract T2 and α from θ1 and θ2. Center—the estimation algorithm was assessed via simulations and brain-like phantom measurements. In these measurements, a realistic signal S was acquired, providing \|S\| and ∠S , from which the T2 and α (or B1 distribution) were estimated. Right—human imaging at 7 T MRI provided high-resolution whole-brain T2 maps, while coping with the B1 distribution. Figure 1. Schematics of the steady state method for T2 and RF field estimation—its design and verification. Starting from simulations, through assessment of the estimation algorithm, via a 3D head-shaped brain-like phantom, to human imaging. Scientific Reports \| (2022) 12:14088 OPEN Figure 2a also shows the dependence of the phase of the signal on the excitation flip angle α. The α dependence curves have an extremum in the vicinity of 15°, i.e., the actual flip angle in that vicinity has a small effect on the phase. As the flip angle in the 3 T implementation was assumed to be given by the scan (due to relatively homogeneous RF field distribution), T2 values could be extracted solely from the phase of the signal. p g In our study, however, the combined (T2, α) dependence of the signal’s phase θ was exploited to cope with the RF field inhomogeneity at ultra-high field MRI. Neglecting, for now, the small T1 dependence of the phase—for T1 values relevant to brain tissues at 7 T, see Fig. 2a—the phase θ of the signal depends on the T2 at the voxel and on the actual flip angle α there. This α is the target flip angle of the scan αscan scaled by the RF field ratio at each voxel: α = αscan∙RFratio, where RFratio is the normalized RF field distribution. As the phase θ(T2, α) (see Fig. 2a) is not a one-to-one map of (T2, α) to θ, at least two measurements, θ1 and θ2, are needed; thus defining a 2D space (θ1, θ2). To extract T2 and α from θ(T2,α), we need a convenient 2D space to represent T2 and α in each voxel. Based on the Bloch simulations, such a 2D space can be generated by two scans with two flip angles, αscan1 and αscan2 = RFA∙αscan1 (RFA is a user set multiplication factor; for example, RFA = 2). Furthermore, we found that varying φinc between the two scans—one scan with (φinc1, αscan1) and a second with (φinc2, αscan2 = RFA∙ αscan1)—provides greater flexibility in controlling the 2D (θ1, θ2) space and its mapping to (T2, α). Figure 2b shows that different combinations of phase increment and flip angle pairs can be useful to adjust the range of viable flip angles and the T2 of interest.h The two phase measurements, θ1 for scan parameters (φinc1, αscan1) and θ2 for scan parameters (φinc2, αscan2), are functions of φinc1, φinc2, T2 and the (actual) flip angles, i.e., θ1 = θ(φinc1, α1, T2) and θ2 = θ(φinc2, α2, T2), where α1 and α2 are the actual flip angles. OPEN Building on the phase-based approach by Wang27,29, which departs from the traditional concepts based on spin-echo, our extension introduces a new T2 mapping solution for ultra-high field MRI. This method can deliver quantitative T2 mapping at 7 T MRI without requiring any additional hardware—such as dielectric pads or multi- channel transmit coil—to reduce the RF field inhomogeneity. Another advantage of this method is that it enables whole-brain imaging with high acceleration factors, as it relies on a 3D k-space acquisition. g g g p q Our study comprised three main steps (Fig. 1). First, we conducted Bloch simulations of the generated steady state signal for different scan parameters (such as RF flip angle, RF phase, and TR). Then, we looked for param- eter combinations that support the range of flip angles, due to the RF field inhomogeneity, in 7 T brain imaging. Next, we developed and assessed an efficient estimation algorithm. Lastly, we performed human brain imaging on a 7 T MRI scanner. The estimation algorithm was assessed on synthetic signals from the Bloch simulations, as well as on actual measurements, in a realistic setting, via a 3D head-shaped phantom, which was designed to model the RF field distribution in the brain30. For both the head-shaped phantom and the human brain imaging the phase-based method was compared with the gold-standard single-echo spin echo (SE–SE). Furthermore, we used the phase-based method to acquire whole-brain T2 maps with sub-millimeter resolution. Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z www.nature.com/scientificreports/ Figure 2. The phase of the steady-state signal as a function of T2 and the flip angle. (a) θ(T2, α, T1) dependence (Bloch simulation results) for a representative small φinc (φinc = 2°). The dependence on T2, α, and T1 is shown in 1D plots and in 2D. (b) T2 and α distributions in the new (θ1, θ2) 2D space. Two examples are shown: Top— (φinc1 = 2°, αscan1) with (φinc2 = 2°, αscan2 = 2αscan1). Bottom—(φinc1 = 3°, αscan1) with (φinc2 = 1.5°, αscan2 = 2αscan1). In each case, α (θ1, θ2) and T2 (θ1, θ2) are shown with the equi-T2 and equi-α lines. Figure 2. The phase of the steady-state signal as a function of T2 and the flip angle. (a) θ(T2, α, T1) dependence (Bloch simulation results) for a representative small φinc (φinc = 2°). The dependence on T2, α, and T1 is shown in 1D plots and in 2D. OPEN (b) T2 and α distributions in the new (θ1, θ2) 2D space. Two examples are shown: Top— (φinc1 = 2°, αscan1) with (φinc2 = 2°, αscan2 = 2αscan1). Bottom—(φinc1 = 3°, αscan1) with (φinc2 = 1.5°, αscan2 = 2αscan1). In each case, α (θ1, θ2) and T2 (θ1, θ2) are shown with the equi-T2 and equi-α lines. Principles of the modified‑SGRE sequence for simultaneous T2 and RF field mapping. The foundation for using phase increments during the RF pulse train was provided by Zur et al. in 198831,32. They showed that an RF pulse train with a quadratic phase φRF(n) = φinc∙(n2 + n)/2 for the n-th pulse—using an appro- priate φinc value in conjunction with a spoiling gradient—can achieve incoherent transverse magnetization, an effective spoiling better than simple gradient spoiling (φinc = 0 case). This is commonly called RF spoiling. Recent work by Wang27 at 3 T provided another keystone, in which the authors showed that small φinc values have the opposite effect; they introduce coherent transverse magnetization, where the phase of the signal possess a strong dependence on T2 (Fig. 2a). Figure 2a also shows the dependence of the phase of the signal on the excitation flip angle α. The α dependence curves have an extremum in the vicinity of 15°, i.e., the actual flip angle in that vicinity has a small effect on the phase. As the flip angle in the 3 T implementation was assumed to be given by the scan (due to relatively homogeneous RF field distribution), T2 values could be extracted solely from the phase of the signal. Principles of the modified‑SGRE sequence for simultaneous T2 and RF field mapping. The foundation for using phase increments during the RF pulse train was provided by Zur et al. in 198831,32. They showed that an RF pulse train with a quadratic phase φRF(n) = φinc∙(n2 + n)/2 for the n-th pulse—using an appro- priate φinc value in conjunction with a spoiling gradient—can achieve incoherent transverse magnetization, an effective spoiling better than simple gradient spoiling (φinc = 0 case). This is commonly called RF spoiling. Recent work by Wang27 at 3 T provided another keystone, in which the authors showed that small φinc values have the opposite effect; they introduce coherent transverse magnetization, where the phase of the signal possess a strong dependence on T2 (Fig. 2a). OPEN Although α1 and α2 are unknown, their ratio must obey α2/α1 = αscan2/αscan1 ≡ RFA. Thus, renaming α1 as α, we have θ1 = θ(φinc1, α, T2) and θ2 = θ(φinc2, RFA∙α, T2), or in a shorthand notation θ1 = θ1(α, T2) and θ2 = θ2(α, T2), where the functions θ1() and θ2() contain the known φinc1, φinc2, and RFA param- eters. One can now map T2 and α to the new (θ1, θ2) 2D space, written as T2(θ1, θ2) and α(θ1, θ2). Figure 2b shows that equi-T2 and equi-α lines are nearly orthogonal, when we are well inside the “balloon” (the support region), which is an indication of the robust estimation for a given set of (θ1,θ2) there. At the “balloon” edges of low or high α values the solution is ill-posed and can provide more than one solution, thus increasing the variability and Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z www.nature.com/scientificreports/ bias of the estimation at that region. Having now the simulated (θ1, θ2) 2D space for T2 and for α, one can point with any measured (θ1meas.,θ2meas.) to that space and provide the expected T2 and α values by a simple interpola- tion. This representation is useful to explore and characterize optimal choices of flip angles and φinc to achieve minimal variability and bias. Figure 2b shows that the set (φinc1 = 3°, αscan1) and (φinc2 = 1.5°, αscan2 = 2αscan1) covers a larger flip-angle range than set (φinc1 = 2°, αscan1) and (φinc2 = 2°, αscan2 = 2αscan1). We performed a detailed analysis to determine the optimal regime for whole-brain imaging, the results of which are summarized in Fig. S1–S4. Variability and bias evaluation + SAR considerations. We examined the variability and bias of the method in the range of flip angles relevant for brain imaging. To do so, noise was added to the simulated signal and the variability and bias of the method were examined as a function of T2 and α. The noise in the simulations was calibrated so the resulting synthetic signal to noise ratio (SNR) matched the measured SNR in agar tubes for the same α and T2, where the agar T2 was in a range matching white matter (WM) and gray matter (GM) at 7T18. The signal dependence on flip angle and phase increment showed that the phase of the signal is high for low φinc (φinc < 10°) (Fig. S1). OPEN It can be seen that the combination (φinc1 = 3°, αscan1) and (φinc2 = 1.5°, αscan2 = 2αscan1) provides a lower variability (i.e., lower std(T2 est.)) and a smaller bias (i.e., lower \|ave(T2 est.) − T2 true\|) for a larger range of flip angles (Fig. S2). We also examined three criteria (Fig. S3): the average estimation variability for 30 < T2 < 50 ms and 5° < α < 17°, and both the minimal and maximal flip angles that provide std(T2 est.) < 5 ms. The result of a combined minimization of the three criteria (shown in Fig. S3d) is a pair of scans with (φinc1 = 3°, αscan1) and (φinc2 = 1.5°, αscan2 = 2αscan1) that provides a good combination of the lowest average std(T2 est.) and supports a flip angle range of 3.7–35° (in which std(T2 est.) < 5 ms).ii l g g Figure S4 shows three additional aspects that were included to establish the final configuration, including the repetition time (TR), the RFA in a realistic experiment and reduction of the cerebrospinal fluid (CSF) signal. Although the combination (φinc1 = 3°, αscan1) and (φinc2 = 1°, αscan2 = 2αscan1) provides a better flip angle range (2.4–35°), in practice, φinc1 = 1° generates a high CSF signal. This can result in an extra signal and a residual artifact in the proximity of the ventricles. To reduce the CSF signal’s effect, it was found worthwhile to use φinc2 = 1.5° (Fig. S4a). As the change in relative variability as a function of TR (Fig. S4b) is insignificant, the choice of TR can be made by balancing between SAR limitations, on the one hand, and scan duration, on the other hand. A TR of 10 ms provided a practical tradeoff. Our examination of the effect of the RFA on the flip angle range showed that the higher the RFA, the better (Fig. S4c). However, to keep SAR within the “Normal” level, it was found that RFA in the range of 1.6–2 (with TR = 10 ms) provides a suitable flip angle range. In case of adopting “First level” SAR limit, one can increase the range of the flip angles. Global phase corrections. In practice, the phase ( ∠S ) of the signal S at a voxel is comprised of the steady- state phase θ(α, T2, T1) plus a global phase θ0. OPEN The global phase θ0 arises from several factors, with a dominant contribution from B0. It can be eliminated by repeating the scan twice, once with + φinc and once with -φinc, and setting θ(α, T2, T1) = ∠ S+ϕinc · conj S−ϕinc /2 (as was shown in Ref.27). The implemented acquisition thus includes four scans: the two scans (φinc1, αscan1) and (φinc2, αscan2) and their repetition with a negative phase incre- ment to remove θ0. Calculating the θ1 and θ2 in this method does not result in phase wrapping, since after the global phase removal, the signals’ phase is in the range of 0° to ~ 50°. Estimation algorithm. The actual estimation algorithm included two main steps, per voxel, namely the removal of the global phase (θ0) and an estimation of T2 and α from (θ1,θ2) using linear interpolation. An addi- tional step was established for low flip angles because low flip angles result in (θ1,θ2) measurement pairs close to the edges of the “balloon” (Fig. 2b), a region where interpolation is an ill-posed problem. Low flip angles are relevant for whole-brain imaging because despite the flip angle of the first scan being set to αscan1 = 15° , the actual whole-brain RF field distribution results in a flip angle in the range of ~ 4° to 22° (even reaching below 4° for some regions, see a representing distribution in Fig. 1). Brain regions where very low flip angles (~ 4–6°) are typically reached are the cerebellum, midbrain, and brainstem, as well as some regions in the temporal lobe. The added step to handle low flip angles takes advantage of two aspects: i) that α changes slowly in space, and ii) that for small flip angles (α < 20°) the phase θ is linear with T2, and that the slope itself is linear with the flip angle α. Detailed description of this step are in the “Materials and methods” section. Figure S5 shows the improve- ment attained using the second step for the low flip angles. Additional steps were also performed to improve the estimation for the expected low values of (θ1,θ2), which, due to noise, results in negative values (see “Materials and methods”). T1 corrections. As mentioned, phase dependence on T1 is small, but it can account for ~ 15% of the final T2 estimation. OPEN To reduce the error due to T1 in human imaging voxels were classified as either “high” or “low” T1 by empirically thresholding Sαscan2 / Sαscan1 . Separate maps—T2(θ1, θ2) and α(θ1, θ2)—were used for each clas- sification, based on T1 = 1 s (representing WM) and T1 = 2 s (the rest). With this correction, the error was further reduced (shown in Fig. S6). A detailed description of the algorithm is provided in the “Materials and methods” section. Results To examine the estimation bias and estimation variability we conducted two imaging experiments with phantoms, one with tubes filled with agarose suspension, the other with a 3D head-shaped phantom. In the first experiment (Fig. 3a), the variability was × 1.4 smaller than with SE–SE (0.5 ms compared to 0.7 ms). The aslope and the relative deviation error (see Eq. 1) calculated between the T2 from this method and the T2 from SE–SE were 1.01 and https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z www.nature.com/scientificreports/ Figure 3. Assessment of estimation bias and variability in phantoms. (a) Comparison of the T2 obtained with the phase-based method and SE–SE in agar tubes. Top—a central slice of the T2 maps and magnitude images. Bottom—estimated T2 for each tube as a function of T2 with SE–SE; aslope = 1.01, relative deviation error = 0.5%. The average standard deviation was 0.5 ms for the phase-based method, and 0.7 ms for SE–SE. (b–d) Comparisons using a 3D-head-shaped brain-like phantom. (b) T2 and α maps estimated by the phase-based method. (c) T2 map estimated with SE–SE. And (d) α map estimated using the vendor’s RF field mapping scan. Two main cross-sections are shown for all cases, Sagittal and Axial. For comparison, the average T2 and standard deviation was calculated in the same region of interest (marked by a blue contour for the phase-based method and a red contour for SE–SE). The average deviation between the α maps of the phase-based method and of the vendor’s RF mapping was calculated to be 0.56° for the Sagittal plane and 0.84° for the Axial plane. Figure 3. Assessment of estimation bias and variability in phantoms. (a) Comparison of the T2 obtained with the phase-based method and SE–SE in agar tubes. Top—a central slice of the T2 maps and magnitude images. Bottom—estimated T2 for each tube as a function of T2 with SE–SE; aslope = 1.01, relative deviation error = 0.5%. The average standard deviation was 0.5 ms for the phase-based method, and 0.7 ms for SE–SE. (b–d) Comparisons using a 3D-head-shaped brain-like phantom. (b) T2 and α maps estimated by the phase-based method. (c) T2 map estimated with SE–SE. And (d) α map estimated using the vendor’s RF field mapping scan. Two main cross-sections are shown for all cases, Sagittal and Axial. Results For comparison, the average T2 and standard deviation was calculated in the same region of interest (marked by a blue contour for the phase-based method and a red contour for SE–SE). The average deviation between the α maps of the phase-based method and of the vendor’s RF mapping was calculated to be 0.56° for the Sagittal plane and 0.84° for the Axial plane. .5%. Thus, the phase-based method provides a small bias and a lower variability compared to SE–SE, while the can duration is × 2.3 faster. In the second experiment, a specially designed 3D head-shaped brain-like phantom was used to examine the capability to cope with an RF field distribution similar to that in the brain. The “brain” had a uniform T2, which helped to separate the two parameters we sought to estimate, α and T2. Our results show low variability in T2 (std(T2 phase-based-method )/std(T2 SE–SE) = 0.46) and an RF field map estimation with little bias (a 4% average deviation from the map acquired with the vendor’s pulse sequence), see Fig. 3b. Even low flip angles, in the ill-posed area of the “balloon”, were well determined using the implemented estimation algorithm (Fig. S8).h g p g ( g ) The contribution of the B1 correction to the T2 estimate can be seen in Fig. 4. It compares T2 maps extracted from a set of four scans (two pairs) to T2 maps extracted from a single pair—as in Ref.27—using either of the pairs (either pair 1: φinc1 = 3° and φinc1 = − 3°, with αscan1; or pair 2: φinc2 = 1.5° and φinc2 = − 1.5°, with αscan2). It can be seen that for both phase increments the RF field inhomogeneity results in either underestimated or overestimated T2 values, depending on the actual flip angle in each voxel (see Fig. 2a for phase dependence on flip angle). The 4-scans result, which combines both phase increments, provides a uniform T2 map of the “brain” tissue in the 3D-head shaped phantom, as expected by the design. p p , p y g Figure 4 also shows the estimated T2 maps, for human imaging, based on either 4-scans or a single scan-pair. Results Although more challenging to observe, due to the heterogeneous T2 distribution in the brain and to the very high T2 values in the CSF regions, it can also be seen that T2, estimated from a single pair, is either underestimated or overestimated compared to 4-scans. This can be observed, for example, in regions such as the cerebellum and the temporal lobes. Table 1 summarizes the results by giving sample T2 values in white matter, grey matter and CSF. For each tissue 2 sampled regions were chosen as shown in Fig. 4—WM1 and WM2 in white matter tissue, GM1 and GM2 in the grey matter tissue and CSF1, CSF2 in the CSF. The table also shows T2 values reported in Ref. 18. Note: the CSF values are underestimated with the current method, as further elaborated in the Discussion section. Continuing with human imaging, Fig. 5 compares the phase-based method with 1.5 mm isotropic voxels to the gold standard SE–SE, for a T2 mapping comparison, and to the vendor RF mapping, for an RF field map- ping comparison. The α map in Fig. 5c was smoothed by 3 × 3 filter to reduce the effect of local CSF signals (see Fig. S13 for original high resolution B1 map). The RF field map extracted with the phase-based approach shows a distribution similar to the separately acquired vendor map with, however, noticeable deviations in the ventri- cles, as well as in some of the CSF region. The ratio of the T2 values and the relative deviation error between the phase-based method and SE–SE is shown in Fig. 6, for the different volunteers. Over all volunteers the T2 ratio https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 www.nature.com/scientificreports/ Figure 4. Comparison of T2 maps extracted with (a) 4-scans, (b) single pair with (φinc1, αscan1) and (c) single pair with (φinc2, αscan2). (d) For each case a plot for a line shown in the Sagittal and Axial scans. The images show 3D-head shaped phantom (left) and human imaging (right). The human axial plane image in (a) shows the regions that were examined and summarized in Table 1. Figure 4. Comparison of T2 maps extracted with (a) 4-scans, (b) single pair with (φinc1, αscan1) and (c) single pair with (φinc2, αscan2). (d) For each case a plot for a line shown in the Sagittal and Axial scans. The images show 3D-head shaped phantom (left) and human imaging (right). Results The human axial plane image in (a) shows the regions that were examined and summarized in Table 1. Table 1. Estimated T2 in sample regions of white matter, grey matter and CSF (see Fig. 4). Single pair (φinc1, αscan1) Single pair (φinc2, αscan2) 4-scans From Ref.18 WM1 19.00 ± 0.86 40.02 ± 2.34 28.83 ± 1.47 33.7 ± 0.7 WM2 16.28 ± 1.21 37.84 ± 4.04 26.74 ± 2.49 GM1 30.02 ± 5.77 60.67 ± 14.52 45.05 ± 10.23 49.2 ± 3.8 GM2 25.37 ± 3.10 59.05 ± 14.97 41.95 ± 9.52 CSF1 341.93 ± 105.73 447.95 ± 31.76 422.75 ± 33.30 CSF2 280.25 ± 3.10 436.36 ± 14.64 408.35 ± 15.73 Table 1. Estimated T2 in sample regions of white matter, grey matter and CSF (see Fig. 4). Single pair (φinc1, αscan1) Single pair (φinc2, αscan2) 4-scans From Ref.18 WM1 19.00 ± 0.86 40.02 ± 2.34 28.83 ± 1.47 33.7 ± 0.7 WM2 16.28 ± 1.21 37.84 ± 4.04 26.74 ± 2.49 GM1 30.02 ± 5.77 60.67 ± 14.52 45.05 ± 10.23 49.2 ± 3.8 GM2 25.37 ± 3.10 59.05 ± 14.97 41.95 ± 9.52 CSF1 341.93 ± 105.73 447.95 ± 31.76 422.75 ± 33.30 CSF2 280.25 ± 3.10 436.36 ± 14.64 408.35 ± 15.73 6 4567890) Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z (T2 phase-based-method/T2 SE–SE) and relative deviation error are 0.80 and 15.45% for WM, and 0.85 and 19.76% for GM (detailed description is in Supplementary Information S4). Finally, high-resolution whole-brain T2 mapping was performed with the phase-based method, with 1 mm and 0.85 mm isotropic voxels. To acquire whole-brain high-resolution images, × 5.11 acceleration was used— combining elliptic sampling and × 2 acceleration in both phase encoding directions. Each of the four scans with 1 mm resolution was 1:13 min giving a total scan time of 4:52 min. For 0.85 mm each scan was 1:42 min long and the total scan time was 6:48 min. Figure 7 shows the estimated T2 maps for the 0.85 mm scan (Fig. S12 Table 1. Estimated T2 in sample regions of white matter, grey matter and CSF (see Fig. 4). Results Single pair (φinc1, αscan1) Single pair (φinc2, αscan2) 4-scans From Ref.18 WM1 19.00 ± 0.86 40.02 ± 2.34 28.83 ± 1.47 33.7 ± 0.7 WM2 16.28 ± 1.21 37.84 ± 4.04 26.74 ± 2.49 GM1 30.02 ± 5.77 60.67 ± 14.52 45.05 ± 10.23 49.2 ± 3.8 GM2 25.37 ± 3.10 59.05 ± 14.97 41.95 ± 9.52 CSF1 341.93 ± 105.73 447.95 ± 31.76 422.75 ± 33.30 CSF2 280.25 ± 3.10 436.36 ± 14.64 408.35 ± 15.73 Table 1. Estimated T2 in sample regions of white matter, grey matter and CSF (see Fig. 4). (T2 phase-based-method/T2 SE–SE) and relative deviation error are 0.80 and 15.45% for WM, and 0.85 and 19.76% for GM (detailed description is in Supplementary Information S4). Finally, high-resolution whole-brain T2 mapping was performed with the phase-based method, with 1 mm and 0.85 mm isotropic voxels. To acquire whole-brain high-resolution images, × 5.11 acceleration was used— combining elliptic sampling and × 2 acceleration in both phase encoding directions. Each of the four scans with 1 mm resolution was 1:13 min giving a total scan time of 4:52 min. For 0.85 mm each scan was 1:42 min long and the total scan time was 6:48 min. Figure 7 shows the estimated T2 maps for the 0.85 mm scan (Fig. S12 https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 \| www.nature.com/scientificreports/ Figure 5. Human imaging—T2 from the phase-based method or SE–SE, and α from the phase-based method or the vendor’s scan. (a) SE–SE Sagittal magnitude image at TE = 30 ms and the estimated T2 maps in three main cross-sections. (b) An α map using the vendor’s pulse sequence. (c) Sagittal magnitude image with φinc = 3 and α = 15°, as well as the estimated T2 and α maps in three main cross-sections. α map shown here was smoothed by a 3 × 3 filter to reduce the effect of local CSF signal. Orange arrows point to the cerebellum and brainstem regions suffering from low flip angles due to B1 inhomogeneity; their inner structure is much more pronounced—and clearly visible—in the phase-based T2 images. Purple arrows point to a region in the CSF that resulted in a low magnitude signal. Figure 5. Human imaging—T2 from the phase-based method or SE–SE, and α from the phase-based method or the vendor’s scan. (a) SE–SE Sagittal magnitude image at TE = 30 ms and the estimated T2 maps in three main cross-sections. Results (b) An α map using the vendor’s pulse sequence. (c) Sagittal magnitude image with φinc = 3 and α = 15°, as well as the estimated T2 and α maps in three main cross-sections. α map shown here was smoothed by a 3 × 3 filter to reduce the effect of local CSF signal. Orange arrows point to the cerebellum and brainstem regions suffering from low flip angles due to B1 inhomogeneity; their inner structure is much more pronounced—and clearly visible—in the phase-based T2 images. Purple arrows point to a region in the CSF that resulted in a low magnitude signal. Figure 5. Human imaging—T2 from the phase-based method or SE–SE, and α from the phase-based method or the vendor’s scan. (a) SE–SE Sagittal magnitude image at TE = 30 ms and the estimated T2 maps in three main cross-sections. (b) An α map using the vendor’s pulse sequence. (c) Sagittal magnitude image with φinc = 3 and α = 15°, as well as the estimated T2 and α maps in three main cross-sections. α map shown here was smoothed by a 3 × 3 filter to reduce the effect of local CSF signal. Orange arrows point to the cerebellum and brainstem regions suffering from low flip angles due to B1 inhomogeneity; their inner structure is much more pronounced—and clearly visible—in the phase-based T2 images. Purple arrows point to a region in the CSF that resulted in a low magnitude signal. Figure 6. Comparison of T2 estimation between the phase-based method and SE–SE. The plot shows the ratio T2 phase-based-method/T2 SE–SE per volunteer, both for WM and for GM. The error bars depict the relative deviation error [see Eq. (1)]. Figure 6. Comparison of T2 estimation between the phase-based method and SE–SE. The plot shows the ratio T2 phase-based-method/T2 SE–SE per volunteer, both for WM and for GM. The error bars depict the relative deviation error [see Eq. (1)]. shows the 1 mm resolution images). To provide even higher robustness following the reduced SNR of the high- resolution datasets, we also incorporated denoising based on a DnCNN deep-learning network33 (provided in MATLAB, The Mathworks, Natick MA, for Gaussian noise removal). This entailed denoising of θ1 and θ2 before the estimation of T2. The denoising greatly improved the observed details of the cerebellum structure, a region with especially low flip angles (Fig. 7b). shows the 1 mm resolution images). Discussionh The expected rewards of pushing the limits and moving to 7 T MRI are increased spatial resolution and shorter scan durations. Both these features are essential for clinical and research imaging, all the more so for quantitative methods. However, scanning at 7 T also poses new challenges, including high power deposition and severe RF field inhomogeneity. The extended phase-based method shown here delivers high-resolution brain T2 imaging while overcoming the above challenges. This is achieved by relying on a modified 3D SGRE sequence, using the phase of the signal to encode the T2 dependence. The 3D SGRE images are also highly robust to B0 inhomoge- neity. This can be seen in the magnitude images of both the phantom example (Fig. 3a) and the human images (Fig. 5). The SE–SE is more distorted both at the edges of the agar tubes and near the nasal areas in the human images. The B0-dependent phase is reliably canceled out by the two scans with opposite phase increments (φinc) of the RF pulse train. However, shifts in the global phase between scans may occur, which will require correc- tions. Similarly, the scans may be sensitive to movements, which will affect the phase. Incorporating a second echo acquisition could be used to correct for both the phase shifts and motion34. Aiming to shorten the total scan duration, one can also consider estimation of the global phase from a single pair, thus reducing the num- ber of scans to three. However, in this case careful analysis and phase unwrapping will be required in the third, non-paired, scan. In this case, phase unwrapping can be especially challenging in regions with short T2 , where B0 changes rapidly, resulting in high local changes in the background phase. Very short T2 may also affect T2 estimation due to limited SNR in such regions.h The current implementation used a non-selective hard pulse for the 3D acquisition. Although this works well for whole brain acquisition as in this study, in other cases it can be a limitation. For faster acquisition and to limit potential aliasing, the use of slab-selective pulses is beneficial. Figure S9 shows that as long as the slab is thick enough, compared to the slice thickness, the estimated T2 is correctly estimated. However, for a single slice-selective acquisition, the simulation by which the T2 and RF field maps are estimated must also account for the slice profile. Results To provide even higher robustness following the reduced SNR of the high- resolution datasets, we also incorporated denoising based on a DnCNN deep-learning network33 (provided in MATLAB, The Mathworks, Natick MA, for Gaussian noise removal). This entailed denoising of θ1 and θ2 before the estimation of T2. The denoising greatly improved the observed details of the cerebellum structure, a region with especially low flip angles (Fig. 7b). https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Human whole-brain T2 maps with a 0.85 mm isotropic voxel. (a) without denoising, (b) with denoising, based on DnCNN model for Gaussian noise removal. Arrows point to the cerebellum region, which especially benefits from denoising. Top row, Sagittal and Coronal planes. Bottom two rows, six slices of the Axial plane, at 10 mm intervals. Figure 7. Human whole-brain T2 maps with a 0.85 mm isotropic voxel. (a) without denoising, (b) with denoising, based on DnCNN model for Gaussian noise removal. Arrows point to the cerebellum region, which especially benefits from denoising. Top row, Sagittal and Coronal planes. Bottom two rows, six slices of the Axial plane, at 10 mm intervals. Scientific Reports \| (2022) 12:14088 www.nature.com/scientificreports/ θ1, however, suffers from pronounced RF field inhomogeneity, which is removed by using two sets of scans (giving θ1 and θ2), as was implemented here, allowing the generation of T2 maps. g g In our study, the estimation algorithm is based on an interpolation procedure, where the simulated data serves as the ground-truth. This method is similar to the dictionary-based approach in MRF, but is based on two measurement points (θ1, θ2) that allow us to represent the parameters of interest, T2 and α, in the (θ1, θ2) 2D space. This offers the advantage of mapping the T2 of interest by a simple linear interpolation. An improvement in the estimation algorithm was implemented in the low flip angles’ range, which extended the viable flip angles (Figs. S5 and S8). In this study, we demonstrated the low variability and small bias of the estimations in both simulations and phantom experiments. In the phantom experiment with agar tubes, the method provides T2 estimation with low variability—a × 3.2 (1.4 × 2.3) lower variability-to-scan-time factor than that of SE–SE. The T2 values were estimated by the phase-based method with a small bias (aslope = 1.01 and relative deviation error of 0.5% compared to SE–SE). p However, the in-vivo T2 ratio of the phase-based method to SE–SE was 0.79 ± 0.16 for WM and 0.86 ± 0.19 for GM. Similarly, there is a ratio of × 0.82 and × 0.88 between the reported values with 4-scans in Table 1 to the values in Ref.18. This result is also similar to the results in Ref.22,34. Possible reasons for the different ratios found for WM and GM are a partial volume of GM and CSF as well as deviations due to T1. Although T1 has a small impact on the phase of the signal and its effect was reduced in our implementation. The ~ 0.8 ratio between the T2 estimated by the phase-based approach and by SE–SE could arise for several reasons, among which are a contribution due to exchange and magnetization transfer35, diffusion36, and different contributions of the fast and slow T2 components to the two methods37,38. For the magnetization transfer no discrepancy was observed between the estimated T2 values in the agarose tubes, although exchange mechanisms are known to be at work in agarose and therefore produce magnetization transfer effects. www.nature.com/scientificreports/ for large movement within a voxel, such as due to flow, the error of the estimated T2 can be significant; reaching 20%, for a velocity of 0.5 mm/s. Two simulations were also performed to analyze possible hardware inaccuracies: (i) a constant error in the actual RF-phase increment, (ii) a randomly distributed error in the actual phase of the RF pulse. In the first case, a constant error of 0.1° resulted in < 4% error, In the second, a randomly distributed error with σ = 0.2° resulted in a negligible error with standard deviation of 0.07 ms in the estimated T2. It is also important to note that the estimation of the T2 in the CSF and other tissues with high T2 values (> 0.5 s) is challenging with this method, since the signal’s phase curve slowly converges for T2 > 100 ms (see Fig. 2a) and so the T2 contours in the (θ1,θ2) space grow denser with T2 (see Fig. 2b). In addition, local intensity drops in CSF voxels, resulting in low SNR voxels, can occur due to fluid movement (purple arrows in Fig. 5 point to such area), thus further limiting T2 estimation of CSF.hh The important advantage of the phase-based approach for T2 mapping is its whole-brain coverage ability. The method shows robust results in the brainstem region and even in parts of the spinal cord (see Fig. 5). These results are achieved without the need for additional hardware to reduce the RF field inhomogeneity, such as dielectric pads or multi-channel transmit coil. Naturally, the method can also benefit from a dielectric pad or multi-channel transmit coils to improve the SNR, especially in regions with low flip angles. The current configuration (φinc1, φinc2, αscan1, RFA, and TR) was designed for the RF field distribution in the brain, and was shown to robustly extract the RF field distribution in the 3D head-shaped phantom (which has a slightly larger RF field inhomogeneity than in vivo). If another region will be of interest, the configuration—the RF pulse phase increments and the scan flip angles—can be adapted accordingly.i l p g p g y It is worth noting that θ1 on its own, calculated from the first pair of scans (with φinc1 = ± 3°), achieves a “T2 weighted” image (see Fig. S10 for the 0.85 mm case), unlike the magnitude of these scans. Discussionh This was already demonstrated in other T2 mapping methods such as balanced SSFP19. ih Another sensitivity of the method that requires discussion is the sensitivity to movement and potential inaccuracy in the RF pulse phase. Although we did not observe noticeable movement in our human scanning, a simulation to examine these vulnerabilities was performed (see Supplementary Information, Section S5). The movement was simulated assuming a constant velocity during the scan, which will result in an additional para- bolic phase term accumulated during the scan. Examining the error due to potential head movement of 1–2 voxels during the scan, it resulted in a small error, less than 1% for a movement of up to 5 mm/min. However, Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z www.nature.com/scientificreports/ Estimation algorithm. The estimation algorithm included the following steps: Estimation step #3: handling of negative θ1 or θ2. For θ1 < 0 (and θ2 > 0), the θ2 from step #1 together with αsmoothed from step #2 were used to estimate T2; using the above simulated θ2(T2,α) for the known α. Similarly, for θ2 < 0 (and θ1 > 0), θ1 and αsmoothed were used to estimate T2. Validation of the estimation algorithm was performed by generating N = 100 noisy repetitions of each point in the simulated datasets of θ1(T2,α) and θ2(T2,α). This was done using a fixed noise which resulted in the SNR varying with T2 and α, depending on the intensity at each point. The noise was fixed to produce an SNR of 180 for the simulated data at T2 = 38 ms and α = 13°; resembling the SNR in the human images acquired with 1.5 mm resolution. The SNR was set as an average SNR over the two signals \|S1\| and \|S2\|. To validate the simulations, the standard deviation of T2 was compared to a measured one in an agar-tubes experiment, both with the same SNR. For this validation two agar-tubes were used—with T2 values of T2 = 34 ms and T2 = 38 ms, representing WM and GM at 7 T. The flip angle distribution in this experiment was uniform (α = 13°). The measured and simulated SNR was 298, resulting in a T2 standard deviation of 0.36 ms in the measurement and 0.32 ms in the simulation, providing comparable results. The variability and bias of the method, under the simulated noise, were examined as a function of T2, α, φinc, TR and RFA. Pulse sequence considerations. The sequence is based on a Siemens 3D GRE sequence that was modi- fied to enable control over both the φinc and the gradient spoiler moment. The RF pulse we used was a hard pulse. An important aspect to consider is the gradient spoiler moment intensity and its effect on the T2 estimation, as well as on image artifacts (in the form of residual signals from spurious echoes). A set of scans was performed to examine the spoiler effect. The gradient spoiler moment needs to provide complete dephasing inside a voxel, which defines a preferable gradient moment size to be ≿ 1/Δr (Δr = √(Δx2 + Δy2 + Δz2)). Estimation algorithm. The estimation algorithm included the following steps: Estimation algorithm. The estimation algorithm included the following steps: Preparatory step #0.1: global phase removal. In practice, the phase ( ∠S ) of the signal S at a voxel is comprised of the steady-state phase θ(α, T2, T1) plus a global phase θ0. The global phase θ0 arises from several factors, with a dominant contribution from B0. It can be eliminated by repeating the scan twice, once with + φinc and once with -φinc, and setting θ(α,T2,T1)=∠ S+ϕinc · conj S−ϕinc /2 (as was shown in Ref.27). The implemented acquisition thus includes four scans: the two scans (φinc1, αscan1) and (φinc2, αscan2), and their repetition with a negative phase increment to remove θ0. Preparatory step #0.2 (optional): denoising. For high-resolution human imaging, a denoising procedure based on a DnCNN deep-learning network33 (provided in MATLAB 2021a, for Gaussian noise removal) was incor- porated. The denoising procedure was implemented on the measured θ1, θ2 with the command denoised_ θ1 = denoiseImage(θ1, net), where the net was set by the command net = denoisingNetwork(’dncnn’). Estimation step #1: T2 and α estimation by interpolation. First, using Matlab’s scatteredInterpolant(), we gener- ated two interpolants, T2(θ1, θ2) and α(θ1, θ2), which map (θ1, θ2) to the desired quantities T2 and α. These inter- polants were then used to estimate T2 and α from any (θ1, θ2) pair, at each voxel.ih p y p As mentioned, phase dependence on T1 is small, but it can account for ~ 15% of the final T2 estimation. Thus, in human imaging, to reduce the error due to T1, voxels were classified as either “high” or “low” T1 by empirically thresholding Sαscan2 / Sαscan1 . Separate maps—T2(θ1, θ2) and α(θ1, θ2)—were used for each classification, based on T1 = 1 s (representing white matter—WM) and T1 = 2 s (the rest). With this correction, the error was further reduced (see simulation results in Fig. S6). Estimation step #2: T2 estimation update for low flip angles. First, the flip angles α found in the previous step were smoothed, generating αsmoothed. For low flip angle voxels with αsmoothed < 4.5°, the flip angles were temporarily set to αtemp = 4.5°, and the matching temporary T2 quantities, T2-temp, were found by interpolation—using αtemp and θ2 (the phase from the scan using the higher flip angle, αscan2 = RFA∙αscan1). The final T2 was found through the linear connection T2 = (αtemp/αsmoothed)∙T2-temp. www.nature.com/scientificreports/ However, different effects of exchange in the living tissue can still be a factor contributing to the acquired complex signal of the steady state acquisition. We also examined potential diffusion contributions to the estimated T2 by scanning a sample of smoked fish (which had and ADC of ~ 0.6 × 10−3 mm2/s, similar to white matter) and did not observe a significant effect (not shown). One of the potential factors is the larger contribution of the fast-relaxing species compared to SE–SE, primar- ily due to much shorter echo times, which was also observed in several previous studies38. Thus, although the estimated T2 was robustly repeated in the volunteers’ data, the resulting ratio between the phase-based method and SE–SE in-vivo still requires further analysis.h q y The fast high-resolution T2 maps of the whole brain that were acquired—1 mm isotropic in 4:52 min and 0.85 mm isotropic in 6:49 min—offer a significant clinical gain. Further acceleration of the method should be possible. One option is to reduce the TR, however, this will require switching the SAR monitoring to the less restrictive “First” level. For this, the effect of the TR on the variability of the estimation was examined (see Fig. S4) and showed that shorter TR result in similar estimation variability. In addition, acceleration methods, tuned to the 3D SGRE acquisition and employing the Compressed Sensing technique, can achieve even higher accelera- tion factors. We also demonstrated the option of employing denoising based on deep-learning techniques that is trained to remove Gaussian noise. This further improves the quality of the images and can be used to further accelerate the scan.i Overall, the extension of the phase-based steady-state method to estimate both T2 and RF field map, dem- onstrated in this work, provides a fast and high-resolution acquisition method for quantitative T2 mapping of the whole brain at 7 T acquired with a single-channel transmit coil. Standardized high-resolution methods are imperative for 7 T MRI to advance multi-site studies and promote personalized medicine. Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z www.nature.com/scientificreports/ Materials and methods Bloch simulations. 1D single voxel simulations based on the Bloch equations were performed with a custom MATLAB (The Mathworks, Natick MA) code39 to examine the signal in steady state. The simulations included an excitation pulse, an acquisition and a net total spoiler (including the area of the acquisition) of 3/Δx (Δx the 1D voxel size). The number of initial repetitions to reach steady-state (“dummy scans”) was set to 500, which was verified to provide reliable steady states. Following the dummy scans, a single acquisition was simu- lated. The simulation was repeated over a grid of flip angles and T2 values, for different values of T1, φinc, and TR. The grid covered T2 from 0 to 200 ms with a resolution of 4 ms, and flip angles from 0° to 70° with a resolution of 1°. The resulting θ(T2, α) map was interpolated prior to its use in the estimation algorithm with 1 ms in T2 and 0.1° in alpha, generating θ1(T2, α) and θ2(T2, α) for relevant φinc and RFA factors. Estimation algorithm. The estimation algorithm included the following steps: www.nature.com/scientificreports/ Both compartments contained 0.1 mM gadopentetate dimeglumine (GdDTPA), for a T1 close to that of human white matter, and consisted of an agarose suspension of 2.5% and 3% for the “brain” and “muscle” compartments, respectively. NaCl (5.5 gr/L) was used to achieve an in-vivo-like RF field distribution. For details, see Ref.30. , α maps from the phase-based method were compared to the equivalent α maps generated by the vendor. As the RF field maps provided by the vendor are scaled to 90°, they were rescaled to the αscan of the phase-based method, before comparison. The average deviation between the α maps by the phase-based method and by the vendor were calculated in two main planes (Sagittal and Axial).h p g The common scan parameters for the phase-based method and SE–SE used in the agar-tube experiments in Fig. 3a) were: FOV 200 × 200 × 104 mm3, resolution 1.1 × 1.1 × 2 mm3, acquired matrix size 176 × 176 × 52. The phase-based method specific parameters were (φinc1 = 3°, α1 = 15°) and (φinc2 = 1.5°, α2 = 30°), TR/TE 10/2.2 ms, using 4 scans with a total scan duration of 6:06 min. The specific scan parameters for SE–SE were: TR—6500 ms, 3 scans with TE = 10,30,50 ms, × 3 in-plane acceleration, with a total scan duration of 19:04 min. The T2 and α maps were estimated based on Bloch simulation with T1 = 2 s.h p 1 The common scan parameters for the phase-based method and SE–SE that were used for the 3D head-shaped phantom in Fig. 3b): FOV 220 × 220 × 144 mm3, isotropic resolution of 1.5 mm, bandwidth per pixel 400 Hz. The phase-based method specific parameters were acquired matrix size 150 × 148 × 96, (φinc = 3, α = 15°) and (φinc = 1.5, α = 30°), TR/TE 10/2.1 ms, using 4 scans with a total scan duration of 9:28 min. The specific scan parameters for SE–SE (Fig. 3c) were: acquired matrix size 144 × 144 × 96, TR—6500 ms, 3 scans with TE = 10, 30, 50 ms, × 3 acceleration, with a total scan duration of 21:12 min. The vendor RF field map scan parameters (Fig. 3d): FOV 220 × 220 × 144 mm3, resolution 2.3 × 2.3 × 4 mm. www.nature.com/scientificreports/ provide dephasing for Δr ≿ 0.9 mm. As shown in Fig. S7b, under this moment, the estimated T2 did not change for the voxel sizes tested. provide dephasing for Δr ≿ 0.9 mm. As shown in Fig. S7b, under this moment, the estimated T2 did not change for the voxel sizes tested. MRI scanning. All scans in this study were performed on a 7 T MRI system (MAGNETOM Terra, Siemens Healthcare, Erlangen) using a commercial 1Tx/32Rx head coil (Nova Medical, Wilmington, MA). When comparing the results of the phase-based method to SE–SE, inside a region, the relative deviation error from the fit was calculated as MRI scanning. All scans in this study were performed on a 7 T MRI system (MAGNETOM Terra, Siemens Healthcare, Erlangen) using a commercial 1Tx/32Rx head coil (Nova Medical, Wilmington, MA). When comparing the results of the phase-based method to SE–SE, inside a region, the relative deviation error thcare, Erlangen) using a commercial 1Tx/32Rx head coil (Nova Medical, Wilmington, MA). When comparing the results of the phase-based method to SE–SE, inside a region, the relative deviation error the fit was calculated as (1) rel. dev. err = 100 · N i=1 T(phase-based-method) 2 −aslopeT(SE-SE) 2 N /ave aslopeT(SE-SE) 2 (1) where aslope is the slope found for each fit, and N is the number of voxels in the comparison. where aslope is the slope found for each fit, and N is the number of voxels in the comparison. e aslope is the slope found for each fit, and N is the number of voxels in the comparison. Phantom imaging. Five tubes with agar concentrations of 1.5, 2, 2.5, 3 and 3.5% were used to compare the phase-based T2 estimation to the gold standard SE–SE, using three TE values (10, 30 and 50 ms). A 3D head- shaped phantom that was designed to model the RF field distribution in the brain was used to examine the T2 and RF field estimation. This phantom was originally designed to include three sub-compartments30, suitable for mimicking brain, muscle and lipid tissues. However, the version used in this study was filled with two “tissue” types: the inner compartment mimicked the “brain” and the outer one, “muscle” (the planned lipid layer was also filled with “muscle”). Estimation algorithm. The estimation algorithm included the following steps: We found it useful to add a parameter to the pulse sequence that directly controls the net gradient spoiler moment (after all previous gradients had been rephased). The net spoiler was set to be equally distributed in all three directions, which was found useful in reducing artifacts. However, our experiments also showed that the gradient moment affected the measured phase, and thus the estimated T2. Figure S7a shows this dependence. Phantom experiments were used to calibrate the gradient spoiler moment to provide the T2 estimate closest to that from SE–SE. Accordingly, the gradient moment was set in all experiments to 0.015 [mT/m∙sec] in each direction. This moment is expected to Scientific Reports \| (2022) 12:14088 \| https://doi.org/10.1038/s41598-022-17607-z ificreports/ provide dephasing for Δr ≿ 0.9 mm. As shown in Fig. S7b, under this moment, the estimated T2 did not change for the voxel sizes tested. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The T2 and α maps were estimated based on Bloch simulation with T1 = 1.5 s (based on estimated T1 of the “brain” tissue). Human imaging. All methods were carried out in accordance with the Weizmann Institute of Science guidelines and regulations. This study was approved by the Internal Review Board of the Wolfson Medical Center (Holon, Israel) and all scans were performed after obtaining informed suitable written consents. Human scanning of six volunteers with isotropic 1.5 mm resolution was acquired for the comparison with SE–SE. The comparison was performed after the SE–SE and the phase-based method images were realigned using SPM12 (https://www.fil.ion.ucl.ac.uk/spm/) to ensure there was no movement between the scans.h ( pi p ) An additional volunteer was scanned with the phase-based method with 1 mm and 0.85 mm resolution. These scans were acquired with an acceleration of × 5.11—using elliptical sampling and × 2 acceleration in both phase encoding directions. The BART40 software was used to reconstruct this dataset. Scan parameters for the phase‑based method and SE–SE comparison with isotropic 1.5 mm voxel. Phase-based method: FOV 220 × 220 × 144 mm3, acquired matrix size 150 × 148 × 96, bandwidth per pixel 400 Hz, TR/TE 10/2.1 ms, (φinc1 = 3°,αscan1 = 15°), (φinc1 = 1.5°, αscan2 = 24–26°) (the αscan2 varied from 24° to 26° according to the specific volunteer’s 100% “Normal” SAR level), with duration of 4 scans—9:28 min. SE–SE: FOV 220 × 220 × 132 mm3, acquired matrix size 144 × 144 × 88, bandwidth per pixel 400 Hz, TR- 6500 ms, TE = 10, 30, 50 ms, using 3 scans with a total scan duration of 21:12 min. Vendor RF field map scan parameters: FOV 220 × 220 × 192 mm3, resolution 3 × 3 × 4 mm. Phase‑based method 1 mm resolution parameters. FOV 220 × 220 × 160 mm3, bandwidth per pixel 400 Hz, TR/ TE 10/2.7 ms, (φinc1 = 3°,αscan1 = 15°), (φinc1 = 1.5°, αscan2 = 25°), duration of 4 scans—4:52 min. Phase‑based method 0.85 mm resolution parameters. FOV 220 × 220 × 163 mm3, bandwidth per pixel 400 Hz TR/TE 10/2.7 ms, (φinc1 = 3°,αscan1 = 15°), (φinc1 = 1.5°, αscan2 = 25°), duration of 4 scans—6:49 min. https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 \| www.nature.com/scientificreports/ References W. On the accuracy and precision of PLANET for mul- tiparametric MRI using phase cycled bSSFP imaging Magn Reson Med 81 1534 1552 (2019) p g 23. Shcherbakova, Y., van den Berg, C. A. 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NeuroImage Clinical 14, 363–370 (2017) g p g 2. Emmerich, J. et al. Data availability ll ll d h y All scans collected in this study were performed according to procedures approved by the Internal Review Board of the Wolfson Medical Center (Holon, Israel). Since this protocol was not defined as an open repository, the data is not provided, to provide the ethics and privacy issues of clinical data. The code will be made available via a request to the corresponding author. Received: 24 January 2022; Accepted: 28 July 2022 Acknowledgements g We are grateful to Dr. Sagit Shushan (Wolfson Medical Center), Dr. Edna Haran and the Weizmann Institute’s MRI technician team—E. Tegareh and N. Oshri—for assistance in the human imaging scans. We thank Dr. Jean- Nicolas Dumez for the Bloch simulation code. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 7. Zhang, J., Kolind, S. H., Laule, C. & MacKay, A. L. 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Reson. 226 22–34 (2013).h 39. Dumez, J.-N. & Frydman, L. Multidimensional excitation pulses based on spatiotemporal encoding concepts. J. Magn. Reson. 226, 22–34 (2013). l d Th k l d d lb h 0. Generalized Magnetic Resonance Image Reconstruction using The Berkeley Advanced Reconstruction Toolbox. https://mrirecon github.io/bart/. 40. Generalized Magnetic Resonance Image Reconstruction using The Berkeley Advanced Reconstruction Toolbox. https://mrirecon. github.io/bart/. References IEEE Trans. on Image Process. 26, 3142–3155 (2017). g g 34. Tamada, D. & Reeder, S. B. Phase-based T2 mapping using RF phase-modulated dual echo steady-state (DESS) imaging. Proc. Intl. Soc. Mag. Reson. Med. 29, 3081 (2021).ll g ( ) 5. Zhang, J., Kolind, S. H., Laule, C. & MacKay, A. L. How does magnetization transfer influence mcDESPOT results?: MT Influence on mcDESPOT. Magn. Reson. Med. 74, 1327–1335 (2015).fll g , ( ) 36. Bieri, O., Ganter, C. & Scheffler, K. On the fluid-tissue contrast behavior of high-resolution steady-state sequences: Fluid-tissue contrast behavior of high-resolution SSFP. Magn. Reason. Med. 68, 1586–1592 (2012). https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 \| Competing interests Competing interests A.S. is employed by Siemens Healthcare Ltd, Israel; all other authors declare no competing financial interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-17607-z. Correspondence and requests for materials should be addressed to R.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 Author contributionsh The theoretical analysis and manuscript drafting were carried out by R.S. and A.S. The data collection and exp ment analysis were carried out by R.S. The theoretical analysis and manuscript drafting were carried out by R.S. and A.S. The data collection and experi- ment analysis were carried out by R.S. © The Author(s) 2022 y 2 g ( ) 8. Deoni, S. C. L., Rutt, B. K., Arun, T., Pierpaoli, C. & Jones, D. K. Gleaning multicomponent T1 and T2 information from steady-state imaging data: 2D relaxometry with steady-state imaging. Magn. Reson. Med. 60, 1372–1387 (2008). Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-17607-z. Correspondence and requests for materials should be addressed to R.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. https://doi.org/10.1038/s41598-022-17607-z Scientific Reports \| (2022) 12:14088 \|
https://openalex.org/W2095216640	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0032631&type=printable	English	null	Metabolic Control by S6 Kinases Depends on Dietary Lipids	PloS one	2,012	cc-by	8,506	Abstract Targeted deletion of S6 kinase (S6K) 1 in mice leads to higher energy expenditure and improved glucose metabolism. However, the molecular mechanisms controlling these effects remain to be fully elucidated. Here, we analyze the potential role of dietary lipids in regulating the mTORC1/S6K system. Analysis of S6K phosphorylation in vivo and in vitro showed that dietary lipids activate S6K, and this effect is not dependent upon amino acids. Comparison of male mice lacking S6K1 and 2 (S6K-dko) with wt controls showed that S6K-dko mice are protected against obesity and glucose intolerance induced by a high-fat diet. S6K-dko mice fed a high-fat diet had increased energy expenditure, improved glucose tolerance, lower fat mass gain, and changes in markers of lipid metabolism. Importantly, however, these metabolic phenotypes were dependent upon dietary lipids, with no such effects observed in S6K-dko mice fed a fat-free diet. These changes appear to be mediated via modulation of cellular metabolism in skeletal muscle, as shown by the expression of genes involved in energy metabolism. Taken together, our results suggest that the metabolic functions of S6K in vivo play a key role as a molecular interface connecting dietary lipids to the endogenous control of energy metabolism. Citation: Castan˜eda TR, Abplanalp W, Um SH, Pfluger PT, Schrott B, et al. (2012) Metabolic Control by S6 Kinases Depends on Dietary Lipids. PLoS ONE 7(3): e32631. doi:10.1371/journal.pone.0032631 Editor: Manuel Tena-Sempere, University of Co´rdoba, Spain Received December 8, 2011; Accepted January 28, 2012; Published March 7, 2012 Received December 8, 2011; Accepted January 28, 2012; Published March 7, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, bui any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by National Institutes of Health grants DK73802 and DK078019 to GT and DK077975 and DK056863 to MHT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The work done by DG was carried out while he was a post-doc at the University of Cincinnati, after which he moved to Resverlogix. Abstract Hence Resverlogix is not affiliated with the work or the materials used in the publication in any way, and there is no competing interest associated with Resverlogix. This does not alter the authors9 adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: matthias.tschoep@helmholtz-muenchen.de Metabolic Control by S6 Kinases Depends on Dietary Lipids Tamara R. Castan˜ eda1, William Abplanalp2, Sung Hee Um3,4, Paul T. Pfluger5, Brigitte Schrott2, Kimberly Brown2, Erin Grant2, Larissa Carnevalli6, Stephen C. Benoit2, Donald A. Morgan7, Dean Gilham8, David Y. Hui9, Kamal Rahmouni7, George Thomas3, Sara C. Kozma3, Deborah J. Clegg10, Matthias H. Tscho¨ p5* 1 Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Du¨sseldorf, Germany, 2 Metabolic Disease Institute, Division of Endocrinology, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 3 Department of Molecular Oncogenesis, Metabolic Disease Institute, University of Cincinnati, Cincinnati, Ohio, United States of America, 4 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Seoul, South Korea, 5 HelmholtzZentrum Mu¨nchen, German Research Center for Environmental Health (GmbH), Neuherberg/Munich, Germany, 6 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Germany Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany, 7 Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa, United States of America, 8 Resverlogix Corporation (TSX:RVX), NW Calgary, Alberta, Canada, 9 Department of Pathology, Centre of Arteriosclerosis Studies, Metabolic Disease Institute, University of Cincinnati, Cincinnati, Ohio, United States of America, 10 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America PLoS ONE \| www.plosone.org Results Consistent with our in vitro findings, male C57BL/6 mice reared on a HFD (60% fat content, n = 7) for 6 wk showed a significantly higher level of phosphorylated S6K in muscle (P,0.01) compared to those that received a FFD (0% fat content, n = 7) (Fig. 2A, B) with no differences in total S6K1 protein levels (Fig. 2C). As expected, mice fed a HFD (n = 8) showed significantly increased bodyweight (45.7761.85 g, P,0.001) and plasma insulin (2.8160.5 ng/ml, P,0.001) compared to the FFD control group (30.9861.16 g and 0.7160.08 ng/ml, respectively; n = 7) (Fig. 2D, E). Fatty acids activate S6K1 in vitro independently of branched-chain amino acids Earlier experiments in rats suggested that exposure to a HFD might activate S6K1 in muscle, liver and mediobasal hypothal- amus [4,5]. We therefore analyzed whether relevant dietary fatty acids such as oleic acid and palmitoleic acid can directly induce S6K phosphorylation in vitro. Incubation of muscle Sol 8 cells or neuronal N-41 cells with oleic acid, palmitoleic acid or linoleic acid consistently activated S6K1 in a dose- and time-dependent manner (Fig. 1A–D and Figs. S1A–B, S3). Regulation of S6 Kinases by Dietary Lipids Regulation of S6 Kinases by Dietary Lipids To further elucidate the molecular mechanisms underlying systemic regulation of energy balance by the mTORC1/S6K pathway, we assessed the role of dietary lipids in determining the metabolic phenotype of mice lacking S6K 1 and 2. The results of this study indicate that the metabolic control of energy balance by S6K is regulated by dietary lipids. dependent on amino acids. Both oleic acid and palmitoleic acid induced S6K1 phosphorylation independently of branched-chain amino acids or indeed any other amino acids (Fig. 1E and Fig. S1C). The S6K1 phosphorylation induced by these fatty acids was enhanced by insulin and prevented by rapamycin, as expected for an effector pathway downstream of mTORC1 (Fig. S1D). Introduction in activating S6K [2,3], and dietary fatty acids are also known to activate S6K in liver and muscle [4] and in the hypothalamus [5] of rats fed on a high-fat diet (HFD). Control of energy balance plays a central role in diseases such as obesity and metabolic syndrome, where pharmacological suppres- sion of appetite alone appears to be insufficient to prevent or reverse weight gain and adiposity. In order to control the balance between food intake (FI) and energy expenditure (EE), however, further insights will be required into the molecular pathways that control key processes such as nutrient sensing and energy metabolism, and in particular the regulatory mechanisms that integrate them. Apart from a potential role in nutrient sensing, S6K1 has also been reported to control systemic energy homeostasis. We have previously demonstrated that global S6K1 deficiency leads to higher EE and a lean phenotype, even when mice are chronically exposed to a HFD with a 60% lipid content [2]. In contrast, S6K over-activation in the hypothalamus decreases EE [6]. The mechanism through which S6K controls energy homeostasis could involve regulation of lipid metabolism, since ex vivo studies in muscle, primary hepatocytes and epididymal white adipose tissue have shown that the combined absence of S6K1 and 2 leads to changes in AMPK activity, mitochondrial biogenesis and beta oxidation [7]. However, it remains unclear whether these effects are retained in vivo or whether the control of systemic energy metabolism by S6K is regulated by dietary lipids. S6 kinases (S6K) 1 and 2 are key effectors of the mammalian target of rapamycin complex 1 (mTORC1) and are well known to play a positive anabolic role in insulin-mediated cell growth and to suppress insulin signaling under conditions of hyperactivation of the mTORC1 pathway [1]. The mTORC1/S6K pathway has also been implicated in nutrient sensing, however. Glucose and leucine are known to function as key dietary components involved March 2012 \| Volume 7 \| Issue 3 \| e32631 1 March 2012 \| Volume 7 \| Issue 3 \| e32631 PLoS ONE \| www.plosone.org The metabolic phenotype of S6K-dko mice depends on dietary lipids chow diet in S6K-dko and wt mice. S6K-dko mice had a significantly lower BW (Fig. 3A, B) and fat mass (Fig. 3C, D) on both standard chow diet (23.3060.45 g and 3.6360.39 g, respectively; n = 12; P,0.001) and HFD (24.0360.57 g and 2.0460.20 g, respectively; n = 9; P,0.001) compared to wt controls (standard chow, 32.5960.51 g and 6.5160.57 g, respec- tively; HFD, 37.3961.1 g and 11.7560.67 g, respectively; n = 12– 13). S6K-dko mice had persistent lower BW than wt mice despite having a higher FI on both standard chow and HFD (Fig. S2A, S2B). Consistent with this observation, EE measured for 48 h and normalized to lean mass was significantly higher in S6K-dko (n = 12, P,0.01) than wt control mice (n = 12) when both were fed on standard chow (Fig. 3E). This difference in EE was amplified considerably upon acute exposure (5 d) to a HFD containing 60% lipids (n = 12, P,0.001) (Fig. 3F). The EE levels remained significantly higher in S6K-dko on a HFD for more than 9 mo (n = 9, P,0.05) when compared to age- and sex-matched wt controls reared under similar conditions (n = 13) (Fig. 3G). chow diet in S6K-dko and wt mice. S6K-dko mice had a significantly lower BW (Fig. 3A, B) and fat mass (Fig. 3C, D) on both standard chow diet (23.3060.45 g and 3.6360.39 g, respectively; n = 12; P,0.001) and HFD (24.0360.57 g and 2.0460.20 g, respectively; n = 9; P,0.001) compared to wt controls (standard chow, 32.5960.51 g and 6.5160.57 g, respec- tively; HFD, 37.3961.1 g and 11.7560.67 g, respectively; n = 12– 13). S6K-dko mice had persistent lower BW than wt mice despite having a higher FI on both standard chow and HFD (Fig. S2A, S2B). Consistent with this observation, EE measured for 48 h and normalized to lean mass was significantly higher in S6K-dko (n = 12, P,0.01) than wt control mice (n = 12) when both were fed on standard chow (Fig. 3E). This difference in EE was amplified considerably upon acute exposure (5 d) to a HFD containing 60% lipids (n = 12, P,0.001) (Fig. 3F). The EE levels remained significantly higher in S6K-dko on a HFD for more than 9 mo (n = 9, P,0.05) when compared to age- and sex-matched wt controls reared under similar conditions (n = 13) (Fig. 3G). Targeted deletion of S6K protects against HFD-induced obesity Body weight (D) and plasma insulin levels (E) were significantly higher in mice on a high-fat diet. Two tailed t test P,0.01, P,0.001. doi:10.1371/journal.pone.0032631.g002 Targeted deletion of S6K protects against HFD-induced obesity Since S6K1 has been reported to function in vivo as a nutrient sensor that responds to branched-chain amino acids such as leucine [3], we further assessed whether S6K1 activation by fatty acids was To uncover the functional relevance of in vivo S6K activation by dietary lipids, we compared the effect of HFD and standard ne Sol 8. Oleic acid increased S6K1 phosphorylation in Sol 8 cells in a dose btained with palmitoleic acid (C and D). S6K1 phosphorylation induced b mino acids in general (E). A.A., amino acid; B.C., branched-chain amino acid ,0.05, ##P,0.01. Figure 1. Fatty acids activate S6K1 in vitro in the muscle cell line Sol 8. Oleic acid increased S6K1 phosphorylation in Sol 8 cells in a dose- dependent (A) and time-dependent (B) manner. Similar results were obtained with palmitoleic acid (C and D). S6K1 phosphorylation induced by palmitoleic acid was independent of branched-chain amino acids and amino acids in general (E). A.A., amino acid; B.C., branched-chain amino acid. One way ANOVA P,0.05, P,0.01, P,0.001; two-tailed t test #P,0.05, ##P,0.01. doi:10.1371/journal.pone.0032631.g001 Figure 1. Fatty acids activate S6K1 in vitro in the muscle cell line Sol 8. Oleic acid increased S6K1 phosphorylation in Sol 8 cells in a dose- dependent (A) and time-dependent (B) manner. Similar results were obtained with palmitoleic acid (C and D). S6K1 phosphorylation induced by palmitoleic acid was independent of branched-chain amino acids and amino acids in general (E). A.A., amino acid; B.C., branched-chain amino acid. One way ANOVA P,0.05, P,0.01, P,0.001; two-tailed t test #P,0.05, ##P,0.01. doi:10.1371/journal.pone.0032631.g001 PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e32631 2 Regulation of S6 Kinases by Dietary Lipids Figure 2. Dietary fatty acids activate S6K1 in muscle. Mice fed a high-fat diet for 6 wk show significantly higher phosphorylated S6K1 (A, B) with no changes in total S6K1 protein levels when compared to control mice fed a fat-free diet (C). Body weight (D) and plasma insulin levels (E) were significantly higher in mice on a high-fat diet. Two tailed t test P,0.01, P,0.001. doi:10.1371/journal.pone.0032631.g002 Figure 2. Dietary fatty acids activate S6K1 in muscle. Mice fed a high-fat diet for 6 wk show significantly higher phosphorylated S6K1 (A, B) with no changes in total S6K1 protein levels when compared to control mice fed a fat-free diet (C). The metabolic phenotype of S6K-dko mice depends on dietary lipids doi:10.1371/journal.pone.0032631.g003 triglycerides (1.7260.1 mg/dl, P,0.01, n = 6) (Fig. 5M) in S6K-dko compared to wt controls (adiponectin, 18976260 pg/ml; resistin, 20136178 pg/ml; triglycerides, 308760.36 mg/dl; n = 7). 6.7560.3 kcal/h/kg BW0.75, and 22.5660.56 kcal/h/kg lean mass). In the absence of dietary lipids (S6K-dko and wt mice on FFD), however, no EE phenotype was detectable (Fig. 4A, C, E, G, I, K). S6K-dko mice S6K-dko mice fed a HFD for 3 mo had significantly lower fat mass gain (1.560.4 g, P,0.001, n = 7) (Fig. 5B), plasma levels of leptin (0.7460.05 ng/ml, P,0.01, n = 6) (Fig. 5D), cholesterol (2.7160.31 mg/dl, P,0.01, n = 7) (Fig. 5F), and triglycerides (2.3360.2 mg/dl, P,0.001, n = 7) (Fig. 5N), and higher plasma levels of NEFAs (0.5260.05 nmol/ml, P,0.05, n = 7) (Fig. 5J) compared with age- and sex-matched wt control mice (fat mass gain, 6.3560.8 g; leptin, 2.0360.30 ng/ml; cholesterol, 6.3660.83 mg/dl; triglycerides, 4.960.49 mg/dl; NEFAs, 0.3760.05 nmol/ml; n = 7). There were no significant differences in plasma adiponectin or resistin levels between S6K-dko mice (n = 6) and wt controls (n = 5–7) (Fig. 5H, L). The metabolic phenotype of S6K-dko mice depends on dietary lipids To address the role of dietary macronutrients in the control of energy metabolism by S6K in vivo, we first analyzed EE in S6K- dko mice that had been fasted for 24 h (indicated with an arrow in Fig. 3H). EE decreased rapidly in S6K-dko (n = 9) to match that of wt controls (n = 13, Fig. 3H). To further analyze whether the metabolic phenotype of S6K-dko mice was limited to dietary fatty acids, we compared the impact of chronic exposure to HFD versus FFD in a new group of age- and sex-matched wt and S6K- dko mice. Caloric content from protein was kept equal between both diets (20%). S6K-dko mice fed a HFD for 3 mo (n = 6) had significantly higher EE than wt controls (n = 7) when normalized for BW (P,0.001, Fig. 4B; 24.462.26 kcal/h/kg BW P,0.01, Fig. 4D), body surface area (P,0.001, Fig 4F; 9.460.9 kcal/h/kg BW0.75, P = 0.0108, Fig 4H) or lean mass (P,0.05, Fig. 4J; 29.362.34 kcal/h/kg lean mass, P = 0.0118, Fig. 4L) compared to the wt control group (16.1160.9 kcal/h/kg BW, PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e32631 3 PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e32631 Regulation of S6 Kinases by Dietary Lipids Figure 3. S6K-dko have higher energy expenditure levels. S6K-dko have significantly lower body weight (A) and fat mass (C) than wt controls even when fed a high-fat diet (B, D). Energy expenditure corrected by lean mass levels is higher in S6K-dko when fed standard chow (E), acute high- fat diet (F) or chronic high-fat diet (G) compared to wt controls. Higher energy expenditure in S6K-dko is reduced with 24 h fasting (H). HFD, high-fat diet. Two-tailed t test P,0.001 and two-way ANOVA P,0.05, P,0.01, P,0.001. doi:10.1371/journal.pone.0032631.g003 g y y p Figure 3. S6K-dko have higher energy expenditure levels. S6K-dko have significantly lower body weight (A) and fat mass (C) than wt controls even when fed a high-fat diet (B, D). Energy expenditure corrected by lean mass levels is higher in S6K-dko when fed standard chow (E), acute high- fat diet (F) or chronic high-fat diet (G) compared to wt controls. Higher energy expenditure in S6K-dko is reduced with 24 h fasting (H). HFD, high-fat diet. Two-tailed t test *P,0.001 and two-way ANOVA P,0.05, P,0.01, P,0.001. Lower fat mass gain and plasma levels of leptin, cholesterol and triglycerides depend on dietary lipids in S6K-dko mice S6K-dko mice fed a HFD for 3 mo exhibited greater glucose tolerance (P,0.001, n = 6) (Fig. 6D) and significantly lower fasting plasma glucose (118.366.8 mg/dl, P,0.001, n = 7) (Fig. 6E) and insulin (0.159960.01 mg/dl, P,0.05, n = 5) (Fig. 6F) compared to wt controls (175.5610.9 mg/dl and 0.416760.11 mg/dl, respec- tively; n = 7). No such differences in glucose tolerance (Fig. 6A) or fasting plasma glucose (Fig. 6B) were detected when mice were fed a FFD. However, S6K-dko mice had significantly lower plasma insulin (0.063360.001 ng/ml, P,0.001, n = 5) (Fig. 6C) com- pared to wt controls (0.116160.007 ng/ml, n = 7) when dietary lipids were absent. Enhanced muscle fatty acid metabolism in S6K-dko mice depends on dietary lipids When the mice were fed a FFD, the only identifiable difference was lower plasma adiponectin (9776104 pg/ml, P,0.05, n = 5) (Fig. 5G), resistin (12436106 pg/ml, P,0.01, n = 5) (Fig. 5K) and Muscle tissue from S6K-dko mice fed on a HFD had significantly higher levels of mRNA for Ucp-3 (P,0.05, n = 5) PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e32631 4 Regulation of S6 Kinases by Dietary Lipids Figure 4. Energy expenditure levels in S6K-dko are diet regulated. Energy expenditure normalized for body weight (B, D), body surface area (F, H) or lean mass (J, L) is higher in S6K-dko than wt mice when fed a high-fat diet. No differences in energy expenditure are observed between S6K- dko and wt control mice fed a fat-free diet (A, C, E, G, I, K). BW, body weight; EE, energy expenditure; FFD, fat-free diet; HFD, high-fat diet. Two-tailed t test P,0.05, *P,0.01 and two-way ANOVA P,0.05, **P,0.001. doi:10.1371/journal.pone.0032631.g004 Figure 4. Energy expenditure levels in S6K-dko are diet regulated. Energy expenditure normalized for body weight (B, D), body surface area (F, H) or lean mass (J, L) is higher in S6K-dko than wt mice when fed a high-fat diet. No differences in energy expenditure are observed between S6K- dko and wt control mice fed a fat-free diet (A, C, E, G, I, K). BW, body weight; EE, energy expenditure; FFD, fat-free diet; HFD, high-fat diet. Two-tailed t test P,0.05, *P,0.01 and two-way ANOVA P,0.05, **P,0.001. doi:10.1371/journal.pone.0032631.g004 (Fig. 7B), Cpt-1b (P,0.05, n = 6) (Fig. 7D), Acca (P,0.01, n = 6) (Fig. 7F) and Pdk-4 (P,0.05, n = 5) (Fig. 7H) compared to muscle from wt controls (n = 6–7). In the absence of dietary lipids, however, no such differences were detected (Fig. 7A, C, E, G). finding that is consistent with leptin-independent, nutrient- activated regulation of this pathway. Based on these studies, and our own in vitro and in vivo observations indicating that fatty acids can directly activate S6K1, we hypothesized that the metabolic phenotype of S6K1 and 2 deficient mice is at least partly dependent on exposure to dietary lipids. BAT thermogenesis does not regulate the dietary lipid- dependent metabolic phenotype of S6K-dko mice When S6K-dko mice were fed a HFD for an extended period, they gained less body fat and exhibited a marked increase in EE compared with wt mice and S6K-dko mice fed on a FFD. S6K- dko mice also had lower fasting leptin levels, lower cholesterol, higher NEFAs and better glucose tolerance on a HFD compared to wt mice. However, none of these features occurred in the absence of dietary lipids. Under these conditions, all mice had normal FI, BW and good health, and long-term FFD exposure did not appear to have any non-specific or adverse effects. Since the metabolic phenotype of S6K-dko mice—involving changes in adiposity, energy metabolism, and glucose tolerance—fails to develop when the mice are fed on a FFD, we conclude that metabolic control by S6K is predominately regulated by dietary lipids. BAT from S6K-dko mice (n = 6) fed a HFD for 5 mo had lower Ucp-1 mRNA levels (n = 5, P,0.05) (Fig. 8B) and higher sympathetic activity (n = 6, P,0.01) (Fig. 8D, F) compared with BAT from wt controls (n = 7). Therefore, it is unlikely that differences in BAT uncoupling explain the observed energy metabolism phenotype. On a FFD, no significant differences in Ucp-1 mRNA levels were detected in BAT from S6K-dko mice (n = 5) (Fig. 8A), compared to wt controls (n = 6–7). BAT sympathetic activity in mice fed a FFD (n = 5, P,0.001) was still higher in the S6K-dko mice than wt controls (Fig. 8C, E). PLoS ONE \| www.plosone.org Discussion The results of this study show that S6Ks are directly activated by fatty acids in vitro and in vivo, a finding that appears to expand the role of S6K as a nutrient sensor. More importantly, our data suggest that the predominant role of S6K in metabolic regulation is in the response to dietary lipids. Hypothalamic S6K over-activation in rats decreases EE [6], whereas S6K1-ko and adipose tissue-specific raptor ko mice show higher EE levels [2,9,10]. Consistent with the results of those studies, S6K-dko mice show high EE. Defining the specific tissue responsible for the considerable EE phenotype is of great interest. Our results indicate that BAT is unlikely to be the only cause of the significantly higher EE levels in response to lipids, since Ucp-1 mRNA expression in S6K-dko mice fed a HFD was lower despite higher BAT sympathetic activity. In fact, adipose tissue has been proposed as a source of higher levels of EE in mice with specific Previously, it has been demonstrated that exposure to a HFD leads to the activation of S6K1 in muscle and liver tissue [4] and also in the hypothalamus [5]. Phosphorylation of S6 (a signaling protein downstream of mTORC1 and S6K1 and 2) has also been observed in the hypothalamus of fed ob/ob and db/db mice [8], a March 2012 \| Volume 7 \| Issue 3 \| e32631 March 2012 \| Volume 7 \| Issue 3 \| e32631 5 Regulation of S6 Kinases by Dietary Lipids Figure 5. S6K-dko plasma biochemistry. S6K-dko mice fed a high-fat diet show significantly lower fat mass gain (B), plasma leptin (D), cholesterol (F) and triglycerides (N), as well as higher free fatty acids (J) compared to wt controls. When fed a fat-free diet, S6K-dko mice still show significantly lower plasma triglycerides (N), as well as significantly lower adiponectin (H) and resistin (K) levels. FFD, fat-free diet; HFD, high-fat diet; NEFAs, non-esterified fatty acids. Two-tailed t test P,0.05, P,0.01, P,0.001. doi:10.1371/journal.pone.0032631.g005 Figure 5. S6K-dko plasma biochemistry. S6K-dko mice fed a high-fat diet show significantly lower fat mass gain (B), plasma leptin (D), cholesterol (F) and triglycerides (N), as well as higher free fatty acids (J) compared to wt controls. When fed a fat-free diet, S6K-dko mice still show significantly lower plasma triglycerides (N), as well as significantly lower adiponectin (H) and resistin (K) levels. FFD, fat-free diet; HFD, high-fat diet; NEFAs, non-esterified fatty acids. Discussion Two-tailed t test P,0.05, P,0.01, P,0.001. doi:10.1371/journal.pone.0032631.g005 Figure 6. S6K-dko glucose tolerance and diet. S6K-dko mice fed a high-fat diet have significantly higher glucose tolerance (D) and lower plasma glucose (E) and insulin (F) compared to wt controls. When fed a fat-free diet, S6K-dko mice do not show better glucose tolerance (A) or lower plasma glucose levels (B) in spite of having significantly lower plasma insulin levels (C). FFD, fat-free diet; HFD, high-fat diet. One-tailed t test P,0.05, two- tailed t test *P,0.001 and two-way ANOVA P,0.001. doi:10.1371/journal.pone.0032631.g006 Figure 6. S6K-dko glucose tolerance and diet. S6K-dko mice fed a high-fat diet have significantly higher glucose tolerance (D) and lower plasma glucose (E) and insulin (F) compared to wt controls. When fed a fat-free diet, S6K-dko mice do not show better glucose tolerance (A) or lower plasma glucose levels (B) in spite of having significantly lower plasma insulin levels (C). FFD, fat-free diet; HFD, high-fat diet. One-tailed t test P,0.05, two tailed t test *P,0.001 and two-way ANOVA P,0.001. doi:10.1371/journal.pone.0032631.g006 Figure 6. S6K-dko glucose tolerance and diet. S6K-dko mice fed a high-fat diet have significantly higher glucose tolerance (D) and lower plasma glucose (E) and insulin (F) compared to wt controls. When fed a fat-free diet, S6K-dko mice do not show better glucose tolerance (A) or lower plasma glucose levels (B) in spite of having significantly lower plasma insulin levels (C). FFD, fat-free diet; HFD, high-fat diet. One-tailed t test P,0.05, two- tailed t test *P,0.001 and two-way ANOVA P,0.001. doi:10.1371/journal.pone.0032631.g006 March 2012 \| Volume 7 \| Issue 3 \| e32631 PLoS ONE \| www.plosone.org 6 Regulation of S6 Kinases by Dietary Lipids Figure 7. Muscle lipid beta oxidation in S6K-dko is diet regulated. S6K-dko mice fed a high-fat diet have higher muscle lipid beta oxidation, as shown by significantly higher mRNA levels of Ucp-3 (B), Cpt-1b (D), Acca (F) and Pdk-4 (H). No such differences were observed in mice fed a fat free diet (A, C, E, G). FFD, fat-free diet; HFD, high-fat diet. Two-tailed t test P,0.05, *P,0.01. doi:10.1371/journal.pone.0032631.g007 Figure 7. Muscle lipid beta oxidation in S6K-dko is diet regulated. S6K-dko mice fed a high-fat diet have higher muscle lipid beta oxidation, as shown by significantly higher mRNA levels of Ucp-3 (B), Cpt-1b (D), Acca (F) and Pdk-4 (H). No such differences were observed in mice fed a fat free diet (A, C, E, G). Regulation of S6 Kinases by Dietary Lipids Regulation of S6 Kinases by Dietary Lipids Regulation of S6 Kinases by Dietary Lipids which the action of S6K links dietary lipids to the control of adaptive thermogenesis. According to such a model, S6K in the arcuate nucleus could be involved in the regulation of skeletal muscle metabolism by the central nervous system. However, tissue-specific gene targeting will be necessary to address whether these differences in energy metabolism in response to dietary lipids are a direct consequence of the lack of S6K activation in skeletal muscle, an indirect effect of S6K modulation in the central nervous system influencing muscle EE via efferent SNS activity, or a combination of the two. deletion of raptor in this tissue [9]. Consistent with our results, expression of uncoupling proteins was similar to or lower than in controls in that study. On the other hand, we found that the expression of genes linked to energy metabolism, including Ucp-3, Cpt-1b, Acca and Pdk-4, was up-regulated in skeletal muscle when S6K-dko mice were fed a HFD. This suggests that skeletal muscle thermogenesis is a more likely cause of the substantial EE response to dietary lipids. This finding is consistent with recently published data in which the absence of S6K or treatment with rapamycin promoted fatty acid oxidation in muscle cells [7,11]. Interestingly, mice lacking raptor in skeletal muscle die early and show muscle dystrophy, lower muscle mitochondrial biogenesis and reduced oxidative capacity [12]. An intriguing effect of null mutations for both S6Ks is the protection against lipotoxicity [24], presumably as a collateral effect of the enhanced muscle fatty acid beta oxidation in mice fed a HFD. Since the mTORC1 pathway mediates cell proliferation [25], as reflected by the smaller size of the S6K-dko mice [26], and lack of S6K1 impairs adipogenesis [27], lipid deposition outside of adipose tissue might be expected in response to chronic HFD exposure. However, higher lipid oxidation in skeletal muscle apparently provides protection against such adverse effects and thereby prevents the detrimental metabolic consequences of limited adipose tissue expansion capabilities in the absence of S6K. This suggests that the absence of S6K not only protects against obesity but also against potential lipotoxicity as a result of collateral hypotrophy. The physiological events associated with the absence of S6K resemble those seen with caloric deprivation, in which insulin levels are low and gluconeogenesis is augmented. Regulation of S6 Kinases by Dietary Lipids In both situations, lipids are used as the main source of energy in order to preserve glucose for the central nervous system and erythrocytes [13,14]. The significantly higher fasting NEFAs and lower plasma triglyceride levels observed in S6K-dko fed a HFD could suggest a higher sensitivity and more efficient response to the perceived lack of dietary lipids. Consistent with this possibility, rapamycin administration in human lymphoma cells alters gene expression in a manner similar to that observed upon starvation [15] and decreased mTOR signaling has been linked to starvation-induced stress, higher mitochondrial respiration and extended life-span in yeast [16,17,18]. In mammals, mTOR signaling has also been linked to longevity [19]. Specifically, levels of phosphorylated S6K1 were reduced in muscle and liver in a mouse model of extended life span [20]. However, it remains possible that the physiological differences observed in our study are also a consequence of altered sensing of dietary sugars, as the HFD and FFD used differed in carbohydrate content, and skeletal muscle energy metabolism depends heavily on insulin-stimulated glucose uptake [21]. In summary, we conclude that S6K deficiency in the absence of dietary lipids has no physiologically relevant metabolic conse- quences even when animals are chronically exposed to consider- able amounts of dietary carbohydrates (Fig. 9). Our findings are consistent with a role for S6K activity in the development of dietary fatty acid-induced metabolic disease. Finally, our results show that S6K deficiency specifically protects against the pathological effects of dietary lipids and suggest that the S6K pathway in skeletal muscle may represent a valuable molecular target for the treatment of metabolic diseases. Further studies will be necessary to elucidate the role of S6Ks in skeletal muscle and central nervous system as a putative molecular interface between dietary lipids and the endogenous control of systemic energy and glucose and lipid metabolism. We found that S6K activation by fatty acids in vitro depends on mTORC1 but not on amino acids. However, the mechanisms underlying this observation remain unclear. mTORC1 transloca- tion to lysosomal membranes and subsequent activation in vitro occurs in response to amino acids [22,23]. However, it is not clear how NEFAs influence this pathway in similar cell models (human embryonic kidney cells and mouse embryonic fibroblasts). Further studies will therefore be required to elucidate the mechanisms underlying fatty acid-dependent, amino acid-independent mTORC1/S6K activation and function. Discussion FFD, fat-free diet; HFD, high-fat diet. Two-tailed t test P,0.05, P,0.01. doi:10.1371/journal.pone.0032631.g007 Figure 8. Brown adipose tissue thermogenesis in S6K-dko is diet independent. S6K-dko mice fed a high-fat diet have significantly lower levels of Ucp-1 mRNA in brown adipose tissue (B) and higher sympathetic activity (D, F) compared to wt controls. When fed a fat-free diet, S6K-dko mice show no significant differences in Ucp-1 mRNA levels in brown adipose tissue (A) but do have significantly higher sympathetic activity (C, E) compared to wt controls. BAT, brown adipose tissue; FFD, fat-free diet; HFD, high-fat diet. Two-tailed t test P,0.01, *P,0.001. doi:10.1371/journal.pone.0032631.g008 PLoS ONE \| www.plosone.org 7 March 2012 \| Volume 7 \| Issue 3 \| e32631 Figure 8. Brown adipose tissue thermogenesis in S6K-dko is diet independent. S6K-dko mice fed a high-fat diet have significantly lower levels of Ucp-1 mRNA in brown adipose tissue (B) and higher sympathetic activity (D, F) compared to wt controls. When fed a fat-free diet, S6K-dko mice show no significant differences in Ucp-1 mRNA levels in brown adipose tissue (A) but do have significantly higher sympathetic activity (C, E) compared to wt controls. BAT, brown adipose tissue; FFD, fat-free diet; HFD, high-fat diet. Two-tailed t test P,0.01, **P,0.001. doi:10.1371/journal.pone.0032631.g008 PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e32631 7 7 Glucose tolerance test Analysis of glucose tolerance was carried out in mice fasted overnight and injected intraperitoneally with 2 g glucose per kg body weight (BW) (50% wt/vol D-glucose [Sigma, St Louis, MO, USA] in 0.9% wt/vol NaCl). Tail-blood glucose levels (mg/dl) were measured with a glucometer (TheraSense Freestyle) and standard glucose strips (Free Style; Abbott Laboratories) before (0 min) and at 15, 30, 45, 60 and 120 min after injection. Real-time quantitative PCR S6K-dko and wt mice were killed following sympathetic nervous system (SNS) recordings performed after approximately 5 mo of exposure to FFD and HFD. Brown adipose tissue (BAT) and gastrocnemius muscle were collected and stored at 280uC prior to analysis of mRNA expression for uncoupling protein 3 (Ucp-3), carnitinepalmitoyl-transferase 1 beta (Cpt-1ß), acetyl coenzyme A carboxylase (Acc) alpha and pyruvate dehydrogenase kinase 4 (Pdk- 4) by real-time quantitative PCR (qPCR) in an iCycler Optical Module (BioRad Hercules, CA, USA) (Table 1). Total RNA was extracted from frozen tissue samples using the RNeasy Lipid Mini Kit (Qiagen, Valencia, CA, USA). cDNA templates for qPCR were synthesized using 0.5 mg of total RNA, 106DNase reaction buffer, DNase 1 amp grade, dNTPs, random primers, and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). qPCR was performed using iQ SYBR Green Supermix (BioRad, Hercules, CA, USA) according to the standard protocol with approximately 70 ng template cDNA. All primers were used at a final concentration of 0.5 mM. A standard curve was used to obtain the relative concentration of each experimental gene; values were normalized to the concentration of the housekeeping gene hypoxanthine–guanine phosphoribosyltransferase (Hprt) in each sample. Energy balance measurements Mice were placed in a customized indirect calorimetry system (TSE Systems GmbH, Bad Homburg, Germany) and FI and EE were monitored simultaneously for 2–3 d after adaptation. FI measurements were taken every 30 min and EE measurements were taken every 45 min at al flow rate of 0.5 l/min. Body composition measurements were taken using nuclear magnetic resonance (Whole Body Composition Analyzer; Echo MRI, Texas, USA). Animals Mice lacking S6K1 and S6K2 (S6K-dko) were generated as described by Pende at al. [26]. Age- and sex-matched S6K1-ko mice were originally generated in a mixed 129/SveJ6C57Bl/6 line and the S6K2-ko mice in a mixed 129/OlaHSD6C57Bl/6 line [26,28]. Male S6K-dko and wild-type (wt) mice were housed in individual cages under a 12:12 h light-dark cycle (7 am light on) Since exposure to dietary fatty acids may directly affect nutrient sensing neurons expressing S6K in the arcuate nucleus in vivo [3], the mediobasal hypothalamus could represent an important site at Figure 9. Model of metabolism regulation by diet in absence of S6K. Mice S6K-dko show lower fat mass gain and higher glucose tolerance, energy expenditure and muscle lipid b-oxidation compared with wt mice when consuming a high fat diet. Those metabolic differences are not present in absence of dietary lipids. doi:10.1371/journal.pone.0032631.g009 Figure 9. Model of metabolism regulation by diet in absence of S6K. Mice S6K-dko show lower fat mass gain and higher glucose tolerance, energy expenditure and muscle lipid b-oxidation compared with wt mice when consuming a high fat diet. Those metabolic differences are not present in absence of dietary lipids. doi:10.1371/journal.pone.0032631.g009 March 2012 \| Volume 7 \| Issue 3 \| e32631 March 2012 \| Volume 7 \| Issue 3 \| e32631 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 8 Regulation of S6 Kinases by Dietary Lipids Luminex100 TM IS analyzer (Luminex Corporation, Austin, TX, USA) and mouse lincoplex kits (Linco Research, St Charles, MO, USA). The intra- and inter-assay coefficient of variation was ,5% and ,12%, respectively, for adiponectin and ,4.5% and ,10.3%, respectively, for resistin. All assays were performed according to the manufacturer’s instructions. with ad libitum access to standard chow (6% kcal from fat, Harlan Teklad Laboratory Diets, Wisconsin, USA), fat-free diet (FFD) (0% kcal from fat, D04112303 Research Diets Inc., New Jersey, USA) or HFD (60% kcal from fat [D12492] and 45% kcal from fat [D12451], Research Diets Inc., New Jersey, USA) and tap water. All animal protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee. For the first round of experiments, mice received standard chow or 60% kcal HFD for 9 mo after weaning; for the second round of experiments, mice were placed on FFD or 45% HFD at 2 mo and continued to receive that diet for 3–5 mo. Regulation of S6 Kinases by Dietary Lipids Regulation of S6 Kinases by Dietary Lipids Regulation of S6 Kinases by Dietary Lipids specified times with palmitoleic and oleic acid (200 mM) preincubated with 0.1% fatty acid-free BSA overnight at 4uC (Sigma, St Louis, MO, USA). Following the different treatments, the cells were lysed in ice-cold RIPA buffer containing protease inhibitors (16PBS, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS with 50 mM NaF, 0.5 M phenylmethylsulfonyl fluoride, 0.1 mM sodium vanadate, aprotinin [10 mg/ml], and leupeptin [5 mg/ml]) (Sigma, St Louis, MO, USA). N-41 cells were also incubated with or without insulin and the mTOR inhibitor rapamycin (20 nM) (Sigma, St Louis, MO, USA). Following the different treatments, the cells were lysed in ice cold RIPA buffer containing protease inhibitors. Lysates were collected in a 1.5-ml tube and cleared by centrifugation at 4uC for 10 min at 7500 rpm. An aliquot of sample was used for determination of protein concentration. Samples were then boiled in 46 lithium dodecyl sulfate/dithiothreitol buffer (Invitrogen, Carlsbad, CA, USA; American Bioanalytical, Natick, MA, USA) for 10 min at 70uC. Cell lysates (50 mg protein) were loaded on 9% (w/v) acrylamide resolving gels, separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). Membranes were blocked in 2% ECL advance blocking agent for 1 h at room temperature and probed with primary antibodies for S6K (Cell Signaling, Beverly, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, primary antibodies were detected using either HRP- conjugated anti-mouse IgG for phosphorylated-S6K (Thr389) or anti-rabbit IgG for GAPDH (Bio-Rad, Hercules, CA, USA). Protein bands were visualized using advance chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA), exposed to CL- Xposure film (Pierce, Rockford, IL, USA) and then quantified using AlphaEase FC software. alpha-chloralose (25 mg/kg initially, followed by 6 mg/kg/h during the protocol). To monitor blood pressure and heart rate, a catheter was inserted into the left carotid artery. Rectal body temperature was maintained at 37.5uC using a lamp and heating pad. A nerve innervating BAT was isolated and placed on bipolar platinum-iridium electrodes for recordings. The nerve signal was amplified 100,0006 at high-frequency and low-frequency cutoffs of 1 kHz and 100 Hz, respectively. Blood pressure, heart rate and BAT sympathetic nerve activity were recorded continuously for the next 20–30 min. Supporting Information Figure S1 Fatty acids activate S6K1 in vitro in the hypothalamic cell line N-41. Palmitoleic acid increases S6K1 phosphorylation in N-41 cells in a dose-dependent (A) and time- dependent (B) manner. Palmitoleic acid-induced phosphorylation was independent of branched-chain amino acids and amino acids in general (C). S6K1 phosphorylation induced by palmitoleic acid was enhanced by insulin and prevented by rapamycin, suggesting a mechanism involving the mTORC1 pathway (D). A.A., amino acid; B.C., branched-chain amino acid. One way ANOVA P,0.05, P,0.01, P,0.001; two-tailed t test #P,0.05. (TIF) Figure S1 Fatty acids activate S6K1 in vitro in the hypothalamic cell line N-41. Palmitoleic acid increases S6K1 phosphorylation in N-41 cells in a dose-dependent (A) and time- dependent (B) manner. Palmitoleic acid-induced phosphorylation was independent of branched-chain amino acids and amino acids in general (C). S6K1 phosphorylation induced by palmitoleic acid was enhanced by insulin and prevented by rapamycin, suggesting a mechanism involving the mTORC1 pathway (D). A.A., amino acid; B.C., branched-chain amino acid. One way ANOVA P,0.05, P,0.01, P,0.001; two-tailed t test #P,0.05. (TIF) To measure S6K activation independently of NEFA, Sol 8 and N-41 cells were treated as described above. After 24 h incubation in serum-free medium, cells were washed in PBS and incubated for 2 h in amino acid-free DMEM basic medium. Cells were then treated with BSA-conjugated palmitoleic acid (200 mM) for 1 h in either amino acid-free, branched-chain amino acid-free or branched-chain amino acid-supplemented DMEM. Cells were lysed and treated as previously described. Figure S2 S6K-dko and food intake. In spite of their lean phenotype, S6K-dko show higher food intake on standard chow (A) or a high-fat diet (B). BW, body weight; HFD, high-fat diet. Two-way ANOVA P,0.05, P,0.01. (TIF) Figure S3 Fatty acids activate S6K1 in vitro in the muscle cell line Sol8. Linoleic acid increases S6K1 phosphor- ylation in Sol8 cells in a dose-dependent manner. One way ANOVA P,0.01. For quantification of S6K in the soleus muscle of male C57BL/ 6 mice, both soleus muscles were collected from animals reared on FFD and 60% HFD and euthanized after 6 wk. Tissues were immediately placed in RIPA buffer containing protease inhibitors, and then samples were lysed with a TissueLyser (Qiagen, Valencia, CA, USA) for 3 min and centrifuged at 4uC for 10 min at 7500 rpm. Supernatant was transferred to a new tube and an aliquot was taken for protein quantification. Supporting Information Phosphory- lated-S6K1 (Thr389) and total S6K1 were quantified using the protocol described above. Acknowledgments We would like to thank Paulo Martins, Randy Seeley and Steve Woods for valuable discussions. Statistical analysis Quantitative data are presented as mean 6 standard error of the mean (SEM). Data were compared by one-way and two-way repeated measures analysis of variance (ANOVA) with post-hoc Bonferroni and Dunnett tests and by one- or two-tailed, unpaired t tests. P,0.05 was considered statistically significant (GraphPad Prism software). Regulation of S6 Kinases by Dietary Lipids At the end of the recording session, the nerve was broken and the background noise was measured and subtracted from baseline activity of the BAT SNS. Sympathetic nervous system recordings Conceived and designed the experiments: MHT DJC DYH SCB GT SCK. Performed the experiments: TRC WA SHU PTP BS KB EG LC DAM DG. Analyzed the data: TRC. Contributed reagents/materials/ analysis tools: MHT GT SK DH KR. Wrote the paper: TRC MHT DJC DYH SCB GT SK. Interpreted data: MHT DJC DYH SCB GT SK. Co- edited manuscript: WA SHU EG LC DG. Conceived and designed the experiments: MHT DJC DYH SCB GT SCK. Performed the experiments: TRC WA SHU PTP BS KB EG LC DAM DG. Analyzed the data: TRC. Contributed reagents/materials/ analysis tools: MHT GT SK DH KR. Wrote the paper: TRC MHT DJC DYH SCB GT SK. Interpreted data: MHT DJC DYH SCB GT SK. Co- edited manuscript: WA SHU EG LC DG. Each mouse was initially anesthetized by intraperitoneal injection with a ketamine/xylazine cocktail (91 mg/kg ketamine and 9.1 mg/kg xylazine). A PE-50 tube was inserted into the trachea for unobstructed spontaneous breathing and a catheter was inserted into the right jugular vein to maintain anesthesia with 3. Cota D, Proulx K, Smith KA, Kozma SC, Thomas G, et al. (2006) Hypothalamic mTOR signaling regulates food intake. Science 312: 927– 930. 2. Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, et al. (2004) Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431: 200–205. 1. Um SH, D’Alessio D, Thomas G (2006) Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab 3: 393–402. 2. Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, et al. (2004) Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431: 200–205. 4. Khamzina L, Veilleux A, Bergeron S, Marette A (2005) Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of 1. Um SH, D’Alessio D, Thomas G (2006) Nutrient overload, insulin resistance, 3. Cota D, Proulx K, Smith KA, Kozma SC, Thomas G, et al. (2006) Hypothalamic mTOR signaling regulates food intake. Science 312: 927– 930. 4. Khamzina L, Veilleux A, Bergeron S, Marette A (2005) Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of Plasma analysis The mouse muscle cell line Sol 8 was a generous gift from Dr. Silvana Obici (University of Cincinnati) and the hypothalamic cell line N-41 was obtained from Cellutions Biosystems Inc. (Toronto, Ontario, Canada). Sol 8 and N-41 cells were grown and maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone Laboratories, Logan, UT, USA) containing 20% and 10% fetal bovine serum (FBS) (GIBCO Laboratories, Grand Island, NY, USA), respectively, with antibiotic/antimycotic mix (GIBCO Laboratories, Grand Island, NY, USA) at 37uC in a 5% CO2 atmosphere. Differentiation of Sol 8 cells was achieved by seeding the cells in 12-well plates and growing them to 50% confluence before switching the medium to DMEM containing 5% horse serum (Sigma, St Louis, MO, USA) for 3 d. For experiments, Sol 8 and N-41 cells were washed with PBS and incubated in serum-free low-glucose DMEM for 24 h prior to and during experiments. Serum-starved cells were then treated for the Mice were killed by decapitation at the end of the study and blood was collected and immediately chilled on ice. Plasma was obtained by centrifugation at 3000 g and 4uC for 15 min and then stored at 280uC. Non-esterified fatty acid (NEFA) levels were measured using a commercially available enzymatic assay kit (Autokit NEFA C, Wako, Neuss, Germany). Plasma cholesterol and triglycerides were determined using Infinity Cholesterol reagent and Infinity Triglyceride reagent b (Thermo Electron, Pittsburgh, Pennsylvania, USA). Plasma glucose was measured using a commercial kit based on the glucose oxidase method (Biomerieux, Marcy l’Etoile, France). Plasma insulin and leptin were measured using a mouse radio-immunoassay (Linco Research, St Charles, MO, USA). The intra- and inter-assay coefficient of variation was 2.7–5.8% and 3.8–10.8%, respectively, for insulin and 4.0–11.2% and 3.3–14.6%, respectively, for leptin. Plasma adiponectin and resistin were measured with a Table 1. Primer sequences for RT-PCR. Mouse gene Accession # Forward sequence Reverse sequence Product Annealing T Ucp-1 NM_009463 59GGGCCCTTGTAAACAACAAA39 59GTCGGTCCTTCCTTGGTGTA39 196 60 Ucp-3 NM_009464 59GTCTGCCTCATCAGGGTGTT39 59CCTGGTCCTTACCATGCAGT39 204 60 Cpt-1b NM_009948 59CAGCGCTTTGGGAACCACAT39 59CACTGCCTCAAGAGCTGTTCTC39 203 60 Acc-a NM_133360 59-GCCTCTTCC TGACAAACGAG-39 59-TGACTGCCG AAACATCTCTG-39 239 60 Pdk4 NM_013743 59CCTTTGGCTGGTTTTGGTTA39 59CCTGCTTGGGATACACCAGT39 294 60 Hprt NM_013556 59TGCTCGAGATGTCATGAAGG39 59TATGTCCCCCGTTGACTGAT39 196 60 doi:10.1371/journal.pone.0032631.t001 Table 1. Primer sequences for RT-PCR. Table 1. Primer sequences for RT-PCR. March 2012 \| Volume 7 \| Issue 3 \| e32631 9 1. Um SH, D’Alessio D, Thomas G (2006) Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab 3: 393–402. Regulation of S6 Kinases by Dietary Lipids obese rats: possible involvement in obesity-linked insulin resistance. Endocri- nology 146: 1473–1481. 17. Kaeberlein M, Powers RW, 3rd, Steffen KK, Westman EA, Hu D, et al. (2005) Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310: 1193–1196. gy 5. Ono H, Pocai A, Wang Y, Sakoda H, Asano T, et al. (2008) Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats. J Clin Invest 118: 2959–2968. 18. Bonawitz ND, Chatenay-Lapointe M, Pan Y, Shadel GS (2007) Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. Cell Metab 5: 265–277. J 6. Blouet C, Ono H, Schwartz GJ (2008) Mediobasal hypothalamic p70 S6 kinase 1 modulates the control of energy homeostasis. Cell Metab 8: 459–467. 19. Finley LW, Haigis MC (2009) The coordination of nuclear and mitochondrial communication during aging and calorie restriction. Ageing Res Rev 8: 173–188. 7. Aguilar V, Alliouachene S, Sotiropoulos A, Sobering A, Athea Y, et al. (2007) S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase. Cell Metab 5: 476–487. 20. Sharp ZD, Bartke A (2005) Evidence for down-regulation of phosphoinositide 3- kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR)-dependent translation regulatory signaling pathways in Ames dwarf mice. J Gerontol A Biol Sci Med Sci 60: 293–300. 8. Villanueva EC, Munzberg H, Cota D, Leshan RL, Kopp K, et al. (2009) Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin and nutritional status. Endocrinology 150: 4541–4551. 21. Abdul-Ghani MA, DeFronzo RA (2010) Pathogenesis of insulin resistance in skeletal muscle. J Biomed Biotechnol. 476279 p. 9. Polak P, Cybulski N, Feige JN, Auwerx J, Ruegg MA, et al. (2008) Adipose- specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. Cell Metab 8: 399–410. 22. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, et al. (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141: 290–303. 10. Pende M, Kozma SC, Jaquet M, Oorschot V, Burcelin R, et al. (2000) Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1- deficient mice. Nature 408: 994–997. 23. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, et al. (2008) The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496–1501. 11. References 4. Khamzina L, Veilleux A, Bergeron S, Marette A (2005) Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e32631 10 Regulation of S6 Kinases by Dietary Lipids Sipula IJ, Brown NF, Perdomo G (2006) Rapamycin-mediated inhibition of mammalian target of rapamycin in skeletal muscle cells reduces glucose utilization and increases fatty acid oxidation. Metabolism 55: 1637–1644. 24. Virtue S, Vidal-Puig A (2010) Adipose tissue expandability, lipotoxicity and the Metabolic Syndrome–an allostatic perspective. Biochim Biophys Acta 1801: 338–349. 12. Bentzinger CF, Romanino K, Cloetta D, Lin S, Mascarenhas JB, et al. (2008) Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab 8: 411–424. 25. Dowling RJ, Topisirovic I, Alain T, Bidinosti M, Fonseca BD, et al. (2010) mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E- BPs. Science 328: 1172–1176. 13. Roche TE, Hiromasa Y (2007) Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer. Cell Mol Life Sci 64: 830–849. 26. Pende M, Um SH, Mieulet V, Sticker M, Goss VL, et al. (2004) S6K1(2/2)/ S6K2(2/2) mice exhibit perinatal lethality and rapamycin-sensitive 59-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol Cell Biol 24: 3112–3124. 14. Jeoung NH, Wu P, Joshi MA, Jaskiewicz J, Bock CB, et al. (2006) Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation. Biochem J 397: 417–425. 27. Carnevalli LS, Masuda K, Frigerio F, Le Bacquer O, Um SH, et al. (2010) S6K1 plays a critical role in early adipocyte differentiation. Dev Cell 18: 763–774. 15. Peng T, Golub TR, Sabatini DM (2002) The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation. Mol Cell Biol 22: 5575–5584. 16. Powers RW, 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S (2006) Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev 20: 174–184. 28. 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https://openalex.org/W3114384164	https://europepmc.org/articles/pmc7823496?pdf=render	English	null	Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deficient Mutant Acclimated to Low and High Temperatures	Biomolecules	2,020	cc-by	13,678	biomolecules Received: 26 November 2020 Accepted: 23 December 2020 Published: 29 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Keywords: brassinosteroids; chloroplast membranes; temperature stress; barley; molecular dynamics; EPR Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deﬁcient Mutant Acclimated to Low and High Temperatures Citation: Sadura, I.; Latowski, D.; Oklestkova, J.; Gruszka, D.; Chyc, M.; Janeczko, A. Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deﬁcient Mutant Acclimated to Low and High Temperatures. Biomolecules 2021, 11, 27. https://doi.org/ 10.3390/biom11010027 Received: 26 November 2020 Accepted: 23 December 2020 Published: 29 December 2020 Citation: Sadura, I.; Latowski, D.; Oklestkova, J.; Gruszka, D.; Chyc, M.; Janeczko, A. Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deﬁcient Mutant Acclimated to Low and High Temperatures. Biomolecules 2021, 11, 27. https://doi.org/ 10.3390/biom11010027 Received: 26 November 2020 Accepted: 23 December 2020 Published: 29 December 2020 Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deﬁcient Mutant Acclimated to Low and High Temperatures 1 Polish Academy of Sciences, The Franciszek Górski Institute of Plant Physiology, Niezapominajek 21, 30-239 Kraków, Poland; ania@belanna.strefa.pl 2 Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; dariusz.latowski@uj.edu.pl 3 Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký University, Šlechtitel˚u 27, 78371 Olomouc, Czech Republic; jana.oklestkova@upol.cz 4 Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia, Jagiello´nska 28, 40-032 Katowice, Poland; damian.gruszka@us.edu.pl 5 Department of Environmental Protection, Faculty of Mathematical and Natural Sciences, University of Applied Sciences in Tarnów, Mickiewicza 8, 33-100 Tarnów, Poland; mrsch7@gmail.com * Correspondence: i.sadura@ifr-pan.edu.pl; Tel.: +48-12-4251833 (ext. 110) Abstract: Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deﬁcient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidiﬁcation than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 ◦C, whereas in the hydrophobic core, they were visible at both 20 and 5 ◦C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 ◦C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts. Citation: Sadura, I.; Latowski, D.; Oklestkova, J.; Gruszka, D.; Chyc, M.; Janeczko, A. Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deﬁcient Mutant Acclimated to Low and High Temperatures. Biomolecules 2021, 11, 27. https://doi.org/ 10.3390/biom11010027 1. Introduction Among the abiotic stresses, temperature stress is a global problem that mainly causes a decrease in the yield of most crop plants in agriculture and horticulture [1,2]. Some cereal species are sensitive to cold (maize), while other species (winter wheat) are sensitive to low temperatures, especially when there is poor snow cover on ﬁelds, which causes frost injuries that lower the yield. On the other hand, high temperatures may be particularly https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 27. https://doi.org/10.3390/biom11010027 Biomolecules 2021, 11, 27 2 of 20 2 of 20 dangerous to plants when there is a water deﬁcit in the summer vegetation season, which has increasingly happened in recent years due to climate changes. Periods of cold (e.g., 5 ◦C) harden plants (e.g., winter cereals) and enable them to better survive frost during winter. In turn, exposure to warmth may increase the ability of plants to survive high- temperature stress (e.g., 40 ◦C). Among the plant strategies for counteracting the negative effects of abiotic stresses (including extreme temperatures) [3], biological membranes are an important element. According to Horvath et al. [4], membranes can be considered to be “thermal sensors”, and are the primary cause of many other metabolic changes within cells and their organelles. During acclimation to changing temperature conditions, the alterations in the ﬂuidity of the biological membranes are connected with changes in the proportion of unsaturated fatty acids, which results in a rearrangement of the membrane structure and its properties. The membrane properties can also be altered by incorporating various components into their structure, such as tocopherols, sterols, steroids [5], or carotenoids [6]. Many of the physiological phenomena that occur in cells could be due to the mul- tidirectional effect of hormones on the plant metabolism, which is also the basis for the adaptability of organisms in order to function in changing environmental conditions. One plant hormone group is the brassinosteroids (BR), which are responsible for regu- lating both plant growth and development as well as their response to stress. BR belong to the steroid phytohormones that were ﬁrst isolated from oilseed rape pollen in the 1970s [7,8]. To date, more than 70 BR are known, including brassinolide, 24-epibrassinolide, 28-homocastasterone, etc. Bajguz and Hayat [9] showed that in plants that had been ex- posed to temperature extremes, BR counteract the inhibition of growth, reduce the biomass losses and increase the plant survival rate. 1. Introduction This is the result of the multidirectional activity of BR at the cellular and molecular level [10]. The function of BR in plants is still being ex- plained and relatively little is known about their impact on the plant membrane properties. Recently, we found that BR may be involved in regulating the accumulation of the proteins that are incorporated in the cell membranes of barley plants (heat shock proteins, proton pumps and aquaporins) [11,12]. Our most recent studies revealed that barley mutants with a BR deﬁciency or a BR insensitivity were characterised by a different fatty acid composition in the cell membranes, which resulted in altered membrane physicochemical properties that could have an impact on the membrane-dependent physiological processes [13]. p p p y g p Studies of the compounds that are incorporated into the membranes have made a huge contribution to understanding the processes that occur in the biological membrane. In the case of thylakoids that had been obtained from the diatom Phaeodactylum tricornutum, studies of the diadinoxanthin cycle have provided information about the molecular dy- namics of the thylakoid membrane and about the inﬂuence of the diadinoxanthin cycle pigment on this effect [14]. Strzałka et al. [15] investigated changes in the physical prop- erties (general membrane lipid ﬂuidity, dynamic orientational order parameter) of the vacuolar membranes compared to the protein body membrane in germinating pumpkin (Cucurbita sp.) seeds. Similar studies were performed on thylakoid membranes that had been isolated from barley during leaf senescence [16]. These measurements are taken using electron paramagnetic resonance (EPR) spectroscopy, which permits the direct study of the systems (including biological systems) that contain unpaired electrons. In biological systems, compounds that contain unpaired electrons are present in the products of bio- chemical transformations, e.g., photosynthesis. EPR can also be used to study systems that do not contain unpaired electrons naturally. In this case, artiﬁcial paramagnetic centres (spin labels) that contain a nitroxyl group, e.g., 5-doxyl stearic acid (5-SASL) or 16-doxyl stearic acid (16-SASL) are incorporated into the studied object. In the case of the biological membranes, the EPR spectra can provide information, among others, about the degree of order (S) and the rotational correlation times (τB and τC) of the molecules that contain a spin label, which is helpful in determining the molecular dynamics of the membranes [14,15]. 2.1. Plant Material, Experimental Design and Sampling 2.1. Plant Material, Experimental Design and Sampling The objects of study were the barley cultivar Delisa (wild type) and its semi-dwarf mutant (522DK), which has disturbances in the BR biosynthesis. The mutant was described in the work by Gruszka et al. [18]. The mutant was obtained via the chemical mutagenesis of the cultivar Delisa. The 522DK mutant has a G > A substitution at position 1130 of the HvDWARF transcript [18] and at position 3031 in the gene sequence [19], which causes the change of the valine-341 residue into isoleucine. The substituted valine-341 is a highly conserved residue that occurs in similar position in the homologous DWARF polypeptides from barley, Arabidopsis, rice and tomato. Since the HvDWARF gene encodes the brassinosteroid C6-oxidase, which is an important factor in the BR-biosynthesis (it catalyses the production of castasterone), the mutant 522DK has a lowered content of castasterone [19] and it also has a decreased level of other BR [20]. The seeds were germinated for three days at 24 ◦C in the dark. After germination, the seedlings were transplanted into pots (40 cm × 15 cm × 15 cm) (about 40 seedlings per pot) containing soil (‘Eco-ziem universal soil’ (Jurków, Poland), soil from the cultivation plots at the University of Agriculture (Cracow, Poland), sand and ‘Substral Osmocote—a universal substrate’ (Scotts Poland sp. z o.o., Warsaw, Poland) at a ratio of 8:4:2:4, respec- tively), after which they were grown for 18 days at 20 ◦C (16 h photoperiod). Then, the plants were divided into two groups. Each group consisted of three pots with plants of the wild-type cultivar and three pots with plants of the mutant. In the ﬁrst group, the temperature was lowered to 5 ◦C (21 days, 8 h photoperiod) and in the second group, it was increased to 27 ◦C (7 days, 16 h photoperiod). Light intensity in the growth chambers was 170 µmol m−2 s−1 and was provided by HPS Philips SON-T AGRO 400 W lamps. Because the current studies are a continuation of our earlier works, the durations of the acclimation and the photoperiods were identical to those that were described in our earlier articles [11,12,20–22]. According to literature data, both cold acclimation and acclimation to higher temperatures enable plants to acquire a greater tolerance to frost or heat, respec- tively [23–25]. 1. Introduction In the presented study we focused on the chloroplast membranes which are crucial In the presented study, we focused on the chloroplast membranes, which are crucial elements of photosynthetic machinery, and affect the level and quality of plant yields [17]. Biomolecules 2021, 11, 27 3 of 20 3 of 20 Bearing in mind the role of membranes as the ﬁrst cellular line to react to changing temper- atures (and also the importance of chloroplasts for the crucial plant process of photosynthe- sis), the aim of the study was to broaden the knowledge about the changes in the molecular dynamics of the chloroplast membranes during the acclimation of a plant to extreme tem- peratures and the inﬂuence of BR on these properties. By studying wild-type barley plants and a BR-deﬁcient mutant, we wanted to answer the following detailed questions: (1) is the content of BR in the chloroplast membranes different in the wild-type plants than in a BR-biosynthesis mutant grown at an optimal growth temperature, i.e., 20 ◦C? (2) How does the temperature of acclimation (5 ◦C and 27 ◦C) modify the accumulation of BR in the chloroplast membranes of the wild type and mutant? (3) Are there differences in the molecular dynamics of the chloroplast membranes from wild-type barley and a mutant that is cultured at 20 ◦C? (4) How does a lower (5 ◦C) or higher (27 ◦C) temperature during plant growth modify the molecular dynamics of the chloroplast membranes? Finally, the role of BR as regulators of the molecular dynamics of the photosynthetic membranes will be discussed in light of the chloroplast content of the photosynthetic pigments and fatty acid composition in the BR-deﬁcient barley mutant and in the respective wild type. 2.3. Isolation of Chloroplasts from Barley Leaves The chloroplasts were isolated based on a modiﬁed protocol of Block et al. [26] and Filek et al. [27]. About 100 g of the aerial part of the barley (mainly leaves) were homogenised using a Camry CE 4050 blender in 400 mL of a chloroplast isolation buffer (CIB) (pH 7.5) that contained 50 mM Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.33 mM sorbitol. The crude extract was ﬁltered and centrifuged for three minutes at 300× g (Hettich zentrifugen Universal 320R, rotor 1494, Tuttlingen, Germany) in order to remove any residues after plant homogenisation. Next, the supernatant was centrifuged for ten minutes at 1200× g (Hettich zentrifugen Universal 320R, rotor 1494, Tuttlingen, Germany). The obtained pellet contained isolated chloroplasts. The purity of the isolated chloroplasts was checked under a microscope (Nikon Eclipse E600, Tokyo, Japan) (Figure S1). The entire isolation process was performed in a cold room (4–6 ◦C). Next, in order to break down the chloroplasts, samples were frozen in liquid nitrogen and thawed in two cycles; this was especially important in EPR studies. Breakdown of chloroplasts was checked under microscope (Nikon Eclipse E600, Tokyo, Japan). Frozen samples were kept at −80 ◦C until the day of analysis. 2.4. Analysis of the Protein Content in Chloroplasts The protein concentration was estimated according to Sedmak and Grossberg [28]. Two microlitres of a 10% water solution of Triton X-100 (Sigma-Aldrich, Pozna´n, Poland) were mixed with 2 µL of the chloroplasts that had been suspended in CIB and 196 µL of a CIB buffer (see Isolation of chloroplasts section) and kept for 15 min on ice. Ten microlitres of each sample were placed into separate wells of a plate (96-well polystyrene titration plate with a ﬂat bottom, FL Medical, Torreglia, Italy) and then, 200 µL of a Bradford reagent (BioRad, Munich, Germany) (diluted with water 1:4) was added. After 10 min, the absorbance was recorded (595 nm) using a SynergyTM2 Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA). The measurements were carried out in three replicates. Bovine serum albumin (BSA) (Sigma-Aldrich, Pozna´n, Poland) was used as the calibration standard. The BSA for spectrophotometric measurements was diluted in the same buffer as the buffer that had been used to isolate the chloroplasts (CIB buffer). 2.2. Leaf Greenness Measurement Leaf greenness, which corresponds to the leaf chlorophyll concentration, was mea- sured using a chlorophyll meter (SPAD 502; Konica Minolta, Tokyo, Japan, (SPAD—Soil Plant Analysis Development)) and the values of greenness are expressed in arbitrary SPAD units. The measurement was taken in the middle part of the fourth fully developed leaf in three technical replications per each leaf and the average value was calculated for each leaf. The leaves from nine plants were measured—three plants from each pot. 2.1. Plant Material, Experimental Design and Sampling Our previous studies [20] showed that after acclimation at 5 ◦C, both the wild type and mutant were characterised by a comparable tolerance to frost (−8 ◦C) while the mutant 522DK had an even better frost tolerance at −6 ◦C compared to the wild-type Delisa. After acclimation at 27 ◦C, the mutant had a much better heat tolerance (tested at 38 and 45 ◦C) compared to the wild type. y Before cutting off the plants, leaf greenness was measured using a chlorophyll meter for the plants that had been grown at 20 ◦C and then in the plants that had been acclimated at 5 ◦C and 27 ◦C. The plants grown at 20 ◦C had four leaves, while the acclimated plants had four well-developed leaves and sometimes a young ﬁfth leaf. The samples to isolate Biomolecules 2021, 11, 27 4 of 20 the chloroplasts were collected from the wild-type and mutant plants that had been grown at 20 ◦C and from the plants that had been acclimated at 5 ◦C and 27 ◦C. It was possible to isolate about 1.5 g (0.3 g per sample) of chloroplasts from 20 to 25 plants that weighed around 40 g. Three samples containing 0.3 g of chloroplasts (each sample from plants from different pot) were collected to analyse the BR. For the other analyses, each sample containing 0.3 g chloroplasts were ﬁrst suspended in a 1 mL chloroplast isolation buffer (CIB) buffer (see Isolation of Chloroplasts section) and these suspensions were used to further analyse the content of proteins, photosynthetic pigments, and the fatty acid composition. The proteins were also analysed in order to standardise the samples to be used for the EPR studies. 2.7. Analysis of the Brassinosteroids in the Chloroplasts Samples of the isolated chloroplasts (0.3 g each) were homogenised in 80% methanol and enriched to the internal standard with deuterium-labelled BR (25 pmol/sample, Olchemim s.r.o., Olomouc, Czech Republic). Next, the samples were centrifuged and the supernatant was passed through Discovery DPA-6S columns (Supelco, Bellefonte, PA, USA) and immunoafﬁnity columns (Laboratory of Growth Regulation, Olomouc, Czech Republic) [31]. The brassinosteroids that had been eluted with cold 100% methanol from the IA columns were dried and resuspended in 40 µL of methanol in order to mea- sure them on a UHPLC using a tandem mass spectrometry (UHPLC–MS/MS) with an ACQUITY UPLC® I-Class System (Waters, Milford, MA, USA) and a Xevo™TQ-S MS triple quadrupole mass spectrometer (Waters MS Technologies, Manchester, UK) [31,32]. The analyses were performed in three repetitions and each repetition included about 0.3 g of the isolated chloroplasts. 2.5. Analysis of the Fatty Acid Composition in Chloroplasts The fatty acids (FA) were extracted by homogenising the isolated chloroplasts. Sam- ples of the chloroplast suspension in the CIB buffer (0.15 mL each sample) were suspended in 0.20 mL of toluene and transferred into screw-capped glass test tubes. Subsequently, Biomolecules 2021, 11, 27 5 of 20 1.50 mL of methanol and 0.30 mL of an 8.0% HCl solution were added. The test tubes were vortexed and then incubated at 45 ◦C overnight in order to turn the extracted fatty acids into fatty acid methyl esters (FAME). After cooling to room temperature, the FAME were extracted by adding 1 mL of hexane and 1 mL of water [29]. The test tubes were vor- texed, and then the hexane layers were analysed using gas-liquid chromatography (GC). The n-hexane extracts were analysed chromatographically on an Agilent 6890N gas chro- matograph (Agilent Technologies, Santa Clara, CA, USA) that was equipped with a ﬂame ionisation detector (FID) and a capillary column. The certiﬁed reference material 37 FAME MIX (Supelco, CRM 47885) and an internal standard (biphenyl) were used to identify and quantify the fatty acid proﬁles. The analyses were conducted using a gas chromatograph equipped with a split/splitless injector. An ionic liquid fused silica capillary column (SLB-IL100, Supelco, 28884-U) (30 m × 0.25 mm ID × 0.2 mm ﬁlm thickness) with a matrix 1,9-di(3-vinylimidazolium)nonane bis(triﬂuoromethanesulfonyl)imide phase was operated under the following programmed conditions: 50–240 ◦C at 3 ◦C min−1 for 30 min (detector and injector temperatures of 240 ◦C), autosampler injection mode and volume of 0.5 µL and split (10:1) with helium 6.0 as the carrier gas (velocity 40 cm min−1). FAME were identiﬁed by comparing them with the standard mixture (Sigma-Aldrich, Pozna´n, Poland) and their retention time. 2.6. Determining the Carotenoid and Chlorophyll Content in Chloroplasts 2.6. Determining the Carotenoid and Chlorophyll Content in Chloroplasts Eighty percent aqueous acetone (2.09 mL) was added to the samples of the suspension of chloroplasts in the CIB buffer (0.01 mL each sample). The samples were vortexed and centrifuged (8 min, 12.100× g) (Eppendorf Mini-Spin, Hamburg, Germany). The UV-ViS (Jasco870, Easton, MD, USA) measurements were taken at wavelengths of 663.2, 664.8 and 470 nm. The measurements were taken in three replicates. The amounts of chlorophylls and carotenoids were calculated according to following Equations [30]: Ca = 12.25·A663.2 −2.79·A664.8 (1) Cb = 21.50·A664.8 −5.10·A663.2 (2) Ca+b = 7.15·A663.2 + 18.71·A664.8 (3) +c = (1000·A470 −1.82·Ca −85.02·Cb)/198 (4) Ca = 12.25·A663.2 −2.79·A664.8 (1) Cb = 21.50·A664.8 −5.10·A663.2 (2) Ca+b = 7.15·A663.2 + 18.71·A664.8 (3) Cx+c = (1000·A470 −1.82·Ca −85.02·Cb)/198 (4) (1) (3) (3) (4) where Ca is the concentration of chlorophyll a; Cb is the concentration of chlorophyll b; Ca+b is the sum of the chlorophyll a and b concentration; and Cx+c is the concentration of carotenoids. 2.7. Analysis of the Brassinosteroids in the Chloroplasts 2.8. Electron Paramagnetic Resonance (EPR) Measurements The molecular dynamics of the cell membranes were determined according to the mod- iﬁed protocol described by Strzałka et al. [15] and was monitored using EPR spectroscopy Biomolecules 2021, 11, 27 6 of 20 with spin labels 5-doxyl stearic acid (5-SASL, Santa Cruz Biotechnology, Heidelberg, Germany) and 16-doxylstearic acid (16-SASL, Sigma-Aldrich, Pozna´n, Poland), which provides information about the molecular dynamics of the region near the polar heads and the membrane interior, respectively. The spin labels were incorporated into the chloroplast membranes at room temperature by vortexing. About 100–300 µL of the chloroplast mem- branes (corresponding to 30 µg of protein) were vortexed for 20 min with 5 µL of 10 mM spin labels in methanol [15]. To remove any traces of the spin labels in the supernatant, the samples were centrifuged three times at 4500× g for two minutes at room temperature. The pellet that was obtained was resuspended in 50 µL of a 10mM CIB buffer (its composition is described in the Isolation of chloroplasts section) in order to obtain a ﬁnal cell membrane concentration yield that corresponded to 30 µg of the protein (the protein content was measured using the Bradford method and no signiﬁcant differences were observed be- tween wild-type Delisa and the 522DK mutant). The EPR spectra of the spin label as a function of the temperature was recorded using an X band EPR spectrometer (Minis- cope, Berlin, Germany) that was equipped with a Temperature Controller (Magnettech, Berlin, Germany) with a temperature range of 0 to 40 ◦C at intervals of 5 ◦C. For the plants that had been acclimated at 5 ◦C, the range of the measurement was between 0 ◦C and 25 ◦C and the interval was 2.5 ◦C. The EPR measurements were performed at microwave powers between 3.2 mW and 10.0 mW with a sweep width of 12–15 mT, a modulation amplitude of 0.1 mT and a microwave frequency of about 9.4 GHz. Two biological replicates with two to three technical replicates were done. The obtained EPR spectra were analysed with MultiPlot 2.0 Software (Magnettech GmBH, Berlin, Germany) (Figure 1). The parameters that were required to calculate the dynamic orientational order pa- rameter (S) and the rotational correlation times (τ2B and τ2C) are presented in Figure 1. 2.9. Statistical Analysis The statistical analysis (ANOVA, post hoc test) was performed using Statistica 13.1 (StatSoft, Tulsa, OK, USA). For the statistical analysis in the study, when the Delisa and 522DK were compared, Student’s t-test was used (p ≤0.05). Values marked with the same letters in Figures 2 and 3 and Table 1 did not differ signiﬁcantly (the comparisons were performed separately for each temperature of plant growth). Moreover, the accumulations of BR (Figure 3) in the Delisa cultivar and mutant at different temperatures were also compared (Student’s t-test, p ≤0.05). The comparisons were performed in pairs (Delisa for 20 ◦C and 5 ◦C; mutant for 20 ◦C and 5 ◦C; Delisa for 20 ◦C and 27 ◦C; mutant for 20 ◦C and 27 ◦C) and any signiﬁcant differences are indicated by an “”. The signiﬁcant differences in the results for the molecular dynamics (Figure 4) between the wild-type Delisa and the mutant are indicated by an asterisk (); the comparisons were performed separately for each EPR measurement temperature. Table 1. Composition of the fatty acids of the chloroplast membranes isolated from the barley wild-type Delisa and its 522DK mutant cultured at 20 ◦C, 5 ◦C, and 27 ◦C. Any signiﬁcant differences between Delisa and the mutant (Student’s t-test, p ≤0.05) for each temperature are indicated by different letters. <LOD—below limit of detection. Table 1. Composition of the fatty acids of the chloroplast membranes isolated from the barley wild-type Delisa and its 522DK mutant cultured at 20 ◦C, 5 ◦C, and 27 ◦C. Any signiﬁcant differences between Delisa and the mutant (Student’s t-test, p ≤0.05) for each temperature are indicated by different letters. <LOD—below limit of detection. 2.8. Electron Paramagnetic Resonance (EPR) Measurements The values of the S parameter were calculated using the following Equation [33]: where S = 0.5407 (A’\|\| −A’⊥)/ao (5) ao = (A’\|\| + 2A’⊥)/3 (6) (5) (6) (5) (6) (5) (6) The rotational correlation times (τ2B and τ2C) were calculated according to the follow- ing Equations [34]: The rotational correlation times (τ2B and τ2C) were calculated according to the follow- ing Equations [34]: τ2B = 6.51 · 10−10 · ∆H0 · ((h0/h−) 1 2 −(h0/h+ ) 1 2 ) [s] (7) (7) and and τ2C = 6.51 · 10−10 · ∆H0 · ((h0/h−) 1 2 + (h0/h+ ) 1 2 −2) [s] (8) (8) 7 of 20 Biomolecules 2021, 11, 27 Figure 1. Schematic electron paramagnetic resonance (EPR) spectra of the SASL-5 (A) and SASL-16 (B) spin labels that had been incorporated into the chloroplast membranes. The spectral parameters that were analysed are indicated (2A’⊥and 2A’\|\|, ∆H0, and h+ , h0 and h−). Figure 1. Schematic electron paramagnetic resonance (EPR) spectra of the SASL-5 (A) and SASL-16 (B) spin labels that had been incorporated into the chloroplast membranes. The spectral parameters that were analysed are indicated (2A’⊥and 2A’\|\|, ∆H0, and h+ , h0 and h−). 2.9. Statistical Analysis Fatty Acids [%mol] Growth Temperature 20 ◦C 5 ◦C 27 ◦C Delisa 522DK Delisa 522DK Delisa 522DK 10:0 <LOD 0.79 0.47 a 0.50 a <LOD 0.35 12:0 4.00 a 3.93 a 4.49 a 4.37 a 4.12 b 4.60 a 14:0 0.28 a 0.25 a 0.23 a 0.19 a 0.29 a 0.21 b 16:0 16.54 b 18.76 a 16.16 a 17.41 a 17.20 a 16.11 a 16:1 6.29 a 4.95 a 4.25 a 4.23 a 6.45 a 3.36 a 18:0 3.53 a 3.24 a 3.37 a 3.47 a 2.96 a 2.59 a 18:1 ∆9 cis 1.05 b 1.84 a 0.93 b 1.15 a 1.36 a 1.54 a 18:2 ∆6 cis 4.24 a 4.32 a 3.05 a 3.01 a 6.85 b 7.64 a 18:3 (3) 63.87 a 61.85 a 66.45 a 65.33 a 60.43 a 63.28 a 20:1 0.19 a 0.06 a 0.34 a 0.34 a 0.34 a 0.31 a 18:3/18:2 15.09 a 14.38 a 22.29 a 21.70 a 8.84 a 8.29 a U/S 3.11 a 2.72 b 3.01 a 2.86 a 3.08 a 3.20 a 8 of 20 Biomolecules 2021, 11, 27 Figure 2. Concentration of chlorophyll (Chl) a (A), chlorophyll b (B), xantophylls and carotenoids (x + c) (C), and total chlorophyll (a + b) (D) in a suspension of chloroplasts from the barley wild-type Delisa and its 522DK mutant. (E) The greenness intensity [SPAD units] of 1st–4th leaf of Delisa and its mutant 522DK. Plants had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C. Mean values (±SE) marked with the same letters (separately for plant growth temperature) did not signiﬁcantly differ according to Student’s t-test (p ≤0.05). Figure 2. Concentration of chlorophyll (Chl) a (A), chlorophyll b (B), xantophylls and carotenoids (x + c) (C), and total chlorophyll (a + b) (D) in a suspension of chloroplasts from the barley wild-type Delisa and its 522DK mutant. (E) The greenness intensity [SPAD units] of 1st–4th leaf of Delisa and its mutant 522DK. Plants had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C. Mean values (±SE) marked with the same letters (separately for plant growth temperature) did not signiﬁcantly differ according to Student’s t-test (p ≤0.05). 9 of 20 Biomolecules 2021, 11, 27 Figure 3. 2.9. Statistical Analysis Content of brassinosteroids in the chloroplasts of the barley wild-type Delisa and the BR-deﬁcient mutant 522DK that had been grown at 20 ◦C and acclimated at 5 ◦C or 27 ◦C. (A) Homocastasterone, (B) 28-norcastasterone, (C) 24–epibrassinolide, (D) Homodolicholide, (E) Castasterone, (F) Brassinolide, (G) Dolicholide, (H) Homodolichosterone, (I) Total BR content. <LOD–below detection limit. Mean values (±SD) marked with the same letters are not signiﬁcantly different according to Student’s t-test (p ≤0.05) (separately for each growth temperature). Additionally, in order to show the impact of temperature on the BR level in Delisa and mutant, the comparisons (Student’s t-test, p ≤0.05) were performed in pairs (Delisa at 20 ◦C and 5 ◦C; mutant at 20 ◦C and 5 ◦C; Delisa at 20 ◦C and 27 ◦C; mutant at 20 ◦C and 27 ◦C) and any signiﬁcant differences are indicated by an “”. Figure 3. Content of brassinosteroids in the chloroplasts of the barley wild-type Delisa and the BR-deﬁcient mutant 522DK that had been grown at 20 ◦C and acclimated at 5 ◦C or 27 ◦C. (A) Homocastasterone, (B) 28-norcastasterone, (C) 24–epibrassinolide, (D) Homodolicholide, (E) Castasterone, (F) Brassinolide, (G) Dolicholide, (H) Homodolichosterone, (I) Total BR content. <LOD–below detection limit. Mean values (±SD) marked with the same letters are not signiﬁcantly different according to Student’s t-test (p ≤0.05) (separately for each growth temperature). Additionally, in order to show the impact of temperature on the BR level in Delisa and mutant, the comparisons (Student’s t-test, p ≤0.05) were performed in pairs (Delisa at 20 ◦C and 5 ◦C; mutant at 20 ◦C and 5 ◦C; Delisa at 20 ◦C and 27 ◦C; mutant at 20 ◦C and 27 ◦C) and any signiﬁcant differences are indicated by an “”. 10 of 20 Biomolecules 2021, 11, 27 Figure 4. Dependence of the dynamic orientational order parameter S that was calculated for the chloroplast membranes that had been isolated from the barley wild-type Delisa and its mutant 522DK with incorporated SASL-5 (A–C) and SASL-16 (D–F) on temperature. Plants had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C. Average data are given ± SE and signiﬁcant differences between the wild type and mutant are indicated with an asterisk (); comparisons were made separately for each EPR measurement temperature. Figure 4. 2.9. Statistical Analysis Dependence of the dynamic orientational order parameter S that was calculated for the chloroplast membranes that had been isolated from the barley wild-type Delisa and its mutant 522DK with incorporated SASL-5 (A–C) and SASL-16 (D–F) on temperature. Plants had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C. Average data are given ± SE and signiﬁcant differences between the wild type and mutant are indicated with an asterisk (); comparisons were made separately for each EPR measurement temperature. 3.1.2. Carotenoid and Chlorophyll Content in the Chloroplast Membranes 3.1.2. Carotenoid and Chlorophyll Content in the Chloroplast Membranes Analysis of chlorophyll a, b and total chlorophylls (a + b) in the isolated chloroplasts showed no differences in the content of these pigments between the wild type and the mutant (Figure 2A,B,D). However, the leaf greenness measurements that were taken using a noninvasive method (chlorophyll meter) showed that for all four of the analysed leaves at all growth temperatures, the mutant was characterised by a more intense greenness (Figure 2E). Moreover, interestingly, according to our previous studies [42], a spectrophoto- chemical analysis of the chlorophyll content (especially chlorophyll b) in the leaf dry matter showed that 522DK had a lower content of chlorophyll than the Delisa cultivar, which was consistent with numerous studies that have proven that BR are important regulators of the chlorophyll accumulation because exogenous BR usually have caused an increase in chlorophyll content especially under stressful conditions [10]. In our current studies, the “greener” leaves of the 522DK mutant was rather a result of the mutant’s semi-dwarfness (more compact cells with a higher density of chloroplasts/chlorophyll per leaf surface), but again an analysis of the pure chloroplast suspension revealed no difference between the wild type and the mutant. In this experiment, the content of chlorophyll in the chloro- plasts was measured because these pigments also play an important role in membrane ﬂuidity [6,43]. Because the chlorophyll contents in the chloroplast membranes of 522DK and Delisa were similar, we can assume that the chlorophylls regulated the ﬂuidity of the chloroplast membranes in comparable manner. p p Similar to chlorophyll, the contents of xanthophylls and carotenoids in the pure chloroplasts were also at the same level in the wild type and the mutant (Figure 2C). Interestingly, our earlier studies [42] showed that the BR-deﬁcient mutant 522DK was characterised by a higher accumulation of carotenoids than Delisa when calculated per leaf dry mass. Contrary to these ﬁndings, Nie et al. [44] reported that carotenoid level increased in Solanum lycopersicum, which had a higher endogenous level of BR compared to its wild type. On the other hand, Koˆcova et al. [45] found that exogenous brassinosteroids did not signiﬁcantly affect the carotenoid content in maize leaves. 3.1.2. Carotenoid and Chlorophyll Content in the Chloroplast Membranes Although the role of BR in the accumulation/biosynthesis of carotenoids seems to be ambiguous, it is certain that carotenoids have huge inﬂuence on the thylakoid membrane ﬂuidity and they are also thought to be responsible for reducing the membrane ﬂuidity of a membrane [6,43]. Because no differences in the carotenoid content in the chloroplasts of the mutant and wild type were observed in this study, we suspect that in the current experiment, the inﬂuence of carotenoids on the chloroplast membrane ﬂuidity was comparable for Delisa and its mutant. 3.1.3. Brassinosteroid Content of the Chloroplasts 3. Results and Discussion 3.1. Characteristic of Selected Chemical Components of the Chloroplast Membranes Isolated from Barley Grown at 20 ◦C, 5 ◦C, and 27 ◦C y 3.1.1. Fatty Acid Composition of the Chloroplast Membranes Only a few FA had differences in content between the wild-type Delisa and its 522DK mutant and even when they were statistically signiﬁcant, they were not huge. Those that were most abundant in the chloroplast membranes were 18:3 (about 60%) and 16:0 (about 16–18%). The content of the other FA, including 18:1∆9cis or 18:2∆6cis, was less than a few percent. What is important is that at all of the tested temperatures, there were no differences between the mutant and Delisa for the main FA (18:3). As for the other FA, for example, at 20 ◦C the 522DK mutant was characterised by a higher content of 16:0 and 18:1∆9cis than the Delisa cultivar, while at 5 ◦C, only 18:1∆9cis content was higher in the 522DK mutant. Biomolecules 2021, 11, 27 11 of 20 11 of 20 For the plants that had been acclimated to a high temperature, the 522DK mutant was characterised by a higher level of 18:2∆6cis than the Delisa cultivar (Table 1). For the plants that had been acclimated to a high temperature, the 522DK mutant was characterised by a higher level of 18:2∆6cis than the Delisa cultivar (Table 1). There were no signiﬁcant differences between Delisa and its mutant at any growth tem- perature for the 18:3/18:2 ratio. Only at 20 ◦C was there a signiﬁcantly higher value of the unsaturated FA to saturated FA ratio (U/S) for Delisa than for the 522DK mutant (Table 1). ( ) ( ) Fatty acids are the main component of the biological membranes and their composi- tion plays a huge role in the membrane properties including membrane ﬂuidity [35,36]. According to Hölzl and Dörmann [37], chloroplasts are the major sites for FA synthesis in plant cells and 16:0, 18:1 ∆9cis, 18:2 ∆9cis,12cis and 18:3 ∆9cis,12cis,15cis FA are present at the highest levels in the chloroplast membranes. Therefore, our data are generally consistent with this [37] and other previous works on chloroplasts that had been isolated from avo- cado fruits and cauliﬂower leaves [38], as well as on the callus of winter oilseed rape [39] and on the leaves of Pisum sativum L, winter wheat, and barley [13,40,41]. 3.1.3. Brassinosteroid Content of the Chloroplasts Interestingly, dolicholide had already been detected by us in the barley leaves of other cultivars [46], but not in Delisa or its 522DK mutant [20]. In our opinion, the differences in the detectable BR proﬁle in the Delisa and 522DK leaves and chloroplasts are due to the fact that in the case of the leaves, 300 mg of fresh leaf material was used for the BR extraction, while in the case of the chloroplast isolation, 40 g of the leaves (25 plants) were used to obtain 1.5 g of the chloroplasts (300 mg pure chloroplasts per sample for the BR analysis). For this reason, the presence of BR that were revealed in the chloroplast samples here was probably below the detection limit in the whole leaves in earlier studies. The detection of BR in the barley chloroplasts conﬁrms our earlier reports (research on wheat [27]) that these compounds accumulate in the chloroplasts. Based on the relatively limited knowledge of BR synthesis/decomposition sites in a cell, it is assumed that all BR biosynthetic enzymes belong to the family Cyt P450s and are located in the reticulum membrane [47]. The fact that BR accumulates in the chloroplasts (probably also in the mem- branes) of both the 522DK mutant and its wild type suggests that these hormones could be very important for the appropriate functioning of the chloroplasts. The phenomenon of the incorporation of BR in the chloroplast structure will require further research, even though some guidance regarding the role of BR was provided by Cai et al. [48], Zhang et al. [49] and Krumova et al. [50]. Exogenous BR prevented injuries to the chloroplast ultrastructure in plants that had been exposed to high and low temperatures [48,49]. However, accord- ing to Krumova et. al. [50], BR have an impact on the functioning and regulation of the photosynthetic apparatus and the architecture of the thylakoid membrane. The authors found that Arabidopsis BR mutants, which overexpress the BR receptor and are deﬁcient in the BR biosynthesis, had increased thylakoid area compared to the wild type, which could be connected with changes in the chloroplast division mechanisms. On the other hand, at 20 ◦C, 5 ◦C, and 27 ◦C, the 522DK mutant had a better (than the wild type) efﬁciency of PSII (photosystem located in chloroplasts) [20]. 3.1.3. Brassinosteroid Content of the Chloroplasts In the chloroplasts, we were able to determine the following BR: brassinolide, castas- terone, 28-homocastasterone, 24-epibrassinolide, 28-norcastasterone, dolicholide, homod- olicholide and homodolichosterone (Figure 3A–H). Biomolecules 2021, 11, 27 12 of 20 Compared to the wild type, the BR-deﬁcient mutant 522DK that had been cultured at 20 ◦C was characterised by a lower content of homocastasterone and 28-norcastasterone but higher content of homodolicholide and homodolichosterone in its chloroplasts. The other BR were at comparable level in the chloroplasts of both genotypes at this temperature. In the mutant that had been grown at 5 ◦C, there was a higher content of homod- olicholide, homodolichosterone and also dolicholide compared to the wild type (Figure 3D,G,H). The homodolichosterone level was even below detection limit in the wild type at 5 ◦C (Figure 3H). As for the other BR at 5 ◦C, there were no signiﬁcant dif- ferences between the mutant and its wild type. Contrary to 20 ◦C, brassinolide was not detected at 5 ◦C in either the mutant or the wild type (Figure 3F). As for the plants that had been acclimated at 27 ◦C, brassinolide was only detected in the chloroplasts of the mutant but not in the wild type (Figure 3F). While the chloroplasts of the 522DK plants at this temperature were characterised by a higher content of homod- olicholide than the wild type, the level of dolicholide and homodolichosterone was below the detection limit in the chloroplasts of the mutant (Figure 3D,G,H). The other BR had a comparable concentration in the chloroplasts of both genotypes at this temperature. p p g yp p As for the total amount of the detected BR (sum of all of the detected BR) (Figure 3I), the obtained results conﬁrmed that the 522DK mutant was generally BR-deﬁcient, but this was only observed at 20 ◦C for the chloroplasts. Interestingly, our earlier analysis of the BR content in the leaves of Delisa and 522DK [20] unambiguously showed that the mutants accumulated less BR in their leaf tissue than the wild type at all three temperatures (20 ◦C, 5 ◦C and 27 ◦C). Moreover, in the leaf material, only brassinolide, castasterone, and homocastasterone were found in that study, while in this study, many more BR were detected in the pure chloroplasts, including dolicholide, dolichosterone, homodolicholide, and also 28-norcastasterone and 24-epibrassinolide. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy To study the molecular dynamics of the chloroplasts that had been isolated from the barley plants, two kinds of spin labels were incorporated into the membranes: SASL-5 and SASL-16. The ﬁrst has a nitroxide group that is bound to the 5th carbon of the stearic acid and that is why it can provide information about the molecular dynamics processes that occur near the membrane surface. SASL-16 has a nitroxide group that is attached to the 16th carbon of the stearic acid and its incorporation into membranes provides information about the dynamic processes that occur inside the membrane [15]. The results of our experiment are presented in Figure 4A–F. It was found that the possibility to measure the spectrum of the spin labels (SASL-5 and SASL-16) was dependent on the ambient temperature of the plant from which the chloroplasts had been isolated. For example, for the chloroplasts that were isolated from the plants acclimated at 5 ◦C with incorporated SASL-16, it was possible to calculate the S parameter in a measurement of the temperature in the range of 0–17.5 ◦C (Figure 4E). On the other hand, for the chloroplasts that were isolated from the plants acclimated at 27 ◦C with incorporated SASL-16, it was possible to calculate the S parameter in the entire measurement temperature range that was used in this study, i.e., 0–45 ◦C. This shows that plant acclimation at 5 ◦C and 27 ◦C resulted in a good adaptation of the studied barley plants to low and high temperatures, respectively. Moreover, the dependence of the dynamic orientational order parameter on the temperature at which the plants were cultivated was also observed (Figure S2). As was mentioned earlier, according to our previous work [20], after acclimation at 5 ◦C and 27 ◦C, the 522DK mutant and its wild-type Delisa developed a tolerance to frost (up to −8 ◦C) and heat (38 ◦C and 45 ◦C). Additionally, in the study of Bojko et al. [51] on thylakoid membranes of T. pseudonana that were cultured at low and high temperatures (12 ◦C and 20 ◦C, respectively), a similar effect of efﬁcient adaptation to acclimating temperatures was observed, which was connected, among others, with the molecular dynamics results. 3.1.3. Brassinosteroid Content of the Chloroplasts Simultaneously (according to the current study), the mutant unambiguously accumulated only one BR—homodolicholide—in the chloroplasts at a higher amount than the wild type at all of the temperatures. Homod- olicholide is a lesser-known BR and is present in relatively small amounts compared to Biomolecules 2021, 11, 27 13 of 20 castasterone or homocastasterone (Figure 3A,D,E). Might it then be responsible for the better performance of the chloroplasts in the mutant that had been exposed to temperature stress? This will also require further studies. castasterone or homocastasterone (Figure 3A,D,E). Might it then be responsible for the better performance of the chloroplasts in the mutant that had been exposed to temperature stress? This will also require further studies. q As was mentioned above, the leaves of the mutant always had less BR than the leaves of the wild type [20]. In the case of mutant chloroplasts, however, we did observe: (1) the occurrence of lower amounts of BR, e.g., homocastasterone at 20 ◦C than in the wild type; (2) no differences in the BR content in the mutant and in the wild type (castasterone at all of the growth temperatures) and (3) a higher amount of BR in the mutant, (e.g., homodolicholide, independent of growth temperature). p g p In our opinion, this fact suggests that the presence of BR in the chloroplasts and perhaps their incorporation into the membranes is driven by accumulation mechanisms that do not have much in common with their biosynthesis process (and ﬁnal BR pro- duction). Perhaps, the purely physicochemical mechanisms are involved (the membrane lipid environment was “beneﬁcial” for the accumulation of speciﬁc steroids—especially homodolicholide in the mutant). Temperature also had an effect on the accumulation of BR in the chloroplasts. Moreover, as was mentioned above, previous studies have shown that the presence of mycotoxins, the application of exogenous brassinosteroids in a culture medium or the cultivar’s tolerance to stress all inﬂuenced/modiﬁed the BR concentration in the chloroplasts in a different manner. A summary of the changes in the BR content in the chloroplast membranes of 522DK at different temperatures compared to its wild type is presented in Table S1. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy The dynamic orientational order parameter (S) that was calculated from SASL-5 and SASL-16 spectra gradually decreased for the chloroplast membranes from both the wild-type Delisa and the mutant along with an increase in the measurement temperature (Figure 4; Figure S2). This effect was connected with an increase in the molecular dynamics of the membranes and a less ordered molecular environment of the spin labels during an increase in the measurement temperature [14]. The obtained values of S are in agreement Biomolecules 2021, 11, 27 14 of 20 14 of 20 with the literature in which the dynamic orientational order parameter S varies between 1 (maximally ordered membranes) and 0 (maximally disordered membranes) [15,52]. An increase in the molecular dynamics is considered to represent an increase in membrane ﬂuidity [53]. For the chloroplast membranes with an incorporated SASL-5 spin label in the mea- surement temperature range between 5 ◦C and 10 ◦C, the 522DK chloroplast membranes from the mutant that had been cultured at 20 ◦C were characterised by higher values of the S parameter than the Delisa’s membranes (Figure 4A). There were no signiﬁcant differences between the chloroplast membranes of Delisa and 522DK for the other measurement tem- peratures (Figure 4A). For the chloroplast membranes of the plants that had been acclimated at 5 ◦C and 27 ◦C, there were generally no noticeable differences between the wild-type Delisa and the mutant (Figure 4B,C); the only exception was at a measurement temperature of 10 ◦C, when the membranes of the mutant were characterised by a higher value of S (Figure 4B). To summarise, near the membrane surface, there were no differences between the Delisa cultivar and its mutant 522DK that had been acclimated at 5 ◦C and 27 ◦C. This is consistent with the fact that the surface area of membranes needs to be quickly ordered in order to support the proper interactions of the membranes with their neighbour area and it is also needed in order to avoid the uncontrolled diffusion of molecules such as oxygen or free radicals, which can cause destructive reactions inside membranes that are not fully adapted to the new conditions of environmental stimuli [14,52,54]. p For the chloroplast membranes with an incorporated SASL-16 spin label, differences in the S parameter were observed between the mutant and the wild type that had been acclimated at 5 ◦C. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy The values of S parameter were signiﬁcantly lower for the Delisa cultivar compared to its mutant in the entire tested temperature range and the differences were almost always statistically signiﬁcant (Figure 4E). The same effect was observed for the membranes of the plants that were cultured at 20 ◦C, but only in the measurement temperature range between 25 ◦C and 30 ◦C (Figure 4D). In this case, interestingly, the S parameters were measurable at a temperature of 35 ◦C only for the chloroplast membranes that had been isolated from the Delisa cultured at 20 ◦C (but not for the mutant). No differences were observed for the membranes of the plants acclimated at 27 ◦C (Figure 4F). To conclude, for measurement of the chloroplasts with an incorporated SASL-16 spin label, we can say that the chloroplast membranes of the mutant 522DK that had been acclimated at 5 ◦C were more ordered (more rigid) than the membranes of the Delisa. Studies using a Langmuir bath led to similar conclusions (author’s unpublished data). The monolayers were built from galactolipids (MGDG, DGDG—the main chloroplast lipid constituents [55]) and also from phospholipids that were isolated from the 522DK mutant and its wild type that had been acclimated at 5 ◦C. In the case of the mutant, the monolayers were characterised by statistically signiﬁcantly lower (than Delisa) values of Alim. Alim represents the area that is occupied by a single molecule in a completely packed layer, provides information about membrane ﬂuidity and decreases with a decrease in ﬂuidity. In case of monolayers built from MGDG, values of Alim [Å2] were 65.6 ± 0.2 and 64.9 ± 0.3, respectively for Delisa and mutant. In case of monolayers built from DGDG values of Alim were 55.2 ± 0.1 and 51.7 ± 0.2, respectively for Delisa and mutant. Usually, greater ﬂuidity of cell membranes is considered as more favourable for a higher frost tolerance, which has been wider discussed in our earlier work [13]. The 522DK mutant, which has a slightly higher frost tolerance than the wild type Delisa [20], is, however, characterized by a greater rigidity of chloroplast membranes than Delisa, as shown in EPR studies. The question remains if/how fact of rigidiﬁcation of chloroplast membranes is related to the frost tolerance of the mutant. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy Interestingly, as for the plasma membrane, it is known that the level of the expression of many cold-inducible genes increases two- to three-fold after the rigidiﬁcation [56]. On the other hand, we have to mention that the 522DK mutant that was studied in the present experiment also had a better high-temperature tolerance than the wild-type Delisa [20], although there was no difference in the membrane molecular dynamics between Biomolecules 2021, 11, 27 15 of 20 15 of 20 the mutant and the wild type after acclimation at 27 ◦C (Figure 4C,F). Interestingly, it is also worth noting here that most of the changes in the FA content were observed at 27 ◦C, but that no differences were observed in the molecular dynamics of the chloroplast membranes of plants that had been acclimated at 27 ◦C in either the hydrophilic and hydrophobic area. p y p y p Analysis of the other three parameters that were calculated from the SASL-16 spectra (∆H0 and rotational correlation times τ2B and τ2C) provided additional information about the movement of a spin label and at the same time about the movement of FA in the interior of the membrane. The ∆H0 parameter provided information on the oscillation and rotation of a spin label and its rotation along the long axis of the molecule [15]. Lower values of ∆H0 indicate a greater freedom of the spin label motion. Our results show that there were generally no differences in the motion of the molecules (∆H0 parameter) of the chloroplast membranes of the Delisa cultivar and its 522DK mutant at the tested temperatures (Figure 5A,B,C). For the other two parameters, τ2B corresponded to the rotation of the spin label molecule along its long axis, while τ2C provided information about the movement of the molecule in the direction perpendicular to the long axis. The rotational correlation times are assumed to reﬂect the local ﬂuidity of a membrane—lower values indicate a greater motional freedom of the spin labelled FA and thus a higher ﬂuidity of a membrane [14]. Figure 5. Dependence of parameters ∆H0 (A–C), τ2B (D–F) and τ2C (G–I), which were calculated for the chloroplast membranes that were isolated from the barley wild-type Delisa and its mutant 522DK with incorporated SASL-16 on temperature. Plants had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy Average data are given ±SE and signiﬁcant differences between the wild type and mutant are indicated with an asterisk (); comparisons were made separately for each EPR measurement temperature. Figure 5. Dependence of parameters ∆H0 (A–C), τ2B (D–F) and τ2C (G–I), which were calculated for the chloroplast membranes that were isolated from the barley wild-type Delisa and its mutant 522DK with incorporated SASL-16 on temperature. Plants had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C. Average data are given ±SE and signiﬁcant differences between the wild type and mutant are indicated with an asterisk (); comparisons were made separately for each EPR measurement temperature. The values of τ2B and τ2C of 16-SASL in the chloroplast membranes from both Delisa and 522DK depended on the temperature of the measurement (Figure 5D–I). It was ob- served that within a temperature range between 0 ◦C and 7.5 ◦C, the chloroplast membranes of the mutant 522DK, which had been grown at 20 ◦C and acclimated at 5 ◦C, was char- acterised by signiﬁcantly higher values of τ2B than its wild type (Figure 5D,E). For the plants that had been acclimated at 27 ◦C, Delisa had a higher value of τ2B than its mutant only around 0 ◦C (Figure 5F). Moreover, the values of τ2C were noticeably higher for the Biomolecules 2021, 11, 27 16 of 20 chloroplast membranes of the 522DK mutant than its wild type, which had been cultivated at 20 ◦C and acclimated at 5 ◦C, within a range of measurement temperatures between 0 ◦C and 10 ◦C (Figure 5G,H). For the chloroplast membranes of the plants that had been accli- mated at 27 ◦C, there were signiﬁcant differences between Delisa and the mutant only at a measurement temperature of 0 ◦C (Figure 5I). When the temperature of the measurement was higher, both parameters had lower values. This decrease in the rate of the molecule rotation along the long axis (τ2B) is in line with an increased rigidity of membranes [14]. g g g y We can assume that the greater motional freedom had FA included in Delisa’s chloro- plast membranes compared to chloroplast membranes of 522DK. Therefore, all of the calculated parameters once again implied that the chloroplast membranes of 522DK at 20 ◦C and 5 ◦C were more ordered/less ﬂuid than the chloroplast membranes of the Delisa cultivar. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy The ﬁnal matter to discuss is what caused the differences in the molecular dynamics of the membranes between the mutant and its wild type. Because the components that are important for membrane structure and properties, such as FA, carotenoids and chloro- phylls were comparable in the chloroplasts of the wild type and mutant (Table 1, Table S1, Figure 2), it is likely that the differences in the membrane molecular dynamics might be connected with other components including BR. Among all of the BR found in current study, the concentration of castasterone and homocastasterone was usually at the highest level in the chloroplasts. In the chloroplasts of the cold-treated plants, these BRs were found in amounts of about 1000–6000 pg per g of F.W. The 522DK mutant that had been accli- mated at 5 ◦C had more rigidiﬁed chloroplast membranes than the wild type, but also had a similar content of these two BR present in high content (Figure 3A,E). This may suggest that these two BR are not the cause of the differences in the membrane molecular dynamics. The chloroplast content of BR from the other groups such as homodolicholide, dolicholide, and homodolichosterone is a different matter. The cold-acclimated 522DK had a signiﬁcantly higher content of these BR in the chloroplasts than the wild type (Figure 3D,G,H); however, their content was generally very low (not more than 30 pg per g of F.W.). Thus, it is difﬁcult to say whether they can be responsible for the changes in the molecular dynamics. In turn, it should be remembered that the molecular dynamics of membranes was quite similar in the mutant and the wild type at 20 ◦C and 27 ◦C, although there were differences in the BR content. To conclude, the role of BR as membrane stabilisers remains an open question. We cannot totally reject the hypothesis about the role of BR in modifying the dynamics of mem- branes because there are other studies that suggest this. Li et al. [57] investigated the effect of exogenous BR on the ﬂuidity of the plasma membrane in mango fruits that had been stored at 5 ◦C. Using the EPR method, the authors found that treatment with BR increased the membrane ﬂuidity in mango fruit. 4. Concluding Remarks y p p (3) Although electron paramagnetic resonance has been used to study the cell mem- brane properties and the inﬂuence of different factors on those properties [60], knowledge about the inﬂuence of BR on the ﬂuidity of cell membranes is still rudimentary. In the current work, it was shown that the main components that are important for the chloro- plast membrane’s structure/properties, such as FA, carotenoids and chlorophylls, were comparable in the chloroplasts of the wild type and mutant 522DK, but it cannot be unam- biguously stated that the BR that were present in the chloroplasts were responsible for the differences in the chloroplast membrane molecular dynamics between the tested genotypes. However, in the hydrophobic (but not hydrophilic) area, the chloroplast membranes of the mutant 522DK that had been acclimated at 5 ◦C were more ordered (more rigid) than the membranes of the wild-type Delisa. Slight differences between the mutant and wild type were also observed after their growth at 20 ◦C. There was practically no difference in the membrane dynamics between the mutant and the wild type after acclimation at 27 ◦C in either the hydrophilic or hydrophobic area. Supplementary Materials: The following are available online at https://www.mdpi.com/2218-273 X/11/1/27/s1, Figure S1: Sample of the freshly isolated chloroplasts from fresh plant material taken to conﬁrm the purity of the chloroplasts, seen under a Nikon Eclipse E600 microscope (Tokyo, Japan), Figure S2: Dependence of the dynamic orientational order parameter S calculated for the chloroplast membranes (with incorporated SASL-5 (A,B) and SASL-16 (C,D)) that were isolated from the barley wild-type Delisa (A,C) and its mutant 522DK (B,D) that had been cultivated at 20 ◦C and acclimated at 5 ◦C and 27 ◦C on temperature. Average data are given ±SE and signiﬁcant differences between the temperatures of growth (20 ◦C, 5 ◦C, and 27 ◦C) are indicated with an asterisk (*); comparisons were made separately for each EPR measurement temperature, Table S1: Changes in the content of FA and BR in the barley chloroplasts of the BR-deﬁcient mutant 522DK (mutation HvDWARF) compared to the wild-type Delisa (↑increase compared to the wild type; ↓decrease compared to the wild type; NC no change compared to the wild type). Author Contributions: I.S. is the coordinator of the project 2018/31/N/NZ9/02430. I.S. and A.J. planned the research. D.L. 4. Concluding Remarks Based on our experiment, the following main ﬁndings can be pointed out: (1) In the chloroplasts that were isolated from the barley wild-type Delisa and the mutant 522DK, regardless of the plant growth temperature, eight brassinosteroids at different concentrations were identiﬁed. Their diverse presence may indicate that they play some role in the functioning of these organelles. (2) Mutant 522DK is a BR-deﬁcient mutant that has a lower accumulation of BR compared to the wild type in its leaf tissue [18,20]. However, in the case of the chloroplasts, this regularity has not been proven in present studies. The mutant’s chloroplasts had a higher level of three brassinosteroids: homodolicholide (regardless of the plant growth temperature), dolicholide (plants at 5 ◦C) and homodolichosterone (plants at 20 ◦C) than the wild-type Delisa. On the other hand, the content of homocastasterone was lower in the mutant’s chloroplasts (plants at 20 ◦C), while castasterone was unchanged (regardless of the plant growth temperature). It is likely that the incorporation of BR into the chloroplast membranes is driven by accumulation mechanisms that do not have much in common with their biosynthesis process or the ﬁnal BR production/concentration in cells. y p p (3) Although electron paramagnetic resonance has been used to study the cell mem- brane properties and the inﬂuence of different factors on those properties [60], knowledge about the inﬂuence of BR on the ﬂuidity of cell membranes is still rudimentary. In the current work, it was shown that the main components that are important for the chloro- plast membrane’s structure/properties, such as FA, carotenoids and chlorophylls, were comparable in the chloroplasts of the wild type and mutant 522DK, but it cannot be unam- biguously stated that the BR that were present in the chloroplasts were responsible for the differences in the chloroplast membrane molecular dynamics between the tested genotypes. However, in the hydrophobic (but not hydrophilic) area, the chloroplast membranes of the mutant 522DK that had been acclimated at 5 ◦C were more ordered (more rigid) than the membranes of the wild-type Delisa. Slight differences between the mutant and wild type were also observed after their growth at 20 ◦C. There was practically no difference in the membrane dynamics between the mutant and the wild type after acclimation at 27 ◦C in either the hydrophilic or hydrophobic area. 3.2. Molecular Dynamics of the Barley Chloroplasts Measured Using EPR Spectroscopy In our earlier studies using the Langmuir bath, we showed that BR are incorporated into the structure of the membrane monolayer from lipids that were isolated from not-hardened and cold-hardened wheat. The changes in the physicochemical parameters of the monolayers such as compressibility were observed [41]. For further investigation of the role of BR as membrane stabilisers studies of EPR on model membranes would be helpful. We can also not exclude the fact that the amount of BR is not crucial here. Perhaps the proportions between each component of membranes including BR as well as sterols, which were not tested here, or some proteins are also important in regulating the chloroplast membrane ﬂuidity. Theoretically, BR can also be located in so-called rafts such as cholesterol in animal cell membranes [58] or phytosterols in plant membranes [59], which affect the membrane properties differently because they are local. Rafts are specialised lipid domains and the role of phytosterols-rich rafts is considered not without merit in maintaining plant membranes in a state of dynamics that is less sensitive to temperature shocks [59]. However, this point of view regarding BR requires further studies. 17 of 20 17 of 20 Biomolecules 2021, 11, 27 4. Concluding Remarks provided valuable suggestions about the research and the interpre- tation of data in the discussions during the writing of manuscript; I.S. isolated the chloroplasts, prepared the samples for the BR analysis, analysed the protein content, performed the leaf greenness measurements, puriﬁed the brassinosteroids on the immunoafﬁnity columns; J.O. measured the brassinosteroid content on UHPLC–MS/MS. I.S. and D.L. performed the EPR measurements; I.S. analysed the data. D.G. provided the seeds of the mutant 522DK and cv. Delisa and characterised them genetically. D.L. and M.C. prepared the samples for the fatty acid analysis and measured the fatty acid content on GC. I.S. analysed the carotenoids content analysis under D.L. coordination; I.S. performed the statistical analysis and prepared the tables and ﬁgures. I.S. wrote the manuscript under the supervision of A.J. All authors have read and agreed to the published version of the manuscript. Biomolecules 2021, 11, 27 18 of 20 18 of 20 Funding: The research costs of the project 2018/31/N/NZ9/02430 were funded by the National Science Centre (Poland). Support was also provided by the ERDF (European Regional Develop- ment Fund) project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0 /16_019/0000827) for the UHPLC–MS/MS analyses of the brassinosteroids. Funding: The research costs of the project 2018/31/N/NZ9/02430 were funded by the National Science Centre (Poland). Support was also provided by the ERDF (European Regional Develop- ment Fund) project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0 /16_019/0000827) for the UHPLC–MS/MS analyses of the brassinosteroids. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article or Supplementary Material. 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https://openalex.org/W2965910820	https://www.econstor.eu/bitstream/10419/196468/1/Article_2788.pdf	English	null	Economic Modeling in the Management of Transition to Bioeconomy	Amfiteatru economic	2,019	cc-by	8,084	Provided in Cooperation with: The Bucharest University of Economic Studies Suggested Citation: Busu, Cristian; Busu, Mihail (2019) : Economic modeling in the management of transition to bioeconomy, Amfiteatru Economic Journal, ISSN 2247-9104, The Bucharest University of Economic Studies, Bucharest, Vol. 21, Iss. 50, pp. 24-40, https://doi.org/10.24818/EA/2019/50/24 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. 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Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. https://creativecommons.org/licenses/by/4.0/ https://creativecommons.o Article Economic modeling in the management of transition to bioeconomy Amfiteatru Economic Journal Busu, Cristian; Busu, Mihail Article Economic modeling in the management of transition to bioeconomy Amfiteatru Economic Journal Provided in Cooperation with: The Bucharest University of Economic Studies Suggested Citation: Busu, Cristian; Busu, Mihail (2019) : Economic modeling in the management of transition to bioeconomy, Amfiteatru Economic Journal, ISSN 2247-9104, The Bucharest University of Economic Studies, Bucharest, Vol. 21, Iss. 50, pp. 24-40, https://doi.org/10.24818/EA/2019/50/24 Busu, Cristian; Busu, Mihail Article Economic modeling in the management of transition to bioeconomy Amfiteatru Economic Journal Provided in Cooperation with: The Bucharest University of Economic Studies Suggested Citation: Busu, Cristian; Busu, Mihail (2019) : Economic modeling in the management of transition to bioeconomy, Amfiteatru Economic Journal, ISSN 2247-9104, The Bucharest University of Economic Studies, Bucharest, Vol. 21, Iss. 50, pp. 24-40, https://doi.org/10.24818/EA/2019/50/24 AE Economic Modeling in the Management of Transition to Bioeconomy * Corresponding author, Mihail Busu ‒ mihail.busu@man.ase.ro Cristian Busu1 and Mihail Busu2* 1)2)The Bucharest University of Economic Studies, Romania Article History Received: 30 September 2018 Revised: 11 November 2018 Accepted: 9 December 2018 Article History Received: 30 September 2018 Revised: 11 November 2018 Accepted: 9 December 2018 Please cite this article as: Busu, C. and Busu, M., 2019. Economic Modeling in the Management of Transition to Bioeconomy. Amfiteatru Economic, 21(50), pp. 24-40. Please cite this article as: Busu, C. and Busu, M., 2019. Economic Modeling in the Management of Transition to Bioeconomy. Amfiteatru Economic, 21(50), pp. 24-40. DOI: 10.24818/EA/2019/50/24 Introduction Evolutionary anthropological studies highlight the challenging factors that society is facing as a whole. Worldwide demographic growth cannot be sustained at the same pace as finite natural resources. Climate change due to environmental pollution caused by industrial development is another major danger, which leads to significant ecosystems imbalances. Faced with these challenges, the strategy proposed by the European Commission in the bioeconomy sector (European Commission, 2012) identifies risks in geopolitics and proposes a series of ambitious goals, including: decreased dependence on finite resources, adaptation to climate change and mitigation, food security and new jobs created in rural areas. Managing the transition to bioeconomy in order to achieve the objectives set by the European Forum entails a collection of data, such as development of knowledge of the sectors generating biomass, feedback on economic interests and data on national government policy as a starting point to a documented analysis to determine the current stage in the transition. Economic modeling is the next step in the Contextual Conversion Scenario to respond to predictable estimations communicated by decision makers. Research activities in the field of bioeconomy will significantly contribute to the knowledge of biomass production potential, identifying the potential barriers to the development of this new industry. Until now, many industrialized countries have developed strategies and policies for a circular economy based on the reuse of biomass. Romania, in turn, has an environmental strategy that allows the future development of bio-economy. Current studies identify the need to determine the intersectoral correlations, respectively the modalities of the relationship between the different sub-sectors of the bioeconomy, which are generating new products. This article aims to develop a model for analyzing sectoral interdependencies, taking into account exogenous variables such as resource production, production expenditures, government policy, investment in technology, R&D and innovation, to assess the prospects of transforming the traditional economy. Abstract The management of the transition to bioeconomy has developed relatively recently, to cope with the challenges of the 21st century. The determinants of transition are related to economic, social, biological and environmental evolution. In particular, the envisaged factors consist in demographic increase, excessive unsustainable consumption, industrial development, climatic changes, depletion of traditional or non-renewable resources. This article presents, first, the conceptual model of the transition process, making a review of the specialized literature. The authors render applicability to the sustainable economic model developed in the primary sector, which is the first biomass-producing industry with potential to generate new products, used in the secondary and tertiary industrial sectors. The second part of the article defines the behavioral research model. The article aims at providing an applicative perspective to the behavioral model, through the economic integration of data collected from the biomass industry. The third part of the article presents the results of the case study, interpreted in the light of the responses to the questionnaire transmitted to the respondents. Data were collected using a questionnaire addressed to management and employees in the biomass industry. Structural equation modeling (SEM) was used, and d splints were analyzed by software-ul Smart PLS 3. One of the main goals of this paper is to identify and assess the relationship between the sustainable development of Bioeconomy through the indicators of performance obtained by producers of biomass in the framework of the economic model of transition to the bioeconomy. The conclusions of our research are in line with the existing literature and confirm the theoretical assumptions, underlining that the performance and sustainable development of the producers is a direct consequence of the association of a number of factors such as innovation and technology, regulation, knowledge and skills of the human factor. Keywords: bioeconomy, behavioral model, technology, research, innovation, environment. JEL Cl ifi ti Q57 O32 C39 C83 Keywords: bioeconomy, behavioral model, technology, research, innovation, environment. Keywords: bioeconomy, behavioral model, technology, research, innovation, environment. JEL Classification: Q57, O32, C39, C83 24 Amfiteatru Economic Keywords: bioeconomy, behavioral model, technology, research, innovation, environment. JEL Classification: Q57, O32, C39, C83 * Corresponding author, Mihail Busu ‒ mihail.busu@man.ase.ro JEL Classification: Q57, O32, C39, C83 Amfiteatru Economic 24 Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE 1. Presentation of the management model in the economic literature Economic literature abounds in models for assessing the transition to bioeconomy. Authors such as Rick Bosman and Jan Rotmans have studied comparatively the two models of management of the transition to bioeconomy in Finland and the Netherlands. The research paper (Bosman and Rotmans, 2016) contains a series of recommendations tailored to the national economic characteristics of the two countries. As such, the authors note a useful suggestion to develop the public-private partnerships in which both public decision makers and the private sector find the right solutions to meet the challenges in managing the transition to bio-economy. An example is provided by the partnership between the European Commission and the Bioindustry Consortium, whereby the state and the private sector invest in innovative technologies for bio-industry to generate new biomass products. This inevitably implies a change in the behavior of the human factor, which understands the need to move Vol. 21 • No. 50 • February 2019 25 AE Economic Modeling in the Management of Transition to Bioeconomy from the traditional economy to the circular economy, by reference to a new sustainable economic model that embraces the innovative factor (Carrez and van Leeuwen, 2015). A management system for the transition to the bioeconomy needs to know in detail what the functional requirements for transforming a traditional economy into the bioeconomy are. Sustainability of analysis models has been the subject of research in economist work (Hockings et al., 2006). The modeling of sectoral interrelationships is constantly evolving, requiring a quantitative and qualitative analysis. For example, it is possible to analyze in the short term the impact on the price evolution of a bio product produced in a secondary sector as a result of the increase in the prices of the basic resources of the primary sector. In addition, the evaluation criteria are varied, belonging to a multitude of disciplinary sciences, from biodiversity to food safety (European Commission, 2012). For this reason, there is no perfectly integrated model of management that aggregates the totality of the exogenous variables, for these reasons as we mentioned related to the continuous development of the interdependence relations and the multitude of variables. Economic modeling requires a permanent adaptation to the specific economic context, being integrated into a scenario adjusted for government policies and private demand for biomass and bioenergy products (Angenendt et al., 2018). Amfiteatru Economic 1. Presentation of the management model in the economic literature The second project was launched by the European Commission in February 2013 and aims at integrating the data collected in the first project and at conceptualizing them in a computer system. The institution that processes this data is the European Observatory for Bioeconomy. The conceptual model used describes the interaction between society and the environmental factors. The management system is defined by the human, demographic, consumerist factor, biophysical processes and the limited natural resources, the technological progress and innovation, and last but not least, the governance policy of the state. of industries with the potential to generate biomass products and identifies barriers to the development of the new industry. The second project was launched by the European Commission in February 2013 and aims at integrating the data collected in the first project and at conceptualizing them in a computer system. The institution that processes this data is the European Observatory for Bioeconomy. The conceptual model used describes the interaction between society and the environmental factors. The management system is defined by the human, demographic, consumerist factor, biophysical processes and the limited natural resources, the technological progress and innovation, and last but not least, the governance policy of the state. The economic modeling of human behavior is influenced by the economic context and cultural affiliation of individuals, in accordance with the principles of the universal theory of multilinear evolution. Thus, condensing the opinions of anthropologists Leslie Alvin White (1975) and Julian Haynes Steward (1972), the change of mentality gradually takes place in a process of awareness of the need for transformation. From the perspective of the transition to bioeconomy, at national level, we observe the potential generated by certain industries, specific to the basic sectors, for the production of biomass, such as agriculture, zootechnics, fish farming, wood processing, secondary sectors such as bio-industry (i.e. biodegradable plastic products, biofuels), the food industry and tertiary sectors, interdependent on the first two, such as the chemical and pharmaceutical industries. The National Strategy for Research, Development and Innovation 2014-2020 identifies, on the basis of a public consultation, the areas of intelligent specialization for the development of bio-economy (GD no. 929 / 21.10.2014). There is a constant need for information and communication on biomass production, support for environmental research and limitation of certification costs, to increase productivity and competitiveness of sectors with the potential to generate new, affordable products. 1. Presentation of the management model in the economic literature This contextual, specific approach is particularly useful in the decision-making process in order to develop a technology appropriate to the development in the biomass-producing sectors and to assess the contributions of these sectors to the production of positive externalities on competitiveness and production in the tertiary sector as well the pharmaceutical industry, the chemical industry, or the biofuels industry. Nowicki et al. (2007) detailed the economic analysis at the level of the economic sub-sectors (for example, horticulture as a sub-sector of agriculture) and, accordingly, the modeling was determined at the level of products that could contain bio-ingredients. From the perspective of the economic models used in the research work, they can be classified into dynamic models of general equilibrium applicable to analyze multi-sector macro interdependencies (i.e. energy ‒ agriculture ‒ bioenergy ‒ bioindustry) developed by economists such as Poganietz (2000), Van Meijl (2006), Banse et al. (2014), economic equilibrium models applicable to certain economic sectors (Izaurralde et al., 2012) defined as partial models and bottom models (Janssen et al., 2010), which respond to contextual questions related to technological, procedural or behavioral development, studying in detail the procedural aspects related to the generation of biomass sources, respectively the production of the economic and ecological effects resulting from these processes , whether and to what extent they meet these expectations. Temporary definition and location in geographic space is of particular significance to conceptualize the management model. This explains why a model performs better in one country than in another or can give clues as to the evolution of the transition to the bio-economy during a period of time. Research studies conducted in the European Economic Area often have common benchmarks for contextual analysis. This is largely due to the two projects launched at the European Union level by the European Commission, which substantiate the framework for assessing the transition to the bio-economy. The first project was launched in November 2012 and refers to the Analysis Toolbox to respond to the EU's Bioeconomic Strategy (System Analysis Tool framework for the EU Bio Based Economy Strategy SAT – BBE within the EU 7th Framework Programme). The first research project makes an inventory Amfiteatru Economic 26 Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE of industries with the potential to generate biomass products and identifies barriers to the development of the new industry. 1. Presentation of the management model in the economic literature Additional investment is needed in technology, so as indicated by the data collected in the research (PwC, 2017). According to the study, the share of GDP gross value added in agriculture is positioning Romania on the first place in the European Union. Given the large number of nurseries in the agricultural sector compared to the European average, the productivity of the sector is relatively low. Also, the yield of agricultural production, relative to arable land, is low compared to the European average due to the fragmentation of agricultural holdings, the level of professional training of farmers, the low level of capitalization and the regulatory framework. The conceptual model is essential to manage the transition to the bio-economy development. The reflective indicators demonstrate the efforts of civil society, the political and business to meet objectives bioeconomy or sustainable resource management, development of new technologies for biomass production, support processes to reuse resources, food security, increase food quality and pharmaceutical, economic and social prosperity by creating new jobs, developing infrastructure for recycling and, last but not least, encouraging sustainable consumption patterns. Economic reforms, environment and research policies adopted, changing forms of organization are necessary elements to turn negative externalities in the bioeconomy benefits. Vol. 21 • No. 50 • February 2019 27 AE Economic Modeling in the Management of Transition to Bioeconomy 2. Econometric model of the of the behavioral model for the management of transition to bioeconomy According to the study by Mariusz Maciejczak (2015) on the determinant productive factors for bioeconomy, it is noted that Romania is the best placed from the EU Member States to produce biomass from waste. Based on this, we have conducted a case study in the biomass industry for enterprises active in the agricultural sector, considering the main biomass sources, namely grain crops. We determine the performance of biomass producers according to the two indicators: the percentage indicator of the variation of biomass production, based on waste reuse, and the variation in the rate of revenue from sales of production relative to operating costs. These will be dependent variables in the model. The independent variables are as following:  Innovation, research and development (indicators: investments in biotechnology or new infrastructure, patents, patents or licenses issued, number of R&D employees, the existence of biotechnology labels or processed organic products needed to inform consumers or distributors – i.e. ecolabels);  Regulation (public funding programs, tax incentives provided in national legislation, incentives for investments in the field);  Knowledge and skills (no information or communications sent by producers on the benefits of using biomass within the production-distribution chain, for awareness of the importance of the field among each participant, no qualified personnel, no training courses for the training of specialists in the field of bio-economy, no participation in bioeconomy discussion forums, for transfer of expertise;  Motivation of employees to determine the extent to which they are stimulated to integrate into the workforce, incentives for outstanding performance, motivation for the transition to bioeconomy;  The performance of the production process (the existence of an integrated management information system, the existence of a circular flow of product reuse, based on their characteristics, i.e. biodegradable products, reintegrated into agriculture as a resource for generating new production, product lifetime.  The performance of the production process (the existence of an integrated management information system, the existence of a circular flow of product reuse, based on their characteristics, i.e. biodegradable products, reintegrated into agriculture as a resource for generating new production, product lifetime. Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE  Energy crops: short rotation woody crops, herbaceous woody plants, herbs, starch crops (corn, wheat and barley), sugar crops (cane and beet), crop crops (herbs, lucerne and clover), soybean, sunflower, saffron);  Aquatic plants: algae, water weeds, hyacinth water, cane and fats. Biomass is the fourth most important global energy source after coal, oil and natural gas it is (Ladanai and Vinterback, 2009). Typically, biomass production can be divided into two categories: bio ENER GIE (the process by which biomass products it is burned to generate heat and electricity) and biofuels (biomass products are converted into liquid fuel using the replacement of petroleum products in transport). Agricultural waste is another segment of biomass. Although Romania has a wide variety of crops, only a few of these are used for energy production, mainly due to the technical limitations of the conversion process (Colesca and Ciocoiu, 2013). The main sources of energy from their crop processing:  Oilseeds (rape, sunflower and soybean) used for biodiesel production;  Oilseeds (rape, sunflower and soybean) used for biodiesel production;  Sugar and starch crops used for the production of bioethanol (wheat, corn, corn, barley, potatoes and sugar beet);  Woods (hells) used for heat and energy production. Unfortunately, in Romania there are not many incentives for the use of modern biomass technologies to transform this into thermal and electrical energy. Thus, the latest available data indicate a total consumption of 3859 ktone/year, of which 3240 ktone /year consumed in the old traditional rural stoves and 619 ktone/year in the consumer industry and tertiary (Bioheat 2016). It terms of bioenergy, Romania has not progressed very much compared to 2007. In 2007 local production of biodiesel and bioethanol started. At the level of 2016, the potential of biodiesel was of about 400,000 tons/year and 120,000 tons/year for bioethanol (NREAP, 2017). Romania's potential to supply biodiesel production (sunflower, soybeans, rape) was around 580 thousand tons / year and bioethanol production around 560 thousand tons/year (consisting of 41 thousand tons / year of corn seeds and 150 thousand tons one / year of wheat germ) (ENERO, 2017). The most promising way for the production of biofuels is rapeseed (EC, 2003). In 2016, circus 161.000 tons of biofuels, accounting for 3.2% of total transport fuels (EBRD, 2017) was produced in Romania. Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy Directive 2003/30 / EC on the promotion of the use of biofuels or other renewable fuels for transport was included in the national legislative framework and in accordance with the law, biofuels shall replace gasoline and gasoline up to 10% by 2020. 2.1. Brief description of the biomass sector in Romania Biomass is part of the alternative energy resources and can be divided into four general categories:  Wastes: agricultural waste, agricultural waste, crop residues, wood waste, urban wood waste and urban waste;  Forest products: wood, wood residues, trees, shrubs and wood scraps, sawdust, bark etc. from clearing forests;  Forest products: wood, wood residues, trees, shrubs and wood scraps, sawdust, bark etc. from clearing forests; 28 Amfiteatru Economic Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy 2.2. Research Methodology In order to achieve an economic modeling in the management of the transition to bioeconomy, the following statistical hypotheses were formulated:  H1: The motivation of the employees has a positive impact on organizational performance;  H1: The motivation of the employees has a positive impact on organizational performance; Vol. 21 • No. 50 • February 2019 29 AE Economic Modeling in the Management of Transition to Bioeconomy  H2: The management quality is positively correlated with the organizational performance;  H3: The strategies and the commercial policies have a positive influence on organizational performance;  H4: Operational integrated processes have a positive impact on the organizational performances;  H5: Employees’ motivation is positively related to the performance of the organization. A survey-based assay was carried out with the help of a questionnaire and the research hypothesis were tested using Partial Least Square model. Data were processed using the SmartPLS 3 software (SmartPLS GmbH, Boenningstedt, Germany) (Ringle et al., 2015). The main purpose of its analysis it was the analysis of the transition to bio-economy of biomass-producing enterprises in Romania. The objective of this study is validating the economic model by analyzing data collected from Biomass energy sector in Romania. The paper also analyzes the impact of the econometric model, represented by innovation, regulation, level of knowledge and skills, production process performance and human factor motivation in the performance of biomass producers. The data collection was done through a survey which was addressed to employees and managers in the biomass production sector. Biomass companies were selected in proportion to their size. The sample chosen was random and the sample size was calculated by multiplying the number of indicators by 5-10 (Kristensen et al., 2010). Thus, the size of the sample should have at least 15 indicators × 5 = 75 respondents. The authors collected data from 90 respondents out of which 80 were valid. The questions were either binary, with answers (Yes = 1, No = 0), or they used a 7- point Likert scale where the choices ranged from "very little = 0" to "very much = 1". The questionnaire has been tested several times to ensure that language, format, and order of questions are appropriate. The following five proxy variables were considered important factors in the implementation of the model (Table no. 1): innovation in the production process (Malerba, 2002; Mainar- Causapé et. 2.2. Research Methodology al., 2017), sector level regulation (Crafts, 2006; Mengat et. al., 2018), knowledge and skills level (Boyatzis, 2006; Müller et. al., 2009), production process performance and motivation of employees. Table no. 1: Description of the variables in the model Innovation (INOV) Inov_1 Existence of investments in biotechnology Inov_2 Labeling for biotechnology or processed organic products Inov_3 The existence of patents or license issued Regulation (REGLEM) Reglem_1 Funding programs from public funds Reglem_2 Tax incentives provided for in national law Reglem_3 Incentives for investment in the field Table no. 1: Description of the variables in the model Innovation (INOV) Inov_1 Existence of investments in biotechnology Inov_2 Labeling for biotechnology or processed organic products Inov_3 The existence of patents or license issued Regulation (REGLEM) Reglem_1 Funding programs from public funds Reglem_2 Tax incentives provided for in national law Reglem_3 Incentives for investment in the field Table no. 1: Description of the variables in the model Amfiteatru Economic 30 Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE Knowledge and competences (KNOWLEDGE) Knowledge_1 Information or communications submitted by producers on the benefits of using biomass in the production-distribution chain Knowledge_2 Training courses for the purpose of training specialists in the field of bioeconomics Knowledge_3 Participation in bioeconomy discussion forums for transfer of expertise Performance of the Production Process (PROD) Prod_1 The existence of an integrated management information system Prod_2 The existence of a circular flow of product re-use based on their characteristics Prod_3 Product life Motivation of the employees (MOTIV) Motiv_1 Motivation employees to determine the extent to which they are stimulated to integrate into the workforce Motiv_2 Incentives given for outstanding performance Motiv_3 Employee motivation for the transition to bio-economy The variables described above will be the independent model latent variables of the model, while the reflective dependent variable is the "biomass producer performance" determined by two proxy variables: "percentage change of biomass production based on waste reuse" and "variation rate of revenue from the sale of production in relation to operating expenses” (Mohr-Jackson, 1998). The dependent variable together with the independent variables described above lead to the construction of the structural model that will be tested and validated in the next chapter (Figure no. 1). Figure no. 2.2. Research Methodology 1: The structural model incorporating the results of managerial decisions on the transition to bioeconomy The questionnaires were addressed to both executive and corporate executives in June 2018 and were finalized at the end of July this year. In Table no. 2 show the distribution of biomass production companies as well as the respondents of the questionnaire. They were selected in proportion to the distribution of enterprises, thus obtaining a sample approaching the optimal theoretical combination. Figure no. 1: The structural model incorporating the results of managerial decisions on the transition to bioeconomy The questionnaires were addressed to both executive and corporate executives in June 2018 and were finalized at the end of July this year. In Table no. 2 show the distribution of biomass production companies as well as the respondents of the questionnaire. They were selected in proportion to the distribution of enterprises, thus obtaining a sample approaching the optimal theoretical combination. Vol. 21 • No. 50 • February 2019 31 AE Economic Modeling in the Management of Transition to Bioeconomy Table no. 2: Distribution of respondents Producers of Biomass Sample SME 82 80 Big Enterprises 8 10 Total 90 90 The analyzed sample consisted of 90 executives and managers from companies operating in the biomass industry in Romania, stratified by sex, age, income, status and level of education, directly interviewed directly, the questionnaire containing 20 of questions. Table no. 2: Distribution of respondents Producers of Biomass Sample SME 82 80 Big Enterprises 8 10 Total 90 90 The analyzed sample consisted of 90 executives and managers from companies operating in the biomass industry in Romania, stratified by sex, age, income, status and level of education, directly interviewed directly, the questionnaire containing 20 of questions. In the elaboration of the questionnaire the stratified sample method was applied. The data were collected during June 1 ‒ July 30, 2018, using an open questionnaire and the main limitation of the data collection process was the lack of co-operation of the individuals surveyed. The main objectives of the study were: to identify respondents' level of knowledge regarding bio-economy based on education level, gender, age and interests’ respondents; assessing innovation and R&D in enterprises; determine the degree of regulation in the sector; assessing the knowledge and skills of employees and assessing the production process of biomass enterprises in the perspective of the transition to bioeconomy. Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE 3.1. The measurement model The measurement model was achieved by the convergent and discriminating validity method. From a statistical point of view, the relation between the reflexive and the dormant variables can be determined by means of structural equation modeling (SEM): {x = Ψx ϑ + εx (1) {y = Ψy η + εy (2) where: (1) {x = Ψx ϑ + εx {y = Ψy η + εy where: (1) (2) (2) where: ɳ - endogenous latent variable; ɳ - endogenous latent variable; ϑ - exogenous variable; x and y - observed variables; Ψx and Ψy - matrices of the systems of equations corresponding to latent variables; Ψx and Ψy - matrices of the systems of equations corresponding to latent variables; εx and εy - the residual variables. εx and εy - the residual variables. εx and εy - the residual variables. 3.1.1. Convergent validity When a latent variable explains a significant part of the variance of its constructs, the convergent validity must be verified (Nakasul, 2017). The convergent validity is examined by the extraction of variance indicator (AVE ‒ average variance Extracted), which measures the variation that a latent variable is capturing its variables associated with the total variance of the variance, including the variance of the error of measurement (Rust Huang, 2012). At a level above 0.5, the latent variable explains more than half of the variance of its latent variables (Čihák et. al., 2013). According to Chin (2010), the variables for which the coefficients are less than 0.5 are excluded. Thus, the following variables will be excluded from the model: inov_1, inov_3 reglem_1 and reglem_3. These are variables whose coefficients are less than 0.5 in figure no. 2. Model I Figure no. 2: PLS-SEM Measurement Model 1 Source: Results determined by the authors valuing the SmartPLS 3 software 3. Results and discussions The degree of significance of the conceptual model variables will be verified before the model is analyzed. This will be done by calculating and interpreting the coefficients "Dillon-Golstein” and "Cronbach's Alpha" (Tenenhaus et al., 2005). The values of these indicators can be seen in table no. 3. Table no 3: Reliability and validity of the variables Construct Dillon Golsteins’ rho Cronbach's Alpha Composite Reliability AVE VIF INOV 1.000 1.000 1.000 1.000 2.221 REGLEM 1.000 1.000 1.000 1.000 1.418 KNOWLEDGE 0.712 0.723 0.821 0.712 1.528 PROD 0.621 0.691 0.679 0.677 2.798 MOTIV 0.678 0.682 0.682 0.651 2.392 Table no 3: Reliability and validity of the variables From the table above, it can be noted that the values associated with the variables in the model are all greater than 0.7, which is why it can be concluded that all five latent variables are significant in our analysis. In addition, the collinearity of the latent variables has been tested. According to Hair et al. (2013) collinearity between independent variables is present if their VIF values are greater than 5. According to the data in the table, we can conclude that the exogenous variables in the model are not collinear. The econometric analysis was carried out by the Partial Least Squares ‒ Structural Equation Modeling (PLS-SEM) method. This consists in the creation of two sub-models, namely the structural model "internal model" and the measurement model, quantification, "external model". The PLS-SEM model was chosen because it is more robust than other similar methods (i.e. CB-SEM), but also less sensitive to small samples, asymmetric distributions or the presence of multicollinearity (Hair et al., 2013). Amfiteatru Economic 32 Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy By comparing the square of the correlation coefficients between the latent variables in the model IU structural and indicator values AVE, discriminant validity is confirmed. Figure no. 3: PLS-SEM Measurement Model 2 Source: Results determined by the authors valuing the SmartPLS 3 software As can be seen from Figure no. 3, all coefficients in the model are greater than 0.5, meaning that they are significant and valid. Thus, the convergent validity of the model is conformed. Model I Figure no. 2: PLS-SEM Measurement Model 1 Source: Results determined by the authors valuing the SmartPLS 3 software Vol. 21 • No. 50 • February 2019 33 33 AE Economic Modeling in the Management of Transition to Bioeconomy After excluding the above variables from the model, a new model was obtained from the previous one. By SEM-PLS a second model resulted (Model II), as outlined in Figure no. 3. Model II Figure no. 3: PLS-SEM Measurement Model 2 Figure no. 3: PLS-SEM Measurement Model 2 3.2. The structural model The structural model (inner model) results from Figure No. 2 in which the relations (path) of exogenous variables (latent) and endogenous (reflective) are described. The structural equation (Zhang, 2009) of the model is as follows: η= Γη+Λϕ+ψζ (3) where: (3) η= Γη+Λϕ+ψζ where: η= Γη+Λϕ+ψζ where: where: η - vector of latent endogenous variables; ϕ - vector of latent exogenous variables; ψ - vector of residual variables; Γ and Λ - the path coefficient matrices. The structural model is evaluated by the values of the coefficients and the R-square value (R_squared). According to figure no. 3, the R-square value is 0.668, which means that about 66.8 % of the variability of biomass producers' performance is explained by the variability of the model. Moreover, the coefficient values of the structural model are all positive, which means that the higher the values the latent variables have, the higher the performance of the manufacturers. Of the five indicators, the variable with the greatest impact is "performance of the production process" (factor 0.722), followed by "knowledge and skills" (factor 0.648) and "innovation" (factor 0.641). Organizational performance with the least impact is given by indicators "degree of regulation of the sector" (factor 0.567) and "employee motivation" (factor 0.345). Indicators reflect emerge of endogenous variable "performance producers of biomass" have values over as 0.5. It follows that these variables in the reflective model (prod_var and income_var) are statistically significant. The high values of the coefficients of the reflexive variables, i.e. the "change in biomass production (factor 0.956) and the " rate of income from the sale of production relative to operating costs" (factor 0.652) shows that the performances of biomass producers are well represented by the two indicators. Multiple factors, in general, increase reliability and improve the performance of the model compared to the unique factors. In our model, the dependent variable is composed of two variables that reflect economic performance, a financial variable, and the other operational variable. In addition, a variable has a higher impact because it has high coefficient values reflecting more powerful measurement paths. This is also underlined by the high values of the two coefficients of endogenous variables. Moreover, hypothesis testing their research described it above, it was made a Bootstrap test on a sample of 300 respondents to generate t-values and standard deviations of the model parameters. Bootstrapping allows the assignment of precision measurements to the sample estimates. Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE 3.1.2. Discriminant validity The AVE indicator can be used to verify the discriminatory validity of the model (Fornell and Larcker, 1981). The authors consider that if the square values of the correlation coefficients between the latent variables is less than the computed value of AVE (Table no. 4), the discriminant validity is confirmed. Table no. 4: Correlation coefficients between the latent variables Latent variables AVE Square of the corr. coefficients of the latent variables Inov Reglem Knowledge Prod Motiv INOV 1.000 1 REGLEM 1.000 .621 1 KNOWLEDGE 0.712 .679 .512 1 PROD 0.677 .528 .491 .531 1 MOTIV 0.651 .513 .324 .552 .621 1 Source: Data analysis was performed by the authors valuing SmartPLS 2 0 M3 software Amfiteatru Economic 34 34 Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy 3.2. The structural model The results are presented in Table no. 5. Vol. 21 • No. 50 • February 2019 35 AE AE Economic Modeling in the Management of Transition to Bioeconomy Table no. 5: Hypothesis testing results Hypothesis Path Coef. Std. Error t-value* P_value H1: INOV -> Biomass Prod Performance 0.643 0.224 3.823 0.013 H2: REGLEM -> Biomass Prod Performance 0.562 0.369 2.701 0.035 H3: KNOWLEDGE -> Biomass Prod Performance 0.684 0.172 3.289 0.027 H4: PROD -> Biomass Prod Performance 0.754 0.131 2.368 0.039 H5: MOTIV -> Biomass Prod Performance 0.222 0.248 1.234 0.282 Note: t-value 2.58 (sig. level = 5 %) Valid hypotheses are those with values of p-value less than 0.05, while the others with p- values higher than 0.05 are not. Therefore, we can conclude that H1, H2, H3 and H4 are valid hypotheses, while H5 is not. These results confirm the economic studies according to which the innovation, the degree of regulation, the level of knowledge and skills and the production process are significant factors for the performance of biomass producers (Campbell et al., 2008; Cardenete et. al., 2014), but argues that motivation is an important factor in the performance of biomass producers (Wagner, 1994; Lewandoski, 2015). Table no. 5: Hypothesis testing results Hypothesis Path Coef. Std. Error t-value P_value H1: INOV -> Biomass Prod Performance 0.643 0.224 3.823 0.013 H2: REGLEM -> Biomass Prod Performance 0.562 0.369 2.701 0.035 H3: KNOWLEDGE -> Biomass Prod Performance 0.684 0.172 3.289 0.027 H4: PROD -> Biomass Prod Performance 0.754 0.131 2.368 0.039 H5: MOTIV -> Biomass Prod Performance 0.222 0.248 1.234 0.282 Note: *t-value 2.58 (sig. level = 5 %) Valid hypotheses are those with values of p-value less than 0.05, while the others with p- values higher than 0.05 are not. Therefore, we can conclude that H1, H2, H3 and H4 are valid hypotheses, while H5 is not. These results confirm the economic studies according to which the innovation, the degree of regulation, the level of knowledge and skills and the production process are significant factors for the performance of biomass producers (Campbell et al., 2008; Cardenete et. al., 2014), but argues that motivation is an important factor in the performance of biomass producers (Wagner, 1994; Lewandoski, 2015). 3.2. The structural model In conclusion, we can state that the degree of innovation, the level of regulation in the sector, the level of knowledge and skills, the performance of the production process have a positive impact on the performance of biomass producers in Romania. Conclusions Using economic modeling in the management of the transition to bioeconomy with SEM- PLS structural equations, the paper identifies and then evaluates the relationship between the determinant characteristics of the conceptual management model of the transition to bioeconomy and the economic performance of the biomass production companies. The results indicate that a significant proportion of variability of endogenous variables is explained by exogenous latent variables. The survey used in our research highlights that integrated business processes used by an enterprise have a positive impact on performance and sustainable development. Important factors that converge towards performance regarding innovation of the production process, the level of knowledge and skills, the performance of the production process and the motivation of employees. In this context, the significant contributions of management processes (including surveillance and inspection) lead to an increase in motivation for the transition to bio-economy of these corporations. This argues the high value of the process- level coefficients of this model. The econometric analysis has shown that the entrepreneurial confidence in government policies for the development of bio-economy is very low. We also note that the principle of Corporate Social Responsibility (CSR) remains an abstract concept without practical Amfiteatru Economic 36 Contributions of the Disciplines Studying the Mechanisms of Human Behavior at Understanding the Transition to Bioeconomy AE application, as suggested by the modest results obtained for the human factor motivation indicator. The results of the behavioral model, analyzed through the structural equations modelling, confirm the conclusions of the studies in the economic literature, emphasizing that innovation, the level of regulation in the sector, the level of knowledge and skills and the performance of the production process are determinants of the management of the transition to the bioeconomy. Some of the recommendations of the Bosman and Rotmans (2016) research study are fully applicable to Romania as regards the need to develop public-private partnerships to meet the needs of the transition to bioeconomy. A limitation of this study is given by the relatively small number of respondents to the survey and by the fact that the paper analyzes a single industry. Other limitations of this research could be related to the subjective answers of the questioned persons and the number of constructs and reflective variables. Conclusions These limitations could be overcome in future research by increasing the sample size and the number of constructs and reflective variables, as well as introducing open questions in the questionnaire. Additional studies in the field of bioeconomy transition on producer performance should be extended to other sectors or industries, and research may, as such, include some likely macroeconomic effects. The analysis could also be developed by analyzing how companies in the industry have funded their efficiency towards a sustainable development of the biomass sector. References Angenendt, G., Zurmühlen, S., Axelsen, H. and Sauer, D. U., 2018. Comparison of different operation strategies for PV battery home storage systems including forecast- based operation strategies. Applied Energy, 229, pp. 884-899. Anon., 2017. National Renewable Energy Action Plan (NREAP). 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https://openalex.org/W3201138321	https://europepmc.org/articles/pmc8448608?pdf=render	English	null	Fabrication and Characterization of a Nanofast Cement for Dental Restorations	BioMed research international	2,021	cc-by	8,764	Research Article Fabrication and Characterization of a Nanofast Cement for Dental Restorations Kh. Yousefi,1 H. Danesh Manesh ,1 A. R. Khalifeh ,1 and A. Gholami 2 1Department of Materials Science and Engineering, School of Engineering, Shiraz University, 71348-51154 Shiraz, Iran 2Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Kh. Yousefi,1 H. Danesh Manesh ,1 A. R. Khalifeh ,1 and A. Gholami 2 1Department of Materials Science and Engineering, School of Engineering, Shiraz University, 71348-51154 Shiraz, Iran 2Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran should be addressed to H. Danesh Manesh; daneshma@shirazu.ac.ir and A. R. Khalifeh; areza1006@gmail.com Correspondence should be addressed to H. Danesh Manesh; daneshma@shirazu.ac.ir and A. R. Khalifeh; arez Received 8 April 2021; Accepted 21 July 2021; Published 10 September 2021 Received 8 April 2021; Accepted 21 July 2021; Published 10 September 2021 Academic Editor: Romeo Patini Copyright © 2021 Kh. Youseﬁet al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study was aimed at fabricating and evaluating the physical and bioproperties of nanofast cement (NFC) as a replacement of the MTA. The cement particles were decreased in nanoscale, and zirconium oxide was used as a radiopaciﬁer. The setting time and radiopacity were investigated according to ISO recommendations. Analysis of color, bioactivity, and cytotoxicity was performed using spectroscopy, simulated body ﬂuid (SBF), and MTT assay. The setting time of cement pastes signiﬁcantly dropped from 65 to 15 min when the particle sizes decreased from 2723 nm to 322 nm. Nanoparticles provide large surface areas and nucleation sites and thereby a higher hydration rate, so they reduced the setting time. Based on the resulting spectroscopy, the specimens did not exhibit clinically noticeable discoloration. Resistance to discoloration may be due to the resistance of zirconium oxide to decomposition. Scanning electron microscopy (SEM), X-ray diﬀraction (XRD), and infrared spectroscopy (FTIR) examinations of the immersed SBF samples showed apatite formation that was a reason for its suitable bioactivity. The results of cell culture revealed that NFC is nontoxic. This study showed that NFC was more beneﬁcial than MTA in dental restorations. Hindawi BioMed Research International Volume 2021, Article ID 7343147, 12 pages https://doi.org/10.1155/2021/7343147 Hindawi BioMed Research International Volume 2021, Article ID 7343147, 12 pages https://doi.org/10.1155/2021/7343147 2. Materials and Methods 2.1. Material. The cement powder used in this study mainly consists of dicalcium silicate, tricalcium silicate (Sanat Avaran Vista, Iran), and zirconia that was used as a radiopa- ciﬁer. Absolute ethanol (molecular weight range 146,000– 186,000 g/mol, degree of hydrolysis > 99:0%; Aldrich, St Louis, MO) was the solution. Particle size analysis (PSA) was performed on the cement powders utilizing VASCO Flex equipment (Cordouan Technologies, Pessac, France). The morphology of the prime powders was studied with ﬁeld emission scanning electron microscopy (FESEM, MIRA3- TESCAN, Czech Republic). Digital images of the cement samples were also taken before and after soaking by a digital camera (Canon, EOS 4000D, Japan). Analysis of color was performed using a spec- trometer (VITA Easyshade Advance 4.0, Germany). Mea- surement was repeated three times by the same operator under steady laboratory light. The equipment was calibrated before each measurement to obtain more accurate results. The diﬀerence in color was calculated by the Commission International Del’eclairage (CIE) system and utilizing the fol- lowing formula [18]: ΔE = Δl ð Þ2 + Δa ð Þ2 + Δb ð Þ2 2 !1/2 , ð1Þ 2.2. Fabrication of Cement. The ﬁrst step was the preparation of the NFC powders. To accomplish this, the cement particles were reduced to nanosize using Wet-Stirred-Media-Milling (WSMM). The WSMM is performed in a vertical cylindrical crushing chamber made of polyethylene. The ceramic beads and cement were added to the grinding chamber. Then, abso- lute ethanol (99.9%, Merck, Germany) was added to the mix- ture up to half the volume of the container. Afterward, the mixture was stirred for 15 hours using a stirrer. The beads were separated from the solution and mixture using a mesh. For obtaining dry cement, the ethanol was evaporated in a drying cabinet for 24 hours and at a temperature of 37°C. After the cement powder was dried, zirconia was added to the cement powder as a radiopaciﬁer. This mixture was named NFC. The PSA method was utilized to measure ﬁnal particle sizes. FESEM was used to observe the morphology of the prepared NFC powders. ð1Þ where ΔE is the color diﬀerence before and after immersion, Δl shows the change in luminosity, Δa shows the change in the red-green parameter, and Δb shows the change in the yellow-blue parameter. 2.6. Bioactivity Property Evaluation. The bioactivity has been evaluated according to ISO 23317/2014 [19]. 1. Introduction time to avoid being washed away by saliva and reduce the possibility of the unset material irritating oral tissues. MTA has been shown to have a long setting time that confers many diﬃculties in handling during dental treatment pro- cesses [7]. The setting time for MTA in normal conditions is more than 2.5 to 3 hours [8], and in some treatments, more than one attempt is required to place a ﬁnal restoration in a tooth. Researchers have made eﬀorts to decrease the cement setting time using some additives and new formula- tions. For instance, several groups have made changes in the formulations of both the solid and liquid phases of MTA cement by adding calcium chloride, potassium chloride, or calcium formate to accelerate the setting time [9, 10]. Using these solutions as the hydration accelerant may enhance the biocompatibility but not compromise MTA’s antibacterial and mechanical properties. Calcium silicate-based cement named MTA was ﬁrst devel- oped as a root-end ﬁlling material, because of its clinical characteristics like bioactivity, biocompatibility, low solubil- ity, adaptation to tooth structures, dimensional stability, and sealing ability [1–3]. MTA mainly consisted of dicalcium sil- icate, tricalcium silicate, tricalcium aluminate, and a small amount of bismuth oxide that has been added for radiopa- city purposes. The limitations, such as poor handling qual- ity, low strength, long setting time, discoloration, and considerable expense, make its use a restoration material complicated [4, 5]. Dental cements are chemically bonded materials that are bonded through chemical reactions without the use of a high- temperature process [6]. One of the most widely used cements is MTA. MTA is a hydrophilic cement powder that produces harden compounds when in contact with water. The process is named hardening or setting. A cement dental ﬁller material should ideally have a relatively short setting The aesthetic appearance of dental treatment is an essen- tial issue for clinicians and patients, especially in the frontal part of the mouth. Hence, the color stability of endodontic materials is considered a critical factor for clinical success 2 BioMed Research International BioMed Research International BioMed Research International [11, 12]. MTA was ﬁrst introduced in a grey form (GMTA) [13]. Discoloration has been reported as the primary problem connected to this dental material. 1. Introduction After viewing the tooth dis- coloration caused by gray MTA, white MTA (WMTA) with reduced Al2O3, MgO, and FeO was developed with an oﬀ- white color to overcome this shortcoming [14]. However, several studies reported that tooth discoloration was induced even by WMTA. Marciano et al. reported that bismuth oxide’s tangency with sodium hypochlorite (NaClO) leads to discoloration of ProRoot WMTA and suggested tangency should be avoided [15]. Many eﬀorts have been carried out to ﬁnd an alternative for bismuth oxide as a radiopaciﬁer. Two products named ENDOCEM and Retro MTA were intro- duced by Kim et al. in which bismuth oxide has been replaced by zirconium oxide [16]. Although the producers claim that these sealer materials do not cause discoloration, there is no evidence to support their claim. the obtained pastes were molded in silicone molds and allowed to set for color stability, bioactivity, toxicity, and radiopacity examinations. The material processing is sche- matically shown in Figure 1. 2.4. Setting Evaluation. The NFC setting behavior was inves- tigated according to ISO 6876/2012 [17]. The freshly pre- pared cement pastes were molded in a stainless steel mold. Then, the samples were incubated under a temperature of 37°C and about 95% relative humidity. Afterward, a ﬂattened indenter with 1 mm diameter and a 400 g load was perpen- dicularly dipped into the paste surface. The process was repeated every 60 seconds. The ﬁnal setting time was recorded when the indenter was not capable of producing an indentation. 2.5. Evaluation of Color Stability. One of the aims of this research was to investigate the color stability of the NFC. To do so, the prepared cement pastes were formed in a cylin- drical silicon mold 10 mm in diameter and 2 mm high. The setting time was 24 hours at 100% humidity and 37°C. The cement samples were then dried and tested. Afterward, the samples were immersed in color stability solutions for 24 hours. The solutions included water (H2O), sodium hypo- chlorite solution (NaClO), and hydrogen peroxide (H2O2). This research was aimed at developing a novel cement base dental material with a short setting time and resistance to discoloration. Additionally, the bioactivity, toxicity, and radiopacity of the acquired cement were examined. 2. Materials and Methods The samples of this examination were 10 mm in diameter and 2 mm in length. After setting, the prepared samples were immersed in simulated body ﬂuid (SBF) for 1 and 14 days. SBF is a solu- tion with an ion composition close to that of the human blood plasma buﬀered at physiological temperature and pH 7.40 with HEPES/NaOH [20]. The chemical composition of the human blood plasma and SBF is given in Table 1. 2.3. Paste Preparation and Molding. In this stage, the nanoce- ment powders were mixed with distilled water, and the mix- ture was gently stirred using a spatula for 30 seconds to attain a homogenous and deformable paste. The prepared paste was used to study the setting behavior. Afterward, 2.6.1. Phase Evaluation and Microstructural Characterizations. The prime NFC nanocomposite samples and tested samples in SBF were analyzed using X-ray diﬀraction (XRD) to ﬁnd the crystalline phases. The XRD patterns were obtained using a diﬀractometer (D8 Advance Model, Bruker, Germany) 3 BioMed Research International 3 Radiopacity Color stability Testing Molding Molding Setting behavior Testing Paste Liquid Bioactivity MTT assay Powder preparation Figure 1: The material processing ﬂow chart. Radiopacity Color stability Molding Figure 1: The material processing ﬂow chart. Table 1: Ion concentration (mM) in simulated body ﬂuid in comparison with blood plasma [21]. Formulation Na+ K+ Ca2+ Mg2+ Cl- HCO3- HPO4 2- SO4 2- Human blood plasma 142 5 2.5 1.5 103 27 1 0.5 SBF 142 5 2.5 1.5 147.8 4.2 1 0.5 using Cu-kα radiation at a wavelength of 1.5 A in the 2θ range of 20°-60°. Identiﬁcation of patterns was performed utilizing JCPDS reference cards and X’Pert HighScore soft- ware. The surfaces of samples before and after immersion in SBF were analyzed using scanning electron microscopy (SEM TESCAN-Vega 3, TESCAN, Czech Republic). The samples were coated with a thin layer of gold (Sputtering coating Q150R-ES, Quorum Technologies, England) for bet- ter electrical conductivity. The samples were then viewed under the SEM operating at 15 kV. Energy-dispersive spec- troscopy (EDS) analysis was performed with a spectrometer (RONTEC, USA) to ﬁnd elemental distribution through the microstructures. cells a stable state. After 24 hours, the culture medium was replaced with the culture medium containing each of the NFC samples at concentrations of 500, 200, 100, 50, 10, and 1 μg/mL, and the plate was returned to the incubator. 2. Materials and Methods To the control well (positive control), the cell culture free of NFC was added. The well free of the cell culture was consid- ered as a witness sample (negative control). After 24 hours, the culture medium was discharged and replaced with 30 μL of MTT medium (4 mg/mL). The plate was incubated for 3 hours, and after charging 100 μL of dimethyl sulfoxide (DMSO), it was added to the wells. The plate was shaken for one minute and was incubated for another 10 minutes to dissolve the crystals in DMSO completely. Finally, the absorption of each well was measured by a microplate reader at a wavelength of 570 nm. The cell viability was measured using the following formula [21]: 2.6.2. Fourier Transform Infrared Spectroscopy (FTIR). To determine the present compounds on the surfaces of sam- ples, FTIR spectra were obtained for NFC samples before and after soaking in SBF using a Tensor II Spectrometer (Germany) and MAUNA-IR 750 equipment. Spectra were acquired in transmission mode in the frequency range of 400 to 4000cm−1. Viability % ð Þ = At −Ab Ac −Ab × 100, ð2Þ ð2Þ where At, Ab, and Ac are absorbance in the test well, absor- bance in the witness well, and absorbance in control well, respectively. 2.7. Cytotoxicity Assessment. The cytotoxicity of NFC was investigated by MTT assay on the periodontal liga- ment(PDL), according to Abbaszadegan et al. [22]. In this method, 106 cells were suspended in 15mL of RPMI% medium containing 10% FBS (Fetal Bovine Serum) and transferred by a sampler, 100μL cell suspension to a 96- well plate, thereby sowing 106 cells. The plate was then incu- bated in an incubator containing 5% CO2 at 37°C to give the 2.8. Radiopacity. Radiopacity is a crucial physical characteris- tic that is desired to cement to be identiﬁed in the root canal. To evaluate the radiopacity property of the NFC, samples were prepared according to the recommendation of ISO 6876/2012 [17]. To accomplish this, the NFC was com- pressed into a ring-shaped mold with an internal diameter BioMed Research International 250 200 150 100 50 0 20 30 40 C3S C3S C3S CS C2S ZrO2 ZrO2 ZrO2 ZrO2 50 60 2𝜃 (degree) a.u Figure 2: The XRD pattern of the primer powder. Figure 2: The XRD pattern of the primer powder. 3.2. Setting Behavior of the Cement Pastes. 2. Materials and Methods In the present study, SPSS IBM sub- sidized software was used to analyze the data. To evaluate the results of biological experiments, the data was displayed in each group as mean ± standard deviation. A signiﬁcant dif- ference between mean groups was analyzed by one-way ANOVA analysis and then Tukey test. In all experiments, an average of 3 measurements was used for each group, and a signiﬁcant level in all statistical tests (P) was considered less than 0.05. 3.4. In Vitro Bioactivity Test. In vitro bioactivity tests of the nanocement were performed by immersion of NFC samples in SBF at 36.5°C for 0, 1, and 14 days. After soaking in SBF, the development of HA on the surface of nanocement was investigated using phase evaluation, EDX analysis, and microstructural investigation. 2. Materials and Methods The setting behav- ior of the cement before milling (primary cement) and after milling (NFC) is presented in Table 2. It can be seen that the ﬁnal setting time of the cement pastes signiﬁcantly dropped from 65 to 15 min (more than 4 times reduction) when the particle sizes decreased from 2723 nm to 322 nm. of 10 mm and a height of 1.0 mm. Then, the samples were kept in an incubator at a temperature of 37°C for 24 hours and under a relative humidity of over 95%. Three specimens of the cement were made for the radiopaque measuring. The samples’ radiography was performed along with an alumi- num step wedge that was used as a reference. The radio- graphs were taken using a dental X-ray system (Gilardoni S.P.A, Italy) operating at 60kV and 4 mA. The exposure time for each examination was 0.3 min, and the target-ﬁlm dis- tance was 30cm. A photographic densitometer (Macbeth Model, Italy) was used to control the density of the radio- graphic images. The signiﬁcant reduction in the NFC setting compared to the primary cement is mainly related to the eﬀect of particle size. The particles’ ﬁneness produces more surfaces to carry out hydration reactions [23] and causes a higher hydration rate in NFC. The mechanism is modeled in Figure 4. 3.3. Evaluation of Color Stability. Evaluation of color stability has been done utilizing visual inspection and spectrometry examination. Figure 5 displays the digital images of NFC samples before and after soaking in diﬀerent solutions. The digital images show that the samples’ color did not change in distilled water, H2O2, and NaClO. The color changes of samples were obtained by the spectrometer and are plotted in Figure 6. The results conﬁrmed that the solutions did not cause realizable discoloration on the NFC samples. The spec- trometry examination shows that ΔE values for NFC are always lower than 5. 2.9. Statistical Analysis. In the present study, SPSS IBM sub- sidized software was used to analyze the data. To evaluate the results of biological experiments, the data was displayed in each group as mean ± standard deviation. A signiﬁcant dif- ference between mean groups was analyzed by one-way ANOVA analysis and then Tukey test. In all experiments, an average of 3 measurements was used for each group, and a signiﬁcant level in all statistical tests (P) was considered less than 0.05. 2.9. Statistical Analysis. 3. Results Figure 8(a) shows the spectra of samples before immersion in SBF. The peaks observed at 430, 502, and 2913 cm−1 correspond to zirconia. Si-O tensile oscillations caused an adsorption band around 990 cm−1 in C-S-H gel, and the peaks around 1488 and 2359 cm−1 are related to those oscillations in the carbonate groups. The broad signals around 3430 cm−1 are due to H-O oscillations in water and calcium hydroxide. compound are weakly observed at angles of 32.4°, 32.9°, and 46.2°. The C-S-H gel, due to its mainly amorphous structure, exhibits weak peaks in the XRD patterns. The diﬀraction pat- terns of zirconium oxide were speciﬁed at 2θ = 24°, 28°, 31.3, and 50.3°. Table 2: Comparison of setting time of the primary cement, NFC, and MTA. Material Initial setting time (min) Final setting time (min) Primary cement 10 65 NFC 5 15 MTA [8] 40 175 Material Initial setting time (min) Final setting time (min) Primary cement 10 65 NFC 5 15 MTA [8] 40 175 Figure 8(b) presents the FTIR spectra of the samples after soaking in SBF. In this sample, besides the sample peaks before immersion (Figure 8(a)), bands at about 961 cm−1 appear (PO4) that may indicate apatite formation on the sur- face of samples [27, 28]. The results attained utilizing FTIR analysis correspond to those taken by X-ray diﬀraction. compound are weakly observed at angles of 32.4°, 32.9°, and 46.2°. The C-S-H gel, due to its mainly amorphous structure, exhibits weak peaks in the XRD patterns. The diﬀraction pat- terns of zirconium oxide were speciﬁed at 2θ = 24°, 28°, 31.3, and 50.3°. compound are weakly observed at angles of 32.4°, 32.9°, and 46.2°. The C-S-H gel, due to its mainly amorphous structure, exhibits weak peaks in the XRD patterns. The diﬀraction pat- terns of zirconium oxide were speciﬁed at 2θ = 24°, 28°, 31.3, and 50.3°. compound are weakly observed at angles of 32.4°, 32.9°, and 46.2°. The C-S-H gel, due to its mainly amorphous structure, exhibits weak peaks in the XRD patterns. The diﬀraction pat- terns of zirconium oxide were speciﬁed at 2θ = 24°, 28°, 31.3, and 50.3°. The XRD results of hydrated NFC after immersion in SBF are presented in Figure 7(b). 3. Results 3.1. Material Characteristics. Phase analysis has been done to ﬁnd components of the prime cement powders. The XRD patterns of the powders are given in Figure 2. The results indicated that the prime powders consist of dicalcium silicate (C2S), tricalcium silicate (C3S), and zirconia (ZrO2). The FESEM images of primary cement and developed cement powders via the WSMM process (NFC) are shown in Figure 3. Microsized cement powders have been transformed into nanoparticles by WSMM (Figures 3(a) and 3(b)). The average particle size of the primary cement and NFC was measured through the PSA method. The results are pre- sented in Figure 3(c). The PSA analysis indicated that the mean particle size of the primary cement was 2723 nm. The measurements revealed that the cement particle sizes were reduced to 322 nm after WSMM operation (Figure 3(d)). 3.4.1. Phase Evaluation. Figure 7 shows the XRD results of the hydrated NFC before and after 14-day immersion in SBF. The XRD results of hydrated NFC before immersion in SBF are presented in Figure 7(a). As illustrated, the hydrated NFC consists of di- and tricalcium silicate, calcium hydroxide, and C-S-H gel. Dicalcium silicate and tricalcium silicate, the prime components of the cement that has not been involved in hydration reactions, exhibit strong multi- peaks at 29.42°, 32°, 41°, and 46.24°. Calcium hydroxide and C-S-H gel are hydration products. The calcium hydroxide peak is clear at 34.1°. The characteristic peaks of the C-S-H 5 5 BioMed Research International (b) (b) (a) (a) (b) (b) 16.00 (c) q (%) 0.0 1.0 10.0 100.0 1000 6000 0.0 Undersize 100.0 Diameter (nm) (c) q (%) 0.0 32.00 (d) 0.0 Undersize 100.0 1.0 10.0 100.0 1000 6000 Diameter (nm) (d) Figure 3: FESM image and particle distribution of the primary cement (a, c), FESM image, and particle distribution of the NFC (b, d). (a) (a) (b) (a) (b) (a) q (%) 0.0 32.00 (d) 0.0 Undersize 100.0 1.0 10.0 100.0 1000 6000 Diameter (nm) (d) 16.00 (c) q (%) 0.0 1.0 10.0 100.0 1000 6000 0.0 Undersize 100.0 Diameter (nm) (c) Undersize q (%) (d) (c) Figure 3: FESM image and particle distribution of the primary cement (a, c), FESM image, and particle distribution of the NFC (b, d). Figure 3: FESM image and particle distribution of the primary cement (a, c), FESM image, and particl pattern of the NFC before and after soaking in SBF was obtained. 3. Results The XRD pattern of this sample is similar to that of NFC before immersion in SBF (Figure 7(a)), except that the new peak of hydroxyapatite appears at 2θ = 25:8° and 32.1° [25, 26]. The XRD pattern of pure hydroxyapatite (Figure 7(c)) conﬁrms this issue. The hydroxyapatite has a similar composition to bone tissue, and the mechanism of its formation will be explained later. 3.4.3. Microstructural Analysis. Figure 9 shows the SEM images of setting samples before and after soaking in SBF for 1 and 14 days. The microstructure of the NFC before immersion is presented in Figure 9(a). As shown, the micro- structure consists of a C-S-H matrix, calcium hydroxide crys- tals, and some pores. These components are products of hydration reactions. Zirconia particles interlocking with the hydration products also are observed in the picture. Figure 9(b) is the SEM image of NFC being immersed in SBF for one day. Nucleation and formation of the apatite on the sample surfaces are visible in the pictures, but it is 3.4.2. FTIR Analysis. To further detect compounds in the cement and prove the hypothesis that the phase formed on the NFC samples is HA, the infrared Fourier spectroscopy BioMed Research International 6 Unhydrated cement Hydrated cement Cement Hydration products Capillary porosity (a) Hydrated NFC Unhydrated NFC (b) Figure 4: Hydration of primary cement (a) and NFC (b). Unhydrated cement (a) (b) Figure 4: Hydration of primary cement (a) and NFC (b). 3.4.4. EDX Analysis. For more clariﬁcation, EDX analysis has been carried out on the cement samples. The results of the elemental EDX analysis of the NFC before and after 1- and 14-day immersion in SBF are shown in Figures 9(a)–9(c). Calcium, silicon, oxygen, and zirconium were found on the samples not immersed in SBF (Figure 9(a)). For the samples immersed in SBF, the results were similar to those of the sam- ples before immersion. The diﬀerence is that the elements of calcium and phosphorus were also seen in the EDX analysis (Figures 9(b) and 9(c) for 1- and 14-day immersion, respec- tively), indicating possible apatite growth. Calcium and phos- phorus concentration originating from the SBF increased considerably over time, as is seen in Figures 9(b) and 9(c). Dry H2O NaCIO H2O2 (a) (b) Figure 5: Digital images of (a) NFC and (b) MTA [24] before and after soaking in various solutions. 3.5. Cytotoxicity. 3. Results According to the American Association of Endodontist’s recommendations, using a new dental material should be based on biological and clinical principles. An end- odontic material must be tested biologically by in vitro exam- inations before implantation in the body to minimize its detrimental eﬀects. Also, biomaterials that will be in perma- nent contact with the body tissues should display low cyto- toxicity. The MTT assay is a proper method for assessing cell viability and determination of biomaterial toxicity [29]. The process is based on mitochondrial activity and the metabolism of cells. In this work, the proliferation/viability of MCF-7 cells was evaluated by the MTT assay, and the results are presented in Figure 10. These results are for the concentration of the cement nanoparticles in the range of 1–500 μg/mL in wells containing the MCF-7 cells and over (b) (a) Figure 5: Digital images of (a) NFC and (b) MTA [24] before and after soaking in various solutions. not signiﬁcant. Figure 9(c) presents the microstructure of NFC immersed in the SBF for 14 days. The picture shows that maintaining the NFC for 14 days in SBF led to the devel- opment of signiﬁcant apatite values on the cement surfaces. BioMed Research International 7 25 20 15 10 5 0 H2O H2O2 NaCIO 2.3 4.9 3.0 0.4 17.2 20.0 𝛥E NFC MTA Figure 6: Discoloration of NFC and MTA [24] after soaking in H2O, H2O2, and NaClO. C3S + H2O ⟶C −S −H + Ca OH ð Þ2 C2S + H2O ⟶C −S −H + Ca OH ð Þ2 ð3Þ 25 20 15 10 5 0 H2O H2O2 NaCIO 2.3 4.9 3.0 0.4 17.2 20.0 𝛥E NFC MTA ð3Þ As hydration reactions progress, the hydrated calcium silicate gel polymerizes and converts to a solid network. The gel is relatively impermeable to water, and so it slows down of further reactions [33]. Nanoparticles provide large surface areas and nucleation sites thereby stimulating the hydration reactions [34]. At the same time, the milling pro- cesses create more reactive surfaces [34]. This has facilitated the progress and completeness of the hydration reactions as schematically shown in Figure 4. Discoloration: the results of experiments revealed that the NFC is resistant to discoloration in contact with H2O, NaClO, and H2O2 solutions. 3. Results Comparison of the results with Keskin et al.’s experiments [24] on MTA in similar condi- tions showed that NFC exhibited higher resistance to color change compared to MTA. The resistance to discoloration is more pronounced when comparing NFC and MTA in con- tact with NaClO solution. No color change is seen for NFC in contact with NaClO, while it led to severe discoloration of MTA (Figures 5 and 6). The resistance of the NFC to discol- oration is related to the use of zirconia instead of bismuth oxide as a contrasting substance. Marciano suggested that discoloration of MTA in the presence of NaClO is due to a change from bismuth oxide to bismuth metal, which is inher- ently black [35]. It seems zirconia is stable in contact with NaClO and is more suitable for endodontic applications than bismuth oxide. The exact mechanism of its color stability needs further investigation. Figure 6: Discoloration of NFC and MTA [24] after soaking in H2O, H2O2, and NaClO. 24 hours. Although the viability percentage declined from 92% to about 63% by raising the concentration from 1 to 500 μg/mL, the viability is acceptable. The results revealed that in dilute solution (concentration below 100 μg/mL), the NFC is nontoxic; in moderate concentration (between 100 and 200 μg/mL), the NFC is nearly nontoxic; and in high concentrations (500 μg/mL), the NFC is slightly toxic. The improvement in the viability of cells in low concentra- tions of NFC may be due to reduced toxic compounds in the cement. 24 hours. Although the viability percentage declined from 92% to about 63% by raising the concentration from 1 to 500 μg/mL, the viability is acceptable. The results revealed that in dilute solution (concentration below 100 μg/mL), the NFC is nontoxic; in moderate concentration (between 100 and 200 μg/mL), the NFC is nearly nontoxic; and in high concentrations (500 μg/mL), the NFC is slightly toxic. The improvement in the viability of cells in low concentra- tions of NFC may be due to reduced toxic compounds in the cement. g Bioactivity: the SBF test of the NFC was performed in this investigation for the evaluation of bioactivity behavior. Apa- tite precipitation on the NFC conﬁrmed by XRD, SEM, and EDX examinations was a strong reason for its proper bioac- tivity. In explaining this issue, we look at how the body reacts to an external implant. 3. Results Generally, implantation of a biomate- rial in the human body may be confronted with four types of reactions: nearly inert, porous, restorable, and bioactive [36, 37]. In a bioactive material, interfacial bonding of biomaterial and bones is created via ion-exchange processes between the implant and surrounding body ﬂuids [38]. The process causes the development of a biologically active component named apatite on the implant surface, similar to the mineral phase in bone concerning chemical and crystallographic characteristics [39, 40]. The XRD, FTIR, and EDAX exami- nations detected the formation of the apatite on the speci- mens. The mechanism’s action can be explained as follows: 3.6. Radiopacity. Radiopacity is a crucial physical characteris- tic needed in restorative dental materials. A minimal radio- pacity value is required for the sealer material to be detected in the root canal and allows the dentist to correct ﬁlling failures before ﬁnal treatment. To understand the qual- ity of zirconium oxide in cement detection in restorative den- tal radiography, the NFC’s radiopacity was obtained. According to the results, the NFC has a radiopacity equiva- lent to 3.3 mm Al. It is higher than the minimum require- ments of dental restorative materials, as stated in ISO 6876/2012 [17]. 3.6. Radiopacity. Radiopacity is a crucial physical characteris- tic needed in restorative dental materials. A minimal radio- pacity value is required for the sealer material to be detected in the root canal and allows the dentist to correct ﬁlling failures before ﬁnal treatment. To understand the qual- ity of zirconium oxide in cement detection in restorative den- tal radiography, the NFC’s radiopacity was obtained. According to the results, the NFC has a radiopacity equiva- lent to 3.3 mm Al. It is higher than the minimum require- ments of dental restorative materials, as stated in ISO 6876/2012 [17]. 4. Discussion Setting behavior: the setting time of NFC was measured about 15 min that is about 4 times shorter than the prime cement and by about 15 times shorter than MTA that was reported by AlAnezi [8] as is given in Table 2. The considerable reduc- tion in the setting time by decreasing the particle sizes can be explained through the hydration reaction mechanisms. The cement consists of tricalcium silicate and dicalcium silicate that react with water to form a basic solution containing OH-, Ca2+, and silicate ions [30]. Due to reactions of these anions and cations in the solution, hydrated calcium silicate gel (C-S-H) and calcium hydroxyl form on the surface of cement particles. The reactions can be expressed as [31, 32] As soon as NFC is immersed in SBF, a solid-liquid inter- face is created between the cement and solution, and hydro- lysis occurs soon after [41, 42]. Ion exchanges are started between hydration products or C3S and C2S, where Ca2+ from the cement exchange with H+ from the aqueous solu- tion form a solid-liquid interface. As a result of the reaction of Ca2+ ions with hydroxyl ions derived from water calcium hydroxide or portlandite, the alkaline condition was created. Cation exchange increases OH- concentration of the solu- tion, and so the surfaces of calcium silicate particles were sur- rounded by the hydroxyl ions of the solution [43]. Another BioMed Research International 8 8 400 350 300 ZrO HA HA 2 ZrO2 C3SZrO2 Ca(OH)2 C3S C2S ZrO2 (a) (b) (c) 250 200 150 100 50 0 20 25 30 35 40 45 50 55 60 2𝜃 (degree) a.u Figure 7: The XRD pattern of (a) NFC before soaking in SBF, (b) NFC after soaking in SBF for 14 days, and (c) pure hydroxyapatite. 400 350 300 ZrO HA HA 2 ZrO2 C3SZrO2 Ca(OH)2 C3S C2S ZrO2 (a) (b) (c) 250 200 150 100 50 0 20 25 30 35 40 45 50 55 60 2𝜃 (degree) a.u Figure 7: The XRD pattern of (a) NFC before soaking in SBF, (b) NFC after soaking in SBF for 14 days, and (c) pure hydroxyapatite. Figure 7: The XRD pattern of (a) NFC before soaking in SBF, (b) NFC after soaking in SBF for 14 days, and (c) pure hydroxyapatite. 4. Discussion Good adaptation and sealing capability of the cement sealer play a crucial role in successfully ﬁlling the ≡SiO−+ Ca2+ ⟶≡SiO−⋯Ca2+ ð5Þ ð5Þ 9 9 BioMed Research International cement dentin interface and preventing the risk of eﬀect of bismuth oxide on the body of osteoblast l keV 10.000 5000 4000 3000 2000 1000 0 0 5 𝜇m CaK𝛼 OK𝛼 CaL𝛼 CK𝛼 AIK𝛼 NaK𝛼 SiK𝛼 ZrL𝛼 ZrL𝛽 CaK𝛽 (a) (a) ZrL𝛼 MgK𝛼 NaK𝛼 PK𝛼 SiK𝛼 AIK𝛼 ZrL𝛽 PK𝛽 CaK𝛽 keV 10.000 0 0 1000 2000 3000 4000 5000 (b) 5 𝜇m OK𝛼 CaL𝛼 CK𝛼 CaK𝛼 (b) 4000 OK𝛼 CaK𝛼 ZrL𝛽 ZrL𝛼 SiK𝛼 MgK𝛼 CaL𝛼 PK𝛽 PK𝛼 CaK𝛽 AIK𝛼 CK𝛼 3500 3000 2500 2000 1500 1000 500 0 0 keV 10.000 5 𝜇m (c) (c) M image and EDX analysis of the sample before soaking in SBF (a), after soaking in SBF for 1 day (b), and after soaking i ke 10.000 5000 4000 3000 2000 1000 0 0 CaK𝛼 OK𝛼 CaL𝛼 CK𝛼 AIK𝛼 NaK𝛼 SiK𝛼 ZrL𝛼 ZrL𝛽 CaK𝛽 (a) 5 𝜇m (a) (a) (a) ZrL𝛼 MgK𝛼 NaK𝛼 PK𝛼 SiK𝛼 AIK𝛼 ZrL𝛽 PK𝛽 CaK𝛽 keV 10.000 0 0 1000 2000 3000 4000 5000 OK𝛼 CaL𝛼 CK𝛼 CaK𝛼 (b) (b) 5 𝜇m (b) (b) 4000 OK𝛼 CaK𝛼 ZrL𝛽 ZrL𝛼 SiK𝛼 MgK𝛼 CaL𝛼 PK𝛽 PK𝛼 CaK𝛽 AIK𝛼 CK𝛼 3500 3000 2500 2000 1500 1000 500 0 0 keV 10.000 5 𝜇m (c) (c) 4000 OK𝛼 CaK𝛼 ZrL𝛽 ZrL𝛼 SiK𝛼 MgK𝛼 CaL𝛼 PK𝛽 PK𝛼 CaK𝛽 AIK𝛼 CK𝛼 3500 3000 2500 2000 1500 1000 500 0 0 5 𝜇m (c) (c) 0 OK𝛼 CaK𝛼 ZrL𝛽 ZrL𝛼 SiK𝛼 MgK𝛼 CaL𝛼 PK𝛽 PK𝛼 CaK𝛽 AIK𝛼 CK𝛼 0 0 0 0 0 0 0 0 0 keV 10.000 (c) (c) (c) Figure 9: SEM image and EDX analysis of the sample before soaking in SBF (a), after soaking in SBF for 1 day (b), and after soaking in SBF for 14 days (c). eﬀect of bismuth oxide on the body of osteoblast-like cells while placed aside dicalcium silicate cement [53, 54]. Studies have shown that bismuth oxide also enhances the cytotoxic- ity in dental pulp cells when added to Portland cement as a radiopaciﬁer. On the other hand, studies have revealed that the body system response to zirconia is nearly neutral. There- fore, the use of zirconia in NFC makes it preferable to MTA concerning its low cytotoxicity. 4. Discussion 4000 3600 3200 2800 2400 2000 1600 PO4 Si-O CO3 Si-O ZrO2 OH (a) (b) ZrO2 1200 800 400 Wave number (cm –1) 0 day 14 days Figure 8: FTIR pattern of the NFC (a) before and (b) after soaking in SBF. Figure 8: FTIR pattern of the NFC (a) before and (b) after soaking in SBF. It is a double layer with an equal opposite charge, which provided suitable conditions for deposits of other substances. On the other hand, the SBF solution contains PO4 3- that is hydrolyzed as follows [46]: result of hydrolysis is the SiO4 4- group that in alkaline condi- tions developed calcium silicate hydrate phase with amor- phous microstructure on the surface of the cement [44]. The calcium silicate hydrate is porous water containing sili- cate gel ﬁlm consisting of silanol or Si-OH groups. After- ward, the Si-OH group in the calcium silicate hydrate phase loses a proton H+ in an alkaline environment, producing a negatively charged surface with the SiO- functional group as [45] H2O + PO3− 4 ⟶≡HPO2− 4 + OH− ð6Þ ð6Þ Immersion of calcium silicate in a phosphate-containing solution, SBF, which includes hydrolyzed HPO4 2-, results in electrostatic interactions between HPO4 2- and Ca2+ on the surfaces of the cement, and it produces inhomogeneous nucleation of apatite as [47] ≡SiOH + H2O ↔≡SiO−+ H3O+ ð4Þ ð4Þ The produced negative functional group attracts the pos- itive calcium ions released in solution under electrostatic interactions, causing the increase of cations on the cement surfaces [46]: ≡SiO−⋯Ca2+ + HPO2− 4 ⟶≡SiO−⋯Ca2+ ⋯HPO2− 4 ð7Þ ð7Þ The formation of apatite on the cement is a reason for its proper adaptation of the NFC in the root canal of the tooth [48, 49]. 4. Discussion gaps at the cement-dentin interface and preventing the risk of restoration failure causing bacterial microleakage and subse- quent clinical problems [50, 51]. The growth of apatite also indicates the high activity of this dental material in the body environment [52] and can be considered as a regenerated material for future investigations. Cytotoxicity: this in vitro study showed that the ability of NFC to induce promotion in cell culture is signiﬁcant. In this novel material, we replaced bismuth oxide with zirconia as a radiopaciﬁer. Investigations revealed the likely cytotoxic Radiopacity: defects in sealer materials should be well detected on the radiographic ﬁlms to prevent the saliva from BioMed Research International 10 0 500 200 100 50 10 1 Concentration (𝜇g/ml) 20 40 60 80 62.7 82.1 88.9 87.17 86.5 92.154 100 120 Viability (%) Figure 10: The cytotoxic eﬀects of NFC on MCF-7 cell lines cells under diﬀerent concentrations. size has been reduced to nanosize via WSMM processes. The ﬁnal setting time of the developed cement considerably reduced from 65 to 15 min by reducing particle sizes from 2723 nm to 322 nm. The discoloration of NFC was investi- gated via spectroscopy examination. It was found that the combination of NFC/zirconia represents acceptable discolor- ation. The phase evaluation and FTIR examination revealed the formation of hydroxyapatite on the soaked samples in SBF for 1 and 14 days. The hydroxyapatite was also detected in microstructural observations. This is evidence of the favor- able bioactivity and biocompatibility of the NFC. The cyto- toxicity of NFC was investigated by MTT assay on the MCF-7 cell line. NFC showed a positive biological property and may be a safe choice for restoration treatment. The radiopacity of the NFC/zirconia dental material was exam- ined using a radiographer. It was found that zirconia pro- vides a radiopacity corresponding to 3.3 mm/Al and meets the standard requirements. Figure 10: The cytotoxic eﬀects of NFC on MCF-7 cell lines cells under diﬀerent concentrations. penetrating and causing infection. Radiopacity examination reveals that defects in combination of NFC/zirconia are well recognizable. Zirconium oxide is used as a radiopaciﬁer in NFC contrary to MTA where bismuth oxide is considered for such performance. The idea for such choice might be due to some investigations which revealed that zirconium oxide presents biocompatible behavior and is shown as a bioinert material with desirable corrosion resistance and mechanical properties [55]. References [1] M. Parirokh and M. Torabinejad, “Mineral trioxide aggregate: a comprehensive literature review–part III: clinical applica- tions, drawbacks, and mechanism of action,” Journal of Endo- dontia, vol. 36, no. 3, pp. 400–413, 2010. [1] M. Parirokh and M. Torabinejad, “Mineral trioxide aggregate: a comprehensive literature review–part III: clinical applica- tions, drawbacks, and mechanism of action,” Journal of Endo- dontia, vol. 36, no. 3, pp. 400–413, 2010. [2] J. Camilleri, F. Sorrentino, and D. Damidot, “Investigation of the hydration and bioactivity of radiopaciﬁed tricalcium sili- cate cement, Biodentine and MTA Angelus,” Dental Materials, vol. 29, no. 5, pp. 580–593, 2013. [3] H. E. Alqaderi, S. A. Al-Mutawa, and M. A. Qudeimat, “MTA pulpotomy as an alternative to root canal treatment in chil- dren's permanent teeth in a dental public health setting,” Jour- nal of Dentistry, vol. 42, no. 11, pp. 1390–1395, 2014. [4] O. Malkondu, M. Karapinar Kazandag, and E. Kazazoglu, “A review on biodentine, a contemporary dentine replacement and repair material,” BioMed Research International, vol. 2014, Article ID 160951, 10 pages, 2014. 4. Discussion Besides, researchers reported evi- dence of bismuth oxide toxicity aside from calcium silicates, as mentioned earlier [56]. The results of toxicity and biocom- patibility tests, as stated in previous sections, showed that zir- conia did not cause toxicity aside from NFC. Acknowledgments We would like to acknowledge the ﬁnancial support pro- vided to this study by Shiraz University (grant No. 96GCU3M1974). The authors also appreciate the Shiraz University of Medical Sciences for their assistance in per- forming the MTT assay examinations. Consequently, NFC was developed to overcome some deﬁciencies of the currently available MTA. By the ﬁneness of the cement particles at the nanoscale (NFC), the setting time was reduced 12 times compared to MTA. Another prob- lem associated with MTA was reported as discoloration. In this investigation, bismuth oxide, which was reported as the main cause of the color change in the presence of MTA, is replaced by zirconia. The results of discoloration indicated that NFC presents a high resistance against solutions such as H2O, NaClO, and H2O2 and is another advantage of this material over MTA. Additionally, other characteristics of endodontic material must be bioactivity, biocompatibility, and nontoxicity. Nanofast cement showed proper bioactive behavior and nontoxic characteristics in all ranges of concen- tration. Endodontic sealer cement also must show proper radiopacity to allow the quality of restoration to be radio- graphically visualized. 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https://openalex.org/W1988532023	https://nottingham-repository.worktribe.com/preview/1006596/Dawn-Marie_Walker--Classrom_Assessment.pdf	English	null	Classroom Assessment Techniques: An Assessment and Student Evaluation Method	Creative education	2,012	cc-by	5,067	Creative Education 2012. Vol.3, Special Issue, 903-907 Published Online October 2012 in SciRes (http://www.SciRP.org/journal/ce) Creative Education 2012. Vol.3, Special Issue, 903-907 Published Online October 2012 in SciRes (http://www.SciRP.org/journal/ce) http://dx.doi.org/10.4236/ce.2012.326136 http://dx.doi.org/10.4236/ce.2012.326136 Assessment Assessment can provide a framework for sharing educational objectives with students and for mapping their progress. For these reasons there is strong support for assessment to be part of the learning process (Dochy & McDowell, 1997). In general, assessment is divided into two concepts: formative and summa- tive. Formative assessment is intended to assist student learning via deep learning approaches. Summative assessment on the other hand, e.g. assessments involving short questions, multiple choice or unseen exams, checks the level of learning at the end of a course/module and often takes the form of an exam or piece of course work which is graded. Exams lend themselves to rote learning, or surface approaches by encouraging students to concentrate on performance goals (passing the test) rather than learning goals (understanding the subject) (Dweck, 1999). This leads some to argue that summative assessment in itself can control, and arbitrarily classify students whilst impairing the student’s own sense of self and leads to a limitation of their educational development (Barnett, 2007). Therefore it is argued that formative assessment should be an integral part of teaching and learning in HE and that it should be systematically embed- ded in curriculum practices (Juwah et al., 2004). Introduction to learning and contributes towards positive student motivation by allowing students to be in charge of their learning environ- ment (Markett et al., 2006). A key strength of classroom inter- action is that it provides scaffolding which allows the student to develop content into context, therefore developing cognitive structures (Moore, 1989). Therefore to promote deep learning, there has to be dialogue and an interactive classroom, and also great care needs to be given when choosing assessment tech- niques to prevent surface learning (Table 1 compares and con- trasts these two approaches to learning). Appropriate assessment in Higher Education (HE) is a topic which has been debated and researched over the years, as not only is assessment respected as a necessary method of quanti- fying students, but it is also required by clients themselves, both students and employers. One of the major problems with assessment is how to make it meaningful, and in a manner which promotes deep learning to develop independent and self motivated thinkers, whilst also fulfilling the assessment criteria. This is often achieved by providing thorough feedback in a timely manner after the assessment, which in large classes can be difficult to the lecturer. Another area of much debate in HE is how to evaluate what is taught. Student evaluation of teach- ing and modules is prone to criticism; therefore many sugges- tions of evaluation methods to improve accuracy have been put forward. The present paper aims to draw on previous theories about: 1) assessment, i.e. summative or formative; 2) feedback; and 3) student evaluated teaching, to propose an assessment method, which also combines an evaluation method. This is often achieved by providing thorough feedback in a timely manner after the assessment, which in large classes can be difficult to the lecturer. Another area of much debate in HE is how to evaluate what is taught. Student evaluation of teach- ing and modules is prone to criticism; therefore many sugges- tions of evaluation methods to improve accuracy have been put forward. The present paper aims to draw on previous theories about: 1) assessment, i.e. summative or formative; 2) feedback; and 3) student evaluated teaching, to propose an assessment method, which also combines an evaluation method. Classroom Assessment Techniques: An Assessment and Student Evaluation Method Dawn-Marie Walker University of Nottingham, Nottingham, UK Email: dawn-marie.walker@nottingham.ac.uk Received August 9th, 2012; revised September 10th, 2012; accepted September 24th, 2012 Some of the challenges that face Higher Education are how to ensure that assessment is meaningful and that feedback is prompt in order to promote learning. Another issue is how to provide lecturers with feed- back regarding their efficacy, in a timely and non-judgmental manner. This paper proposes that Class- room Assessment Techniques (Angelo and Cross, 1993), maybe a good way of answering both of those issues. They are quick and easy tasks set within the lecture, which tests the students’ knowledge, provid- ing an immediate opportunity for further elaboration if needed by the lecturer, therefore providing imme- diate feedback to the students. It also ensures that the lecturer has delivered the most salient messages, therefore also providing feedback to the lecturer. Keywords: Assessment; Student Evaluation; Feedback; Classroom Assessment Techniques Copyright © 2012 SciRes. Feedback Feedback is information about how the student’s present state (of learning and performance) relates to the desired goals and standards (Nicol & Macfarlane-Dick, 2006) and systematic reviews show that effective feedback leads to learning gains (Black & William, 1998). Lecturer feedback serves as an au- thoritative external reference point against which students can evaluate, and self-correct their progress and their own internal goals (Juwah et al., 2004). Hence the main aim of feedback is in developing self regulated students, which requires them to internalize personal goals against which their performance can be compared and assessed by themselves (Nicol & Macfarlane- Dick, 2006). However providing meaningful feedback in a timely manner can be difficult. Although there has been ex- panding numbers of students attending HE, the actual resource allocated per student in the largest classes may be much less than ten years ago (Gibbs, 2006). The work load of lecturers is often calculated by “class contact hour” which ignores class size, therefore assessment loads are sometimes ignored (Gibbs, 2006). These time constraints, together with modularization of degrees, often without any increase in staffing, can increase the utilization of summative assessment (Gibbs, 2006) and there- fore leads to a decrease in timely and relevant feedback which would have enhanced learning. Therefore HE establishments are wrong if they quantify teaching effectiveness on SET, or see students as customers, and shape their educational provision to meet their wishes or evaluations, as students objectives centre around getting the highest grades with the least amount of effort, or time (Chad- wick & Ward, 1987). Therefore good lecturers who use tech- niques to promote deep approaches to learning, which are by their nature, often harder work and more difficult tasks than surface approaches, may be looked upon less favorably than a teacher who “spoon feeds” information to the students (Platt, 1993). The author has some unpublished data from staff and stu- dents at the University of Nottingham where she is based re- garding the SET procedure. Significantly more students than staff thought the SET aimed to maintain/improve teaching stan- dards, and to help initiate dialogue between the staff and stu- dents. Although is within the SET remit, the fact that staff are less likely to agree with these statements, means that the SET is not fulfilling its capabilities. Student Evaluated Teaching The need for greater accountability and improvement in the quality of teaching has become a major issue in HE in recent years (Coaldrake & Stedman, 1998; Ballantyne et al., 2000). Therefore SET has become an integral part of HE’s approach to maintaining teaching standards via a summative method: to gain data for administrative purposes, to provide information to students and to meet government guidelines; and a formative purpose: giving diagnostic feedback to lecturers about their teaching effectiveness (Marsh, 1987). SET is often the only measure of teaching effectiveness (Perry, 1997), so it is of pa- ramount importance that the students give meaningful input. Literature however, suggests that SET is currently not fulfilling all its objectives as there doesn’t appear to be a consensus as to what “effective teaching” includes (Shelvin et al., 2000). For example, Lowman and Mathie (1993) identifies lecturers’ ef- fectiveness, as comprising 1 intellectual excitement; and 2 in- terpersonal rapport; whilst Swartz et al. (1990) view it as com- posing: a) clear instructional presentation; and b) good man- agement of student behavior. However in reality it’s probably all of these items compounded with others such as encouraging students to have self worth (Covington, 1997), etc. Other prob- lems with this system relates to the validity of the student evaluations as it is human nature to be subjective in voting, for example Shelvin et al. (2000) found that student evaluation frequently measures other factors such as 63% of the variance of the “lecturer effectiveness” score being accounted for by charisma. quired of them to gain a top mark and enables them to gain feedback, via reflection on their own work when compared with the criteria, and so will encourage deep learning (Norton et al., 2001). Feedback is an extremely important part of learning and the assessment process. For any assessment to be useful to the student in their personal development there needs to be a timely feedback loop that will encourage the student to learn from the process, to reflect on their work and to assimilate the knowl- edge for future practice. When assessment (often formative) encompasses a feedback loop, it results in positive benefits on learning and achievement across all content areas; knowledge, skills and levels of education (Black & William, 1998). Table 1. Table 1. Compare and contrast deep learning with surface learning (based on Ramsden, 1992). standing of expectations and standards, to correct misunder- standings and to get an immediate response to difficulties (Freeman & Lewis, 1998). It can also inform the lecturers as to whether they are teaching appropriately and whether it is at the right level, therefore providing an immediate opportunity for realignment of their teaching. A common method of closing the loop and providing feedback to the lecturer is “Student Evalu- ated Teaching (SET)”. Deep learning Takes a broad view Surface learning Takes a narrow view Looks for meaning Relies on rote learning Focuses on the concepts and arguments to solve the problem Focuses on the formula to solve the problem Relates new knowledge to previously learnt knowledge Focuses on learning unrelated bits of a task Relates knowledge across mod- ules/courses Information is memorized solely for assessment Relates theory to practice Theory is not reflected upon in real life Evidence and argument between theories is developed No cross referencing between theories Emphasis is student centered Emphasis is external, i.e. assessment driven Copyright © 2012 SciRes. Approaches to Learning The deep approach to learning which is what HE strives to achieve, involves the critical analysis of new ideas, with the student relating their own previous knowledge to the new knowledge, theoretical ideas, and evidence. This in turn leads to understanding and long-term retention of concepts so that they can be used for problem solving in unfamiliar contexts. The surface approach to learning is the unquestioning acceptance and memorization of information as isolated and unlinked facts which lead to rote learning for examinations, most of which are promptly forgotten about following the exam (Marton & Saljo, 1976; Biggs, 1987; Biggs, 1993; Ramsden, 1992), i.e. “brain dump”. Deep learning is driven by challenging, open-ended problems with lecturers acting as facilitators in an interactive classroom. An interactive classroom promotes deep approaches To optimize the learning from the assessment procedure the marking criteria for that assessment should be transparent and explicit, as this will enable students to understand what is re- 903 D.-M. WALKER Table 1. Classroom Assessment Techniques The use of Classroom Assessment Techniques (CATs) is one way of resolving all of these problems. CATs offer an egalitar- ian and productive method of student evaluation, gives the op- portunity for immediate formative feedback to both students and staff, and is also a formative assessment, therefore pro- motes deep learning techniques, and thus enhancing knowledge and motivation. CATs were first presented and described in detail in a book by Angelo and Cross in 1993. CATs are quick and simple activities which are designed to give both the lec- turer and the students’ useful, immediate feedback. They also assess the teaching-learning process rather than other con- founding issues such as the charisma of the lecturer, or how easy the course is. They are defined as “small-scale assessments conducted continually in college classrooms by discipline- based teachers to determine what students are learning in that class”, (Cross & Steadman, 1996: p. 8). There are various types of CATs one can adopt (Table 2) al- though perhaps the most commonly used one is the “one-min- ute paper,” where students are asked to write down answers to questions such as, “What was the most important thing you learned during this class?” or “What questions do you still have on this topic?” This type of technique enables the lecturer to discover how the students are processing and synthesizing the presented material as well as which points need to be reiterated or elaborated on before progressing. Therefore this method assesses student knowledge and also offers the lecturer imme- diate feedback regarding whether the students have grasped the most salient pieces of information from the lecture giving an opportunity to recap on any misunderstood items. Although, arguably, some CATs could be regarded as summative in nature, such as the minute paper, because of the immediate feedback and dialogue ensuing, they therefore become formative. CATs differ from tests and other forms of student assessment in that it provides timely opportunity for course improvement, with the goal of understanding the students’ learning and therefore im- proving teacher effectiveness. Another benefit of this system is CATs are sometimes called test-feedback cycles, and imple- mentation of them allows teachers and students to share, on a regular basis, their conceptions about both the goals and proc- esses of learning (Stefani & Nicol, 1997) thereby opening up dialogue opportunities. Feedback Another telling analysis is that SET procedures do not seem to be followed, such as students are significantly less likely to believe that enough time has been set aside for this task, and that the feedback loop is not closed with dialogue from the lecturer to the students. Due to the lack of feedback, it appears that the students believe that SET is just It is also important that the feedback is in a loop and is part of a dialogue which encourages engagement. Dialogue between the lecturer and student will help develop the student’s under- Copyright © 2012 SciRes. 904 D.-M. WALKER to fulfill government requirements, although they maintain that teaching needs to be evaluated significantly more than the staff. So it appears that students value this process, but become disil- lusioned by the lack of feedback/impact, and not giving enough time to complete the form thoughtfully. students to take them seriously (Enerson et al., 2007). CATs rely on self-assessment, thus promoting the internal resources necessary for lifelong learning, and autonomy which enhances the learning process. In an evaluation of CATs use forty-five out of forty-six faculties in a university setting reported that there were no negative experiences associated with their use of CATs (Catlin & Kalina, 1993), although there is still some de- bate regarding their efficacy as Cottell & Harwood (1998) found no difference in grades, participation, or perceptions of learning between students who used CATs and those who didn’t. Classroom Assessment Techniques They are usually not graded to enable the student to interact with the feedback, rather than become obsessed with the grade. However some authors argue that even making CATs count for 1% of the final grade will encourage Table 2. Table 2. Examples of CATs (adapted from a table on the National Teaching and Learning Forum, 2008). CAT Method Feedback Effort Knowledge probe At the beginning of class as students to answer preset questions (open, or multiple choice) to assess students existing knowledge. If multiple choice, use vote pads for immediate discussion. Note any weaknesses in knowledge for elaboration. If open ended, could also utilize peer assessment. Prep: low In class: medium Analysis: low Minute paper At the end of class ask student to write “what is the most important point you learned today?” and “What is the least clear to you?” Collect and review responses. Ensure that they have obtained the correct message. In the next class comment on the findings. Or, ask for peer review and swop with partner. Discuss any discrepancies. Prep: low In class: low if collected, higher if peer assessed Analysis: low One-sentence summary Can be used at any time during class to test knowledge about an important topic you expect them to be able to summaries. Ensure the students have the message. Can be done with a vote pad. Prep: low In class: low Analysis: low Directed paraphrasing Ask students to write a layman’s summary of any principle taught. This assesses their ability to comprehend and transfer concepts. Peer or teacher assessed. Ensure the salient points are covered. Prep: low In class: medium Analysis: medium Application cards Ask students to write down on real-world application for a theory, principle or procedure you have just covered. Collect and pick out a broad range of examples to present to the class. Or peers assess and discuss. Prep: low In class: low Analysis: med Muddiest point Ask students to write down the “muddiest point” of the lecture, i.e., the concept they feel they haven’t understood. Collect written answers or get them to discuss with their peers. Or have them vote on predetermined items using hand held voting systems Prep: low In class: medium Analysis: low Table 2. Examples of CATs (adapted from a table on the National Teaching and Learning Forum, 2008). At the beginning of class as students to answer preset questions (open, or multiple choice) to assess students existing knowledge. Collect and review responses. Ensure that they have obtained the correct message. In the next class comment on the findings. Or, ask for peer review and swop with partner. Discuss any discrepancies. Table 2. Prep: low In class: low if collected, higher if peer assessed Analysis: low At the end of class ask student to write “what is the most important point you learned today?” and “What is the least clear to you?” 905 Copyright © 2012 SciRes. D.-M. WALKER groups of students, so that they can design a suitable CAT for their target lecture. This involves meeting with the students shortly after their target lecture to discuss what they believe were the salient points and how they might assess the student’s grasp of them. Working in their small groups they then design the CAT they feel would be the most appropriate (or design a new one), and carry it out at the start of the next lecture. They then collect the data back from the students and review it in their groups and report back to the author with any deficits in the learning identified. If any deficits are observed, some ideas from them about how these could be remedied such as re- sources which they could be referred to for further reading, typed up study notes, etc. are required from them. These re- sources are then given back to the class by the students, and feedback obtained e.g. whether other students knew of any further useful resources which were omitted, etc. To ensure com- mitment, the author has allocated 5% to this task. This task not only promotes deep learning (to both “teachers” and “students”), but also encourages team work and hones their teaching/public speaking ability. For the author, it also ensures some student designed teaching, from which she can also learn. Although it seems to have worked out well, it does take some organization, such as getting students to form groups, and staggering them throughout the module. However the feedback from all students was positive so far. that there is very little time investment when compared to more traditional assessment such as essays or exams, especially when one bears in mind the time taken to provide feedback. This method also fosters open dyadic communication and good rapport. CATs can be used within any size of classroom, large lectures or small seminars, and can personalize learning and lend themselves to peer led teaching/feedback. They have also been used in e-learning/distance formats (Henderson, 2001) and so are extremely versatile. Table 2. They are well suited to the ad- vent of the Interactive Voting Systems that many universities have adopted. These are systems that can be built into Power- Point presentations and which use individual voting pads. The lecturer can then build a CAT into their presentation, ask the students to vote with their key pad and the system will then calculate the results immediately, presenting them on the screen for the lecturer and students to analyze. Mobile phones and SMS technology have also been shown to work when used in this manner (Markett et al., 2006). Using media in this way can enhance the learning experience as it is interactive therefore promoting deep learning. For example, Laurillard (1996) claims that by changing the media used in class, the student activity is changed and hence improves the learning situation i.e. “peda- gogical re-engineering” (enhancing learning by changing the balance or combination of the components used) (Collis, 1996). Conclusion In the author’s own teaching, she has used CATs with great effect. In a module consisting of approximately 10 lectures, she built in around 5 CATs. The students were not told which lec- tures the CATs would appear in prior. This ensured that atten- tion was maintained throughout the module. She used a CAT when there was an important theory/fact for the students to understand due to the ensuing lectures/work developing on from it. As she wanted to make the use of CATs fun, she de- cided not to mark them, but rather got students to debate around the topics with students next to them. Dependent upon when the author needed feedback about her teaching, and which impor- tant theory the students needed to grasp, informed which CAT was used, and also where in the lecture. She found that using voting systems built into the PowerPoint presentation engaged the students and gave immediate feedback about whether they were correct or not. She also found that students discussing CATs in small groups, such as the one-sentence summary pro- moted deep learning. The students would then write down the agreed answer on a card anonymously which were then col- lected so that the author would get feedback regarding the ef- fectiveness of her teaching. CATs encourage the view that teaching and learning is a formative process that evolves over time. By being able to react swiftly to student answers, they provide the opportunity for immediate feedback to the lecturer which can be promptly acted upon, therefore giving the chance to the teacher to close the feedback loop. It encourages self-assessment by the student and reflection amongst both the lecturers and students. How- ever care must be taken in choosing the appropriate CAT and also allowing enough time in class to ensure that they are worthwhile. It may also be a good idea to give the CATs a nominal grade of 5% or 10% to ensure that the students value them. Tips for successful use of CATs (Angelo & Cross, 1993):  Don’t ask for feedback on things you can’t or won’t change;  Don’t collect more feedback than you can analyze and re- spond to by the next lecture;  Before you use a CAT, ask yourself: How might responses to this question(s) help me and my students improve? Conclusion If you can’t answer that question, don’t do the assessment;  Don’t use too many different CAT techniques in one se- mester. Student responses are more useful when the stu- dents are comfortable with a particular technique and un- derstand it (Martin, 2011).  Don’t use too many different CAT techniques in one se- mester. Student responses are more useful when the stu- dents are comfortable with a particular technique and un- derstand it (Martin, 2011). With virtual learning environments (VLE) become more in- tegrated into HE teaching, the author has also used CATs within the VLE used at the University of Nottingham. Along- side putting the PowerPoint slides and associated handouts up online, she has found success with an online survey replicating the knowledge probe CAT which asks one or two questions. It appears that the students value the engagement that using CATs offer, as the SET scores for her modules are always high and the pass rate of assessments are also high. The author also val- ues the timely feedback on her teaching, so she can detect any problems early and give her opportunity to approach the theory in another manner, encourage peer teaching and learning, and identify key items of literature for them. doi:10.1016/j.compedu.2005.11.014 Biggs, J. (1993). What do inventories of students’ learning process really measure? A theoretical review and clarification. British Jour- nal of Educational Psychology, 83, 3-19. Marsh, H. W. (1987). Students’ evaluation of university teaching: Re- search findings, methodological issues and directions for future re- search. 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https://openalex.org/W2511710684	https://orca.cardiff.ac.uk/id/eprint/89879/1/360SP.PDF	English	null	International Ocean Discovery Program Expedition 360 Scientific Prospectus Southwest Indian Ridge Lower Crust and MohoThe nature of the lower crust and Moho at slower spreading ridges (SloMo-Leg 1)	Scientific prospectus	2,015	cc-by	13,559	International Ocean Discovery Program Expedition 360 Scientific Prospectus Southwest Indian Ridge Lower Crust and Moho The nature of the lower crust and Moho at slower spreading ridges (SloMo-Leg 1) Published by International Ocean Discovery Program Published by l O i Published by y International Ocean Discovery Program y International Ocean Discovery Program Disclaimer Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the participating agencies, Texas A&M University, or Texas A&M Research Foundation. Portions of this work may have been published in whole or in part in other IODP documents or publications. This IODP Scientific Prospectus is based on precruise Science Advisory Structure panel discussions and scientific input from the designated Co-Chief Scientists on behalf of the drilling proponents. During the course of the cruise, actual site operations may indicate to the Co-Chief Scientists, the Staff Scientist/Expedition Project Manager, and the Operations Superintendent that it would be scientifically or operationally advantageous to amend the plan detailed in this prospectus. It should be understood that any proposed changes to the science deliverables outlined in the plan presented here are contingent upon the approval of the JRSO Science Services Director. Publisher’s notes This publication was prepared by the JOIDES Resolution Science Operator (JRSO) at Texas A&M University as an account of work performed under the International Ocean Discovery Program (IODP). Funding for the program is provided by the following international partners and implementing organizations: National Science Foundation (NSF), United States Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan European Consortium for Ocean Research Drilling (ECORD) Ministry of Science and Technology (MOST), People’s Republic of China Korea Institute of Geoscience and Mineral Resources (KIGAM) Australian Research Council (ARC) and GNS Science (New Zealand), Australian/New Zealand Consortium (ANZIC) Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan Ministry of Earth Sciences (MoES), India Coordination for Improvement of Higher Education Personnel, Brazil Citation: Dick, H.J.B, MacLeod, C.J., and Blum, P., 2015. Southwest Indian Ridge lower crust and Moho: the nature of the lower crust and Moho at slower spreading ridges (SloMo-Leg 1). International Ocean Discovery Program Scientific Prospectus, 360. http://dx.doi.org/10.14379/iodp.sp.360.2015 Copyright Except where otherwise noted, this work is licensed under a Creative Commons Attribution License. Unrestricted use, distribution, and reproduction is permitted, provided the original author and source are credited. Abstract International Ocean Discovery Program (IODP) Expedition 360 will form the first leg of a multiphase drilling project that aims to drill through the crust/mantle boundary at the ultraslow-spreading Southwest Indian Ridge and investigate the nature of the International Ocean Discovery Program (IODP) Expedition 360 will form the first leg of a multiphase drilling project that aims to drill through the crust/mantle boundary at the ultraslow-spreading Southwest Indian Ridge and investigate the nature of the Mohorovičić seismic discontinuity (Moho). Expedition 360 is expected to drill ~1300 m into lower crustal gabbro and is unlikely to penetrate the crust–mantle tran sition or recover a significant amount of peridotite. Drilling will be sited at Atlantis Bank, on an elevated wave-cut platform on the east flank of the Atlantis II Transform. Previous drilling and mapping shows that Atlantis Bank is a large oceanic core com plex, exposing a tectonic window of deep crustal and lithospheric mantle exhumed on the footwall of an oceanic detachment fault. The shallowest part of Atlantis Bank, at 700 m water depth, consists of a ~25 km2 wave-cut platform rimmed by a thin bio clastic limestone cap. The platform is part of a continuous gabbro massif ~40 km long by 30 km wide, overlying granular mantle peridotite that forms the lower slopes of the eastern wall of the Atlantis II Transform. Mapping shows that basement on the wave-cut platform consists largely of shallow-dipping amphibolitized gabbro mylo nite generated by detachment faulting. This fault rooted near-continuously into par tially crystalline gabbro for >4 million years. The mylonite exposed on the platform, and by cross-faulting and landslips on the sides of Atlantis Bank, both cut and are cut by steeply north dipping greenschist-facies diabase dikes. Thus, the gabbro crystal lized at depth was uplifted into the zone of diking at the ridge axis, creating, in effect, the equivalent to the base of a dike–gabbro transition seen in many ophiolites. Previous Ocean Drilling Program (ODP) operations at Atlantis Bank drilled the 1508 m deep Hole 735B and 150 m deep Hole 1105A, both recovering long sections of gabbro. During Expedition 360, we propose to drill to a nominal depth of 1.3 km at a site on the northern edge of the Atlantis Bank platform, ~1 km north-northeast of Hole 1105A and ~2 km northeast of Hole 735B. ISSN World Wide Web: 2332-1385 February 2015 Expedition 360 Scientific Prospectus Abstract A future drilling expedition, SloMo-Leg 2, aims to deepen the hole to ~3 km, with the overall goal of penetrating the crust–mantle transition, which is believed to be ~2.5 km above the seismically de termined Moho. Specific objectives of Expedition 360 include establishing the lateral continuity of the igneous, metamorphic, and structural stratigraphies previously drilled to the southwest, testing the nature of a magnetic polarity transition, and in vestigating the biogeochemistry of the lower crust. 3 Expedition 360 Scientific Prospectus Schedule for Expedition 360 Expedition 360 is based on Integrated Ocean Discovery Program (IODP) drilling Pro posal 800-Full (available at www.iodp.org/ active-proposals). Following ranking by the IODP Scientific Advisory Structure, the expedition was scheduled for the R/V JOI DES Resolution, operating under contract with the JOIDES Resolution Science Operator (JRSO). At the time of publication of this Scientific Prospectus, the expedition is scheduled to start in Colombo, Sri Lanka, on 30 November 2015 and to end in Port Louis, Mauritius, on 30 January 2016 (total days = 61). Accounting for 5 days of port call and 14 days of transit, a total of 42 days will be available for the drilling, coring, and downhole measurements described in this prospectus (for the current detailed schedule, see iodp.tamu.edu/ scienceops/). Further details about the facilities aboard the JOIDES Resolution can be found at www.iodp.tamu.edu/ publicinfo/ drillship.html. Introduction The IODP Science Plan 2013–2023 sets a number of basic challenges for the next de cade of planetary exploration. It recognizes that in order to understand the inherent connections between the Earth’s interior and its surface environment we must ad dress fundamental questions about basic plate tectonic processes. Central among these questions are how seafloor spreading and mantle melting lead to the creation of oceanic lithosphere at mid-ocean ridges, and what controls the architecture of the ocean crust thus formed (IODP Science Plan Challenge 9). Constraining the compo sition, diversity, and architecture of the lower ocean crust and shallow mantle is crit ical to understanding the global geochemical cycle, particularly the exchange of heat, mass, and volatiles between the Earth’s interior, oceans, and atmosphere. Crust formed at mid-ocean ridges extends across three-fifths of the Earth’s surface, comprising ~60% by area and ~30% by volume of the Earth’s crust. The ocean crust, as determined by seismic refraction studies, is typically ~6–7 km thick and apparently relatively uniform. The lower portion, corresponding to seismic Layer 3, is widely as sumed to consist of gabbroic rocks formed in a magma chamber beneath the ridge axis. Layer 3 is separated from the mantle beneath by the Mohorovičić seismic dis continuity (Moho) and is conventionally regarded as the boundary between the igne ous crust above and tectonized mantle peridotite beneath. Alternative interpretations are, however, possible: seawater penetration into peridotite causes partial alteration 4 Expedition 360 Scientific Prospectus to serpentinite and reduces its velocity to that comparable to gabbro; hence, the seis mic structure of Layer 3 in itself gives us little information as to its lithology. The Moho could equally represent an alteration front boundary between altered and un altered peridotites and need not coincide with the crust/mantle boundary at all. In stead, the Moho could lie well within Layer 3. If this is the case, we know less about the architecture and composition of the ocean lithosphere than we thought we did and very much less about global magmatic, volatile, and heat budgets. Until the advent of ocean drilling, the stratigraphy of Layer 3, the lower crustal layer, was largely unknown and widely misinterpreted. Even to date, only two penetrations of the lower crust >200 m have been attempted, and these ended no deeper than ~1508 m. Our direct knowledge, therefore, remains remarkably limited. Introduction What we have learned from deep drilling in the Indian Ocean and the Atlantic (e.g., Dick et al., 2000; Blackman et al., 2011), when compared to the shallow (~100–200 m deep) sec tions drilled at the East Pacific Rise (EPR; Gillis, Mével, Allan, et al., 1993; Gillis et al., 2014), is that the mechanisms of formation of the gabbroic lower crust at slow- and fast-spreading ridges differ profoundly (e.g., Sinton and Detrick, 1992; Coogan, 2014). At slow and ultraslow ridges, which represent ~60% of the global ridge system, tectonic stretching is recognized to be an integral part of the seafloor spreading pro cess (e.g., Tucholke and Lin, 1994; Tucholke et al., 1998; Escartín and Canales, 2011). A consequence of this discovery is that tectonic windows exposing lower crust on the seafloor are widespread at slow and ultraslow ridges. In practice, they are the only places at which the crust/mantle boundary is likely to be accessible by drilling with current technology. Rationale for the SloMo project The aim of SloMo (shorthand for “The nature of the lower crust and Moho at slower spreading ridges”) is simple: to drill as deeply as possible through Layer 3 in order to set the stage for the first ever penetration of the crust/mantle boundary (SloMo-Leg 2). Thus SloMo will test the hypothesis that the Moho, at least at slow and ultraslow ridges, represents an alteration boundary rather than the igneous crust–mantle tran sition. In doing so it will also be able to address key questions, including determining how magnetic reversal boundaries are expressed in the lower crust, assessing the role of the lower crust and shallow mantle in the global carbon cycle, and constraining the extent and nature of life at deep levels within the ocean lithosphere. 5 Expedition 360 Scientific Prospectus Deep drilling on Atlantis Bank will also provide an important step toward the long- term objective of drilling a total crustal penetration in fast-spreading ocean crust in the Pacific Ocean by providing critically needed experience in engineering a deep hole in lower crustal rocks. SloMo is not simply about drilling the crust/mantle boundary but is as much about the journey on the way there. By recovering a near-complete section of the igneous lower crust and crust–mantle transition at Atlantis Bank, we will be able to address many of the important questions posed in the IODP Science Plan’s Challenge 9, de veloped further in Scientific objectives. At slow- and ultraslow-spreading ridges, the lower crust uniquely preserves that critical link where magmatic and tectonic pro cesses directly reflect plate dynamics, melt input, and the pattern of mantle flow. We now understand that at such ridges a substantial portion of plate spreading is accom modated in the lower crust by tectonic extension due to faulting and, in places, duc tile deformation. Large-offset “detachment” normal faults exert strong control on melt distribution and transport in the lower crust and delivery to the seafloor. This is in marked contrast to fast-spreading ridges, where it is accepted that the crust princi pally undergoes magmatic accretion by the injection of melt into the lower crust, dik ing, and eruption of magmas on the seafloor; thus, rollover and corner flow by ductile flow accompanying mantle upwelling and plate spreading is believed largely limited to the mantle. Rationale for the SloMo project However, at slower spreading ridges, which are cooler and support only ephemeral magma chambers, the lower crust can potentially support a shear stress. As a consequence, with lower rates of magma supply, and colder, stronger lithosphere formed directly beneath the ridge axis, slower spreading ridges have very different morphologies and crustal architectures. Thus, as stated in Challenge 9, a full picture of crustal architecture and accretion can only be drawn if both fast- and slow-spread ing environments are addressed. Geological setting The Southwest Indian Ridge (SWIR) lies at the slow end of the mid-ocean-ridge spreading-rate spectrum, with a full spreading rate of 14 mm/y. The ridge trends ap proximately southwest–northeast for most of its length, spreading almost due north– south. Between ~52°E and 60°E, it is offset by a series of closely spaced, long-offset transform faults. One of these, the Atlantis II Transform at 57°E, offsets the SWIR si nistrally by 200 km (Engel and Fisher, 1975) (Figure F1). Between this and the Novara Transform, ~140 km to the east, the spreading axis is divided into two segments, sep 6 6 Expedition 360 Scientific Prospectus arated by a nontransform discontinuity at 57°35′E (Hosford et al., 2003). The spread ing rate here is asymmetric: at the short westerly Segment AN-1, adjacent to the northern ridge/transform intersection of the Atlantis II Transform, magnetic anoma lies reveal rates of 8.5 mm/y to the south and 5.5 mm/y to the north (Hosford et al., 2003); in consequence, the transform has been lengthening by 3 mm/y for at least the past 20 million years (Dick et al., 1991b; Hosford et al., 2003). At ~20 Ma, a 10° coun terclockwise change in regional spreading direction put the Atlantis II Transform into transtension for a period of ~12 million years (Dick et al., 1991b, Hosford et al., 2003; Baines et al., 2007, 2008). Immediately to the east of the Atlantis II Transform, parallel to it and on a flow-line directly south of spreading Segment AN-1, is an elevated transverse ridge (Figure F1) consisting of a series of uplifted blocks connected by saddles that rise to as little as 700 m below sea level (Engel and Fisher, 1975; Dick et al., 1991b). Atlantis Bank, at 32°43′S, 57°17′E, forms the shallowest and northernmost portion of this ridge, 95 km south of the axis of the SWIR. The bank consists of a raised dome ~40 km long by ~30 km wide, rising from 5700 m depth at the base of the transform wall to 700 m on a ~25 km2 flat-topped platform at its crest and then drops to 4300 m on its eastern flank across two prominent transform-parallel east-dipping normal faults (Baines et al., 2003; Hosford et al., 2003) (Figures F1, F2). Geological setting ROV survey results show the central pavement to be bare rock, locally knobby igneous outcrop surrounded on its periphery by a pavement of limestone (in some places ripple marked) and carbonate-cemented pebble conglom erate. Using the British Geological Survey’s 5 m diamond rock drill and 1 m BRIDGE ori ented corer, MacLeod et al. (1998) drilled 42 successful cores on the surface of this platform region, recovering 33 igneous rocks (Figure F3). They reveal continuous gab bro outcrop on the platform surface, including at proposed drill Site AtBk6 (Figure F4) but pass into serpentinized peridotite and pillow-basalt breccia at its southern tip, where the flat platform narrows into a north–south spine (32°44.5′S). The dredging and submersible studies, conducted over a broader ~700 km2 area, showed that gab bro is present for >35 km parallel to the spreading direction (Figure F2B), suggesting that accretion of a continuous gabbroic layer persisted for at least 4 million years (Dick et al., 1991a; Matsumoto et al., 2002). Many of the gabbroic rocks in the shallow drill cores and seen in ROPOS ROV footage from the surface of the Atlantis Bank platform are mylonitic, displaying intense crys tal-plastic deformation with subhorizontal fabrics. In Hole 735B, amphibolite facies crystal-plastic deformation of oxide gabbro and olivine gabbro was likewise found to be strong in the uppermost 500 m but diminished markedly downhole (Robinson, Von Herzen, et al., 1989; Cannat et al., 1991; Dick et al., 1991a; Miranda and John, 2010). Considered together, it is apparent that the upper surface of Atlantis Bank rep resents a detachment fault zone, effectively a high-temperature “plutonic growth fault” active (for >4 million years) during the accretion of the gabbroic lower crust and responsible for its exhumation. This “hot”’ detachment must have rooted in or near a melt lens at the top of a continuously replenished magma chamber/mush zone, close to the midpoint of the AN-1 spreading ridge (Dick et al., 1991a, 2000; Nat land and Dick, 2001, 2002). Oceanic detachment faults are now known to be widespread along slow- and ul traslow-spreading ridges (e.g., Escartín et al., 2008; Sauter et al., 2013), often respon sible for exhuming elevated oceanic core complex massifs that expose mantle peridotite and/or gabbro on the seafloor. Oceanic core complexes at the Mid-Atlantic Ridge (MAR) typically display prominent spreading-direction-parallel corrugations on their flat-topped or domal upper surfaces (Cann et al., 1997; Tucholke et al., 1998). Geological setting Immediately to the east of the Atlantis II Transform, parallel to it and on a flow-line directly south of spreading Segment AN-1, is an elevated transverse ridge (Figure F1) consisting of a series of uplifted blocks connected by saddles that rise to as little as 700 m below sea level (Engel and Fisher, 1975; Dick et al., 1991b). Atlantis Bank, at 32°43′S, 57°17′E, forms the shallowest and northernmost portion of this ridge, 95 km south of the axis of the SWIR. The bank consists of a raised dome ~40 km long by ~30 km wide, rising from 5700 m depth at the base of the transform wall to 700 m on a ~25 km2 flat-topped platform at its crest and then drops to 4300 m on its eastern flank across two prominent transform-parallel east-dipping normal faults (Baines et al., 2003; Hosford et al., 2003) (Figures F1, F2). Hole 735B is located at the southwest corner of the flat surface of the Atlantis Bank platform, at 731 m water depth (32°43.395′S; 57°15.959′E) (Figures F2, F3). During ODP Legs 118 and 176, the hole was drilled to a total 1508 m below seafloor (mbsf), with 87% core recovery, all in gabbro (sensu lato) ~11 Ma in age (Robinson, Von Her zen, et al., 1989; Dick et al., 2000). Further operations during Leg 179 drilled the 160 m deep Hole 1105A in the center of the platform (32°43.13′S; 57°16.65′E) (Petti grew, Casey, Miller, et al., 1999), also in gabbro and with similar overall core recovery rates. Site surveys of the Atlantis Bank area have included multibeam, magnetic, and gravity surveys and rock sampling using seabed rock coring, remotely operated vehicle (ROV), submersible dives, and dredging (Dick et al., 1991b; MacLeod et al., 1998; Arai et al., 2000; Kinoshita et al., 1999; Matsumoto et al., 2002). A high-resolution bathy metric map of the ~25 km2 summit region compiled from narrow-beam echo sound ings (Dick et al., 1999) shows the platform to consist of a broadly flat pavement ranging from ~750 to 689 m water depth). Proposed deep drill Site AtBk6 is located at the center of the platform, ~1 km north-northeast of Hole 1105A and 2 km northeast 7 Expedition 360 Scientific Prospectus of Hole 735B (Figure F3). Geological setting Sampling of fault rock from the detachment fault zones themselves shows that defor mation typically localized on weak, low-temperature secondary minerals such as talc, 8 Expedition 360 Scientific Prospectus derived primarily from alteration of peridotite (Dick et al., 2001, 2008; MacLeod et al., 2002; Escartín et al., 2003; Schroeder and John, 2004). Where gabbro is present in the detachment footwall it may be unaffected or barely affected by crystal-plastic defor- mation (e.g. 15°45′N on the MAR: MacLeod et al., 2002, and Escartín et al., 2003; At- lantis Massif: Ildefonse et al., 2007), whereas elsewhere it is heavily deformed, as at Kane Megamullion on the MAR (Dick et al., 2008) and at Atlantis Bank. Submersible observations at Atlantis Bank (Kinoshita et al., 1999; Matsumoto et al., 2002) show that the detachment fault surface is preserved over large regions in the deeper waters on both sides of the platform. The damage zone and underlying gabbro are well exposed by high-angle normal faulting on the eastern side of the complex and in headwalls of large landslips on the western flank. Here, lower temperature fault rocks are present in addition to the amphibolite facies mylonite. These chlori- tized and weathered fault gouge and talc-serpentine schists, preserved locally on the fault surface (Dick et al., 2001; Miranda and John, 2010), are closely comparable to fault rocks found on the MAR core complexes (e.g., MacLeod et al., 2002; Escartín et al., 2003, Schroeder and John, 2004; Dick et al., 2008). The absence of the low-temperature fault rocks on the flat surface of the Atlantis Bank platform compared to its flanks suggests they have been removed, a deduction en- tirely consistent with the long-held belief (Dick et al., 1991b; Palmiotto et al., 2013) that Atlantis Bank was once an ocean island, and its flat top results from erosion, be- fore later subsiding to its present level. Shallow drilling and dredging on the summit and uppermost flanks of the Atlantis Bank platform amply verified this. Indurated carbonates were drilled at 24 sites from the periphery of the Atlantis Bank platform and were also recovered in dredges from its flanks (MacLeod et al., 1998, 2000; Palmi- otto et al., 2013). Although some (probably recent) carbonate sand was recovered, most of the sediment is indurated bioclastic limestone (skeletal packstone to wacke- stone; MacLeod et al., 2000) of Miocene to Pleistocene age (Palmiotto et al., 2013). Geological setting This sediment contains an abundant macrofauna, primarily bryozoans, mollusks, al- gal nodules, and echinoids but also some solitary corals. Benthic and, in some in- stances, large planktonic foraminifers are common. Whereas Palmiotto et al. (2013) suggest a water depth of ~100–200 m based upon assemblages in dredged carbonates from the flanks of the platform, green algal (dasyclad) assemblages in drill cores from the platform summit (G. Della Porta and V.P. Wright, pers. comm., 1999) indicate wa- ter depths at wave base or shallower, demonstrating that the platform was at sea level, and probably above. How much material has been removed by subaerial erosion is not known, potentially 100–200 m with reference to the inferred thickness of the de- 9 9 Expedition 360 Scientific Prospectus tachment fault damage zone. On the basis of pitted gabbro outcrops to ~1500 m be low sea level that may reflect preferential subaerial weathering of plagioclase, Palmiotto et al. (2013) speculate that the Atlantis Bank island could have been ele vated to as much as 1 km above sea level. ROV observations of steep sides up to 10 m, locally even 50 m, at the flanks of the gabbro pavement on the summit of the plat form may represent coastal cliffs and small sea stacks resulting from the dispersal of wave energy on the flanks of the island during erosion. The Atlantis II Transform transverse ridge, on which Atlantis Bank lies, is clearly anal ogous to oceanic core complex massifs found on the MAR (e.g., Cann et al., 1997; Tucholke et al., 1998; Dick et al., 2008), although here, as elsewhere along the SWIR (Cannat et al., 2006; Sauter et al., 2013), the prominent spreading-direction-parallel corrugations that are characteristic of the flat surfaces of the Atlantic oceanic core complexes are not so obvious and potentially present only on the down-dropped ter race on the eastern side of Atlantis Bank (Figure F2A). The Atlantis II Transform trans verse ridge has, furthermore, clearly been uplifted far above the surrounding seafloor. Whereas flexural uplift of detachment fault footwalls to form oceanic core complexes is typically 1 ± 0.5 km (Tucholke et al., 1998; Lavier et al., 1999), the Atlantis II Trans form transverse ridge has been uplifted by 3 km relative to surrounding seafloor of similar age (Baines et al., 2003). Dick et al. (1991b) and Baines et al. Geological setting (2003) propose that the original detachment-related uplift at the ridge/transform intersection was ac centuated by an additional phase of flexural uplift, imparted upon the transform in response to the change of spreading direction on the SWIR at 19.5 Ma, and accom modated by the reactivation as normal faults of originally transform-related north– south structures. One consequence of the relative uplift of the Atlantis II transverse ridge is that the crust/mantle boundary, as mapped by dredging and submersible traverses, is exposed along its western wall for a distance of nearly 40 km (Figure F2B). This boundary was traversed at two locations by Shinkai 6500 Dives 466 and 458 at 4500 and 4650 m wa ter depth, respectively. Dives 467 and 459 immediately above each of them found the detachment surface at 3000 and 1755 m, respectively, potentially indicating gabbro layer thicknesses as little as 1500 and 2895 m. Elsewhere along the western flank of Atlantis Bank, serpentinized peridotite was recovered in dredges from depths as shal low as 2000 m water depth (Dredge JR31-DR8), though in other nearby places only at >3000 m (Figure F2B). At the southern end of the platform itself, serpentinized harz burgite was drilled at 839 mbsf (Site JR31-BGS12). Peridotite sampled at several loca tions above the contact mapped on the transform wall consists largely of talc- 10 Expedition 360 Scientific Prospectus serpentine schist that likely overlies the gabbro massif. This and serpentine pebbles found in beach conglomerates overlying gabbro near the crest of the platform indi cate that a discontinuous talc-serpentine sheet was associated with the detachment fault at shallow levels and is now locally preserved on the detachment surface. The serpentinite drilled at the southern end of the platform, however, is relatively massive harzburgite with well-preserved pseudomorphs of pyroxene. Possible origins of these include a peridotite screen in gabbro, similar to those drilled at Atlantis Massif in Hole U1309D, an enclave of less deformed and altered serpentinized peridotite in the orig inal detachment fault shear zone, or juxtaposition through a northward-dipping ridge-parallel normal fault that demarcates the southernmost end of the flat platform at 32°44.4′S (Figure F3). A wide-angle seismic refraction survey of the Atlantis II Transform region found the Moho at 5 ± 1 km beneath Atlantis Bank (Muller et al., 1997, 2000; Minshull et al., 1998) (Figure F5). Geological setting The direct geological constraints outlined above offer strong sup port to the inference that the seismic Moho cannot therefore represent the crust/ mantle boundary in this region. Whereas Muller, Minshull, and colleagues also con cluded that it was likely a serpentinization front, they based this conclusion on the geochemical argument that the original igneous crustal thickness there was originally ~4 km (based upon rare-earth element inversions), and with the basaltic carapace re moved by detachment faulting, the remainder was likely to be ~2–2.5 km thick. How ever, they admit this interpretation is nonunique, primarily because of the overlap in P-wave seismic velocity between gabbro and ~20%–40% partially serpentinized peri dotite (e.g., Miller and Christensen, 1997). At what level is the crust/mantle boundary likely to lie beneath Atlantis Bank? Pro jecting the detachment surface to the locations of the traversed crust/mantle bound ary described above indicates that the crustal thickness at these points prior to mass wasting on the transform wall was significantly <2000 m, whereas the depth to Moho below the transform wall remains ~5 km (Figure F5). Given that the elevated core complex massif is produced by flexure during uplift into the rift-mountains due to a spreading direction change (Baines et al., 2003), it seems reasonable to conclude that the mapped boundary is most likely to project approximately subhorizontally be neath the center of Atlantis Bank, consistent with the igneous crustal thickness of 2– 2.5 km suggested by Minshull et al. (1998). 11 11 Expedition 360 Scientific Prospectus 2. How much mantle is incorporated into the lower crust? An unanticipated finding in the cores from Hole 1309D at the Atlantis Massif in the north Atlantic was the incorporation of significant volumes of hybridized mantle per idotite (Blackman, Ildefonse, John, Ohara, Miller, MacLeod, and Expedition. 304/305 Scientists; Blackman et al., 2011; Drouin et al., 2009) (Figure F6). Comparable screens of mantle rock were not found in either Holes 735B or 1105A. This could reflect dif ferences in accretion due to different magma budgets, or it could simply be happen stance, whereby further drilling will encounter equivalent ultramafic horizons at Atlantis Bank. 1. What is the igneous stratigraphy of the lower ocean crust? The drilling will determine if the igneous, metamorphic, and structural stratigraphy found for Holes 735B and 1105A is laterally continuous across the wave-cut platform on Atlantis Bank. In combination with Holes 735B and 1105A and the existing sur face mapping, this will provide a four-dimensional view of the lateral continuity and evolution of the lower crust and the process of emplacement in space and time. 2. How much mantle is incorporated into the lower crust? Scientific objectives Expedition 360 represents the first leg of the SloMo Project, which seeks to use the tectonic window into the lower crust at Atlantis Bank to recover the full lower section of the ocean crust, core through the igneous crust–mantle transition, determine if Moho represents a serpentinization front and, ultimately, core through the Moho (~5.5 km at an ultraslow-spreading ocean ridge). SloMo has two phases. Phase I uses the JOIDES Resolution to drill to 3 km to test the hypothesis that the Moho represents a serpentinization front and to recover the full lower crustal section and crust–mantle transition zone. Phase II will utilize the riser D/V Chikyu to drill down ~5.5 km through the seismic Moho itself. In order to achieve the Phase I objective, two expe ditions will be needed. Expedition 360 will need to properly establish the borehole for Leg 2 and thereafter drill as deep as possible. Given optimal conditions, a depth of at least 1300 mbsf, with continuous coring, should be achievable. Operations during Expedition 360 should allow us to address the following: 3. What are the modes of melt transport into and through the lower crust? Several different modes of melt transport were identified in the Hole 735B cores. These included small intrusive bodies of cumulates, larger intrusive units on the scale 12 Expedition 360 Scientific Prospectus of hundreds of meters, anastomosing channels produced by focused flow and melt- rock reaction, and compaction of late iron titanium–rich melts into shear zones where they hybridized olivine gabbro to iron titanium oxide–rich gabbro and gab bronorite. The continuity and scale of these features cannot be determined from a sin gle deep hole. Thus, drilling during Expedition 360 seeks to further document these features in a younger section of the massif. 4. How does the lower crust shape the composition of mid-ocean-ridge basalt (MORB)? The complex stratigraphies seen in Holes 735B and U1309D (Figure F6) differ in sig nificant ways. What common factors, however, influence the composition of MORB? Recent work on gabbroic sections from the EPR has shown that the lower crust acted as a reactive porous filter homogenizing diverse melts that were intruded into it and modifying their trace element contents prior to their eruption to the seafloor (Lissen berg et al., 2013). To what extent do such processes operate at slower spreading rates? . What is the strain distribution in the lower crust during asymmetric seafloor spreading? 5. What is the strain distribution in the lower crust during asymmetric seafloor spreading? Asymmetric spreading produced by detachment faulting is now recognized as one of three major accretionary modes at slow-spreading ridges (symmetric, asymmetric, and amagmatic rifting). Although each of these is important, little is known of how magmatism and tectonism interact, and hence how lower crustal accretion differs in these environments. At present, two deep holes in oceanic core complexes, Holes 735B and U1309D, allow us to determine strain distribution with depth. The distri bution of strain documented in these two locations differs significantly, with far more intense crystal-plastic deformation being found in the upper 500 m of Hole 735B than in Hole U1309D. We cannot assess the nature, extent, and role played by high- temperature deformation during lower crustal accretion with isolated, one-dimen sional sections. Offsetting and drilling to the north of Hole 735B will allow us to de termine the continuity of the strain distribution from Hole 735B across the Atlantis Bank core complex and thereby assess the role and broader significance of synmag matic deformation in this tectonic environment. 6. What is the nature of magnetic anomalies? Magnetic anomaly “stripes” are detectable across the Atlantis II Transform transverse ridge, including the Atlantis Bank platform itself (Dick et al., 1991b; Allerton and Tivey, 2001; Hosford et al., 2003; Baines et al., 2007, 2008). Whereas most of the plat form is reversely magnetized, as are the gabbro intervals of Holes 735B and 1105A (Ki 13 Expedition 360 Scientific Prospectus kawa and Pariso, 1991), a narrow normally polarized zone ~2 km wide is present near its northern end. This is equated to Anomaly C5r.1n (11.476–11.531 Ma) (Allerton and Tivey, 2001; Baines et al., 2008) (Figure F7). It is detectable on surface (Dick et al., 1991b; Hosford et al., 2003) and deep-towed magnetic profiles (Allerton and Tivey, 2001) and in the JR31 seabed drill cores; however, the precise location of the bound- ary in each case is slightly offset (Figure F7). Because the sea-surface magnetization signal is derived from a much greater volume of rock than the near-bottom signal in the cores, Allerton and Tivey (2001) deduced from the sense of offset that the mag- netic anomaly boundaries are inclined. They modeled the southern limit of the anomaly as a planar boundary dipping ~25° toward the south, suggesting that the sur- face represented either an isotherm corresponding to the Curie point or the edge of an intrusion (Figure F7). Alternatively, the form of the southern edge of the anomaly may have been modified by faulting: extensive brittle deformation and cataclasis is observed in shallow drill cores from the location of the southern reversal boundary, probably corresponding to a northward-dipping normal fault at the kink/narrowing of the platform summit at 32°41.8′S. If so, the reversal boundary is likely to dip more steeply than the 25° proposed by Allerton and Tivey (2001). An important objective of Expedition 360 is to drill through normal polarity Chron C5r.1n to determine the nature of its boundaries and what controls magnetic anom- alies in plutonic rocks. 7. Is there life in the lower crust and hydrated mantle? A primary objective of the SloMo Project is to determine the microbiology of the lower crust, the potential serpentinized mantle above Moho, and the underlying mantle, to address IODP Science Plan Challenge 6 “What are the limits of life in the subseafloor?” For this purpose, Expedition 360 will assess the microbiology of the up- per portion of the lower crust. 8. What is the role of the lower crust and shallow mantle in the global carbon cycle? Serpentinization and weathering of ultramafic rocks as well as alteration of basalts are known, under the right conditions, to cause the formation of carbonates. Such car- bonates are present in the form of extensive veining in serpentinized peridotites at the southern edge of the Atlantis Bank platform (shallow drill Site JR31-BGS-12 and contingency drill Site AtBk4). 14 Expedition 360 Scientific Prospectus The extent of these reactions in the lower crust is, to date, largely unknown. Although we do not expect that carbon sequestration will be a significant process in the lower ocean crust based on Holes 735B and U1309D, the possibility exists that Expedition 360 may penetrate the base of the lower ocean crust and thus offer the first opportu nity to determine if carbon sequestration is significant below it. Drilling and coring strategy The JOIDES Resolution will move directly to proposed Site AtBk6, lower the subsea camera, and do a short survey to find a flat and solid-looking plutonic rock surface suitable for deployment of a reentry system. Prior site survey data indicates a flat bare- rock gabbro pavement at this precise location (proposed Site JR31-BR-8) (Figure F4). If the weather is suitable (<1 m heave), we will deploy a reentry system and complete the primary hole as described below. If the weather is initially unsuitable for deploy ment of a reentry system but acceptable for drilling, we will instead proceed to con tingency proposed Site AtBk4 at the southern end of the platform, where prior seabed rock drilling during the JR31 site survey cruise cored serpentinite. There we would ini tiate a bare-rock spud in and drill a single-bit hole until weather improves or until bit destruction. In the event that the weather remains unsuitable for the deployment of the reentry system, we will proceed to the second contingency proposed Site AtBk2 immediately east of the Atlantis Bank wave-cut platform and drill a single-bit hole where the original, uneroded detachment surface was down-dropped by a 1000 m off set east-facing normal fault. This would recover the uppermost section of the core complex that is missing at proposed Site AtBk6 due to erosion. If unsuitable weather continues, we will proceed to the third contingency proposed Site AtBk5 and drill an other single-bit hole until the weather improves. At the primary proposed Site AtBk6, deployment of a reentry system will be initiated as soon as weather permits. Reentry system Plan A begins with drilling-in a reentry cone with a 15 m long conductor casing (13.375 inches) using a mud motor. Two such systems will be carried on board. The conductor casing will be cemented in place and the cement plug will be drilled out before coring begins. If Plan A fails, we will proceed with Plan B: drilling an 18.5 inch hole, lowering a 16 inch casing into the open hole (15 m), and then deploying a free-fall hammer funnel (hard rock reentry system [HRRS]-type) over the 16 inch casing. Following deployment of the reentry 15 Expedition 360 Scientific Prospectus system, we will core with the rotary core barrel (RCB) to 220 m, log the hole, open the hole to a 12.5 inch diameter, and install 200 m of 10.75 inch casing. Drilling and coring strategy We will then drill as deep as time allows, reserving ~2 days for logging prior to departure. system, we will core with the rotary core barrel (RCB) to 220 m, log the hole, open the hole to a 12.5 inch diameter, and install 200 m of 10.75 inch casing. We will then drill as deep as time allows, reserving ~2 days for logging prior to departure. From 220 m downhole, our intention is to core continuously. However, the primary objective of SloMo Project Phase I is to drill to 3 km, requiring that we drill as deep a hole as possible on Expedition 360 to enable a second expedition (SloMo-Leg 2) to achieve this objective. A conservative estimate of what can be drilled with continuous coring during Leg 1 is 1300 m, which is short of what is needed on Leg 1 to achieve the objectives of Leg 2. Accordingly, once penetration to a few hundred meters below the magnetic transition is achieved, and if time is an issue to achieve the primary ob jective, we will consider alternating four-cone coring bit runs with tricone drilling bit runs to achieve greater depth, alternating coring and drilling without coring between bit runs. A rough estimate is that drilling without coring would be nearly twice as fast due to the heavier weight that can be placed on a more robust tricone drill bit and no wireline time to retrieve core. If the stratigraphy of the lower kilometer is similar to Hole 735B, this would not greatly affect the overall science objectives, as the lower half of Hole 735B exhibited far less variability than the upper portion. If and when the hole reaches 1400 m, we would resume continuous coring as we entered depths deeper than Hole 735B. Logging/downhole measurements strategy Wireline logging will form an essential component of the operations at Atlantis Bank during Expedition 360. Downhole measurements of the in situ physical properties of the formation will complement and extend those measured on cores, assist in char acterizing the nature of incompletely recovered intervals, and thereby help in refin ing the igneous, metamorphic, and structural stratigraphies of the borehole(s). Borehole wall imaging will be of great importance in establishing the true geometry of features such as lithological contacts, veins, and fractures; furthermore, by match ing distinctive features in cores with their representations on borehole wall images, core pieces may subsequently be reoriented to geographical coordinates. This can in turn allow spatially anisotropic properties of cores, such as magnetic remanence di rections, igneous petrofabrics, fault kinematics, and so on, to be considered in the geographic reference frame and allow upscaling and integration of data from the drill site with regional-scale geological and geophysical information. 16 Expedition 360 Scientific Prospectus At the primary proposed Site AtBk6, we intend to conduct wireline logging operations in two phases. The first phase will be after coring the hole to 220 mbsf and before de ploying 200 m of 10.75 inch casing. This first set of logs from ~30 to ~220 mbsf is pro jected to take <1 day. The second phase of downhole logging will take place at the end of Expedition 360 and will cover the interval from 200 mbsf to the bottom of the hole, projected to be at ~1300 mbsf. This should take ~2 days. At the beginning of each logging phase, the hole will be filled with freshwater; based on experience from previous expeditions this enhances the quality of Formation MicroScanner (FMS) electrical borehole wall imagery (Blackman, Ildefonse, John, Ohara, Miller, MacLeod, and Expedition. 304/305 Scientists, 2006). In both phases of downhole logging, we anticipate making three separate logging runs with the following tool configurations, in order: (1) triple combination (triple combo), (2) FMS-sonic, and (3) Ultrasonic Borehole Imager (UBI). In the second log ging phase, the Versatile Seismic Imager (VSI) will be added as a fourth run. The triple combo tool string consists of six separate probes: • The Accelerator Porosity Sonde (APS) uses an electronic neutron source to mea- sure the porosity of the formation. • The Hostile Environment Litho-Density Sonde (HLDS) measures bulk density. Logging/downhole measurements strategy The tool employs a single-arm caliper to push the sonde against the borehole wall and, by so doing, giving a measurement of borehole size. • The Hostile Environment Natural Gamma Ray Sonde (HNGS) measures the nat- ural radioactivity of the formation, including indications of the concentrations of Th, U, and K. • The High Resolution Laterolog Array Tool (HRLA) measures the electrical resis- tivity of the formation at two different invasion depths. • The Phasor Dual-Induction Spherically-Focused Resistivity Tool (DIT). • The Magnetic Susceptibility Sonde (MSS-B) measures borehole magnetic suscep- tibility at two different vertical resolutions and depths of investigation. The FMS-sonic tool string combines the FMS, Dipole Sonic Imager (DSI), and General Purpose Inclinometry Tool (GPIT) tools. It also carries an HNGS sonde to allow direct correlation with the data from other logging runs. The FMS consists of four orthogo The FMS-sonic tool string combines the FMS, Dipole Sonic Imager (DSI), and General Purpose Inclinometry Tool (GPIT) tools. It also carries an HNGS sonde to allow direct correlation with the data from other logging runs. The FMS consists of four orthogo 17 Expedition 360 Scientific Prospectus nal pads with 16 electrodes on each pad mounted on arms that are pressed against the borehole wall. Resistivity measurements from the electrodes are combined to gen erate high-resolution electrical images of strips of the borehole wall (a single pass im ages ~22% of the circumference of the hole). In practice FMS images are particularly useful for the identification of (the orientation of) planar features such as faults, frac tures, veins, borehole breakouts, and lithologic variations in the borehole wall. The images can resolve millimeter-scale features if they have sufficient resistivity contrast. They are oriented to the geographical reference frame by means of the GPIT. If dis tinctive planar features in the core can be matched directly to their representations on the FMS imagery, the cores may be reoriented to geographical coordinates. The DSI records a full set of acoustic waveforms to measure the compressional and shear-wave velocity of the formation (VP and VS). VP can be combined with the density log to gen erate synthetic seismograms and provide high-resolution seismic-borehole integra tion. The UBI tool measures the amplitude and transit time of an acoustic wave propagated into the formation. It provides medium–high resolution acoustic images of the bore hole wall, which are geographically oriented by means of a GPIT sonde. Logging/downhole measurements strategy Although of lower resolution than the FMS images, UBI data have the advantage of providing 100% coverage of the borehole wall. UBI and FMS images are utilized in a similar manner. The VSI is a well seismic geophone used in conjunction with an air gun source at the surface to help constrain the velocity structure of the vicinity of the borehole. Mea surements are made with the tool held at different depths within the borehole, typi cally 50 m apart. In addition to the above, it is possible that borehole fluid temperatures may be mea sured on one or more occasions by deploying the Sediment Temperature Tool (SET), holding the drill bit just above the bottom of the hole, and allowing temperatures to equilibrate. This may be considered near the end of the expedition to ascertain for mation temperatures at the base of the hole. We may also consider deploying the core orientation tool ; although useless in hard rock/RCB coring operations for orienting the core itself, the information would give a direct measure of borehole deviation. More information about the capabilities of the downhole logging tools is available at iodp.tamu.edu/tools/logging/index.html. 18 Expedition 360 Scientific Prospectus Risks and contingency The biggest imponderable and probably greatest risk to the successful implementa tion of the drilling plan is the weather. Although at a latitude of 32°S, Atlantis Bank lies to the north of the edge of the Antarctic Circumpolar Current and Southern Ocean and can be affected by storms and poor sea states at any time of year. This has occasionally affected scientific operations during previous research cruises. As de tailed in Operations plan, we have identified several contingency sites where we could drill single-bit bare-rock spud-in rotary-cored holes until the weather improves. These operations should be possible in sea states greater than the ≤1 m heave condi tions required to set the hard-rock drill-in reentry system at the primary proposed Site AtBk6. It should be noted that these contingency sites are not envisaged as alternate deep-penetration sites. In marginal conditions there is a risk that the HRRS will not be drilled in perfectly vertically, which may cause problems for the setting of the cas ing strings and, ultimately, successful deep penetration during Expedition 360 and fu ture drilling expeditions. A complete backup drill-in reentry system will be carried, as well as a less sophisticated drilling/casing system (Plan B), as outlined in Operations plan. It should be noted that once the reentry system and casing strings have been installed, coring operations should be much less vulnerable to adverse weather con ditions. Hole conditions and core recovery in Holes 735B and 1105A on Atlantis Bank have been exceptional (average 87% recovery over >1500 m in Hole 735B); accordingly, we do not anticipate the difficult hole conditions encountered at almost all other ODP/ IODP ocean basement drill sites. Overall, we have allowed 3.8 days for weather contingency. If this is not required during Expedition 360, we will simply drill deeper at the primary proposed Site AtBk6. Site survey data The supporting site survey data for Expedition 360 are archived at the IODP Site Sur vey Data Bank. 19 Expedition 360 Scientific Prospectus References Allerton, S., and Tivey, M.A., 2001. Magnetic polarity structure of the lower oceanic crust. Geophysical Research Letters, 28(3):423–426. http://dx.doi.org/10.1029/2000GL008493 Arai, S., Dick, H.J.B., and Scientific Party, 2000. Cruise Report, Mode 2000 (Kairei/Kaiko KR00- 06): Yokosuka, Japan (Japanese Agency for Marine-Earth Science and Technology). Baines, A.G., Cheadle, M.J., Dick, H.J.B., Hosford Scheirer, A., John, B.E., Kusznir, N.J., and Matsumoto, T., 2003. Mechanism for generating the anomalous uplift of oceanic core complexes: Atlantis Bank, Southwest Indian Ridge. Geology, 31(12):1105–1108. http:// dx.doi.org/10.1130/G19829.1 Baines, A.G., Cheadle, M.J., Dick, H.J.B., Hosford Scheirer, A., John, B.E., Kusznir, N.J., and Matsumoto, T., 2007. The evolution of the Southwest Indian Ridge from 55°45′E–62°E: changes in plate-boundary geometry since 26 Ma. Geochemistry, Geophysics, Geosystems, 8(6):Q06022. http://dx.doi.org/10.1029/2006GC001559 Baines, A.G., Cheadle, M.J., John, B.E., and Schwartz, J.J., 2008. The rate of oceanic detach ment faulting at Atlantis Bank, SW Indian Ridge. Earth and Planetary Science Letters, 273(1–2):105–114. http://dx.doi.org/10.1016/j.epsl.2008.06.013 Blackman, D.K., Ildefonse, B., John, B.E., Ohara, Y., Miller, D.J., Abe, N., Abratis, M., Andal, E.S., Andreani, M., Awaji, S., Beard, J.S., Brunelli, D., Charney, A.B., Christie, D.M., Col lins, J., Delacour, A.G., Delius, H., Drouin, M., Einaudi, F., Escartín, J., Frost, B.R., Früh- Green, G., Fryer, P.B., Gee, J.S., Godard, M., Grimes, C.B., Halfpenny, A., Hansen, H.-E., Harris, A.C., Tamura, A., Hayman, N.W., Hellebrand, E., Hirose, T., Hirth, J.G., Ishimaru, S., Johnson, K.T.M., Karner, G.D., Linek, M., MacLeod, C.J., Maeda, J., Mason, O.U., McCaig, A.M., Michibayashi, K., Morris, A., Nakagawa, T., Nozaka, T., Rosner, M., Searle, R.C., Suhr, G., Tominaga, M., von der Handt, A., Yamasaki, T., and Zhao, X., 2011. Drill ing constraints on lithospheric accretion and evolution at Atlantis Massif, Mid-Atlantic Ridge, 30°N. Journal of Geophysical Research: Solid Earth, 116(B7):B07103. http:// dx.doi.org/10.1029/2010JB007931 Blackman, D.K., Ildefonse, B., John, B.E., Ohara, Y., Miller, D.J., MacLeod, C.J., and the Expe dition 304/305 Scientists, 2006. Proceedings of the Integrated Ocean Drilling Program, 304/ 305: College Station, TX (Integrated Ocean Drilling Program Management International, Inc.). http://dx.doi.org/10.2204/iodp.proc.304305.2006 Cann, J.R., Blackman, D.K., Smith, D.K., McAllister, E., Janssen, B., Mello, S., Avgerinos, E., Pascoe, A.R., and Escartin, J., 1997. Corrugated slip surfaces formed at ridge–transform intersections on the Mid-Atlantic Ridge. Nature, 385(6614):329–332. http://dx.doi.org/ 10.1038/385329a0 Cannat, M., Mével, C., and Stakes, D., 1991. Normal ductile shear zones at an oceanic spread ing ridge: tectonic evolution of Site 735 gabbros (southwest Indian Ocean). Sampling and data sharing strategy Shipboard and shore-based scientists participating in the Expedition 360 project should refer to the IODP Sample, Data, and Obligations Policy and Implementation Guidelines posted on the Web at www.iodp.org/program-documents/. This docu ment outlines the policy for distributing IODP samples and data to research scientists, curators, and educators. The document also defines the obligations that sample and data recipients incur. The Sample Allocation Committee (SAC), composed of Co- Chief Scientists, EPM/Staff Scientist, and IODP Curator (on shore) and curatorial rep resentative (on board ship) will work with the entire scientific party to formulate a formal expedition-specific sampling plan for shipboard and postcruise sampling. Shipboard scientists are expected to submit research proposals and associated sample requests (web.iodp.tamu.edu/sdrm/) ~3–6 months before the beginning of the ex pedition (exact timeline to be announced by the EPM/Staff Scientist). Based on the shore-based and shipboard sample requests submitted by this deadline, the SAC will prepare a tentative expedition sampling plan, which will be subject to modification depending upon the actual material recovered and collaborations that may evolve be tween scientists during the expedition. Modification of the sampling strategy during the expedition must be approved by the SAC. The archive section half will not be sampled, except for a limited number of whole rounds if these are required to meet primary project objectives. Shipboard samples will be taken from the working section half, sparing the equivalent of one half of the section half (a quarter round) in all intervals if possible. All sample frequencies and sizes must be justified on a scientific basis and approval will depend to some degree on core recovery, the full spectrum of other requests, and the cruise objectives. Some redundancy of measurement is unavoidable or even desirable, but minimizing the du plication of measurements among the shipboard party and identified shore-based col laborators will be a factor in evaluating sample requests. If critical intervals with particularly high sampling demand are recovered, special de cisions may be required, including reduced sample size, collaborations and sharing of samples and/or measurements results. A sampling plan coordinated by the SAC is typ ically required before critical intervals are sampled. The actual sampling of critical in tervals may be postponed and carried out in the shore repository after the cruise. 20 Expedition 360 Scientific Prospectus References In Von Her zen, R.P., Robinson, P.T., et al., Proceedings of the Ocean Drilling Program, Scientific Results, 118: College Station, TX (Ocean Drilling Program), 415–429. http://dx.doi.org/ 10.2973/odp.proc.sr.118.157.1991 Cannat, M., Sauter, D., Mendel, V., Ruellan, E., Okino, K., Escartin, J., Combier, V., and Baala, M., 2006. Modes of seafloor generation at a melt-poor ultraslow-spreading ridge. Geology, 34(7):605–608. http://dx.doi.org/10.1130/G22486.1 Coogan, L.A., 2014. The lower oceanic crust. In Rudnick, R.L. (Ed.), Treatise on Geochemistry (2nd ed.) (Vol. 4): The Crust. Holland, H.D., and Turekian, K.K. (Series Eds.): New York (Elsevier), 497–541. http://dx.doi.org/10.1016/B978-0-08-095975-7.00316-8 21 Expedition 360 Scientific Prospectus Dick, H.J.B., Arai, S., Hirth, G., John, B.J., and KROO-06 Scientific Party, 2001. A subhorizon tal cross-section through the crust mantle boundary at the SW Indian Ridge. Geophysical Research Abstracts, 3. Dick, H.J.B., MacLeod, C.J., Robinson, P.T., Allerton, S., and Tivey, M.A., 1999. Bathymetry of Atlantis Bank—Atlantis II Fracture Zone: Southwest Indian Ridge. In Dick, H.J.B., Nat land, J.H., Miller, D.J., et al., Proceedings of the Ocean Drilling Program, Initial Reports, 176: College Station, TX (Ocean Drilling Program), 1–13. http://dx.doi.org/10.2973/ odp.proc.ir.176.104.1999 Dick, H.J.B., Meyer, P.S., Bloomer, S., Kirby, S., Stakes, D., and Mawer, C., 1991a. Lithostrati graphic evolution of an in-situ section of oceanic Layer 3. In Von Herzen, R.P., Robinson, P.T., et al., Proceedings of the Ocean Drilling Program, Scientific Results, 118: College Station, TX (Ocean Drilling Program), 439–538. http://dx.doi.org/10.2973/ odp.proc.sr.118.128.1991 Dick, H.J.B., Natland, J.H., Alt, J.C., Bach, W., Bideau, D., Gee, J.S., Haggas, S., Hertogen, J.G.H., Hirth, G., Holm, P.M., Ildefonse, B., Iturrino, G.J., John, B.E., Kelley, D.S., Kikawa, E., Kingdon, A., LeRoux, P.J., Maeda, J., Meyer, P.S., Miller, D.J., Naslund, H.R., Niu, Y.-L., Robinson, P.T., Snow, J., Stephen, R.A., Trimby, P.W., Worm, H.-U., and Yoshinobu, A., 2000. A long in situ section of the lower ocean crust: results of ODP Leg 176 drilling at the Southwest Indian Ridge. Earth and Planetary Science Letters, 179(1):31–51. http:// dx.doi.org/10.1016/S0012-821X(00)00102-3 Dick, H.J.B., Natland, J.H., Miller, D.J., et al., 1999. Proceedings of the Ocean Drilling Program, Initial Reports, 176: College Station, TX (Ocean Drilling Program). http://dx.doi.org/ 10.2973/odp.proc.ir.176.1999 Dick, H.J.B., Schouten, H., Meyer, P.S., Gallo, D.G., Bergh, H., Tyce, R., Patriat, P., Johnson, K.T.M., Snow, J., and Fisher, A., 1991b. Tectonic evolution of the Atlantis II Fracture Zone. In Von Herzen, R.P., Robinson, P.T., et al., Proceedings of the Ocean Drilling Program, Scien tific Results, 118: College Station, TX (Ocean Drilling Program), 359–398. References http:// dx.doi.org/10.2973/odp.proc.sr.118.156.1991 Dick, H.J.B., Tivey, M.A., and Tucholke, B.E., 2008. Plutonic foundation of a slow-spreading ridge segment: oceanic core complex at Kane Megamullion, 23°30′N, 45°20′W. Geochemis try, Geophysics, Geosystems, 9(5). http://dx.doi.org/10.1029/2007GC001645 Drouin, M., Godard, M., Ildefonse, B., Bruguier, O., and Garrido, C.J., 2009. Geochemical and petrographic evidence for magmatic impregnation in the oceanic lithosphere at Atlantis Massif, Mid-Atlantic Ridge (IODP Hole U1309D, 30°N). Chemical Geology, 264(1–4):71–88. http://dx.doi.org/10.1016/j.chemgeo.2009.02.013 Engel, C.G., and Fisher, R.L., 1975. Granitic to ultramafic rock complexes of the Indian Ocean Ridge system, western Indian Ocean. Geological Society of America Bulletin, 86(11):1553– 1578. http://dx.doi.org/10.1130/0016-7606(1975)86<1553:GTURCO>2.0.CO;2 Escartín, J., and Canales, J.-P., 2011. Detachments in oceanic lithosphere: deformation, mag matism, fluid flow, and ecosystems. Eos, Transactions of the American Geophysical Union, 92(4):31. http://dx.doi.org/10.1029/2011EO040003 Escartín, J., Mével, C., MacLeod, C.J., and McCaig, A.M., 2003. Constraints on deformation conditions and the origin of oceanic detachments: the Mid-Atlantic Ridge core complex at 15°45′N. Geochemistry, Geophysics, Geosystems, 4(8):1067. http://dx.doi.org/10.1029/ 2002GC000472 22 Expedition 360 Scientific Prospectus Escartín, J., Smith, D.K., Cann, J., Schouten, H., Langmuir, C.H., and Escrig, S., 2008. Central role of detachment faults in accretion of slow-spreading oceanic lithosphere. Nature, 455(7214):790–794. http://dx.doi.org/10.1038/nature07333 Gillis, K., Mével, C., Allan, J., et al., 1993. Proceedings of the Ocean Drilling Program, Initial Reports, 147: College Station, TX (Ocean Drilling Program). http://dx.doi.org/10.2973/ odp.proc.ir.147.1993 Gillis, K.M., Snow, J.E., Klaus, A., Abe, N., Adrião, Á.B., Akizawa, N., Ceuleneer, G., Cheadle, M.J., Faak, K., Falloon, T.J., Friedman, S.A., Godard, M., Guerin, G., Harigane, Y., Horst, A.J., Hoshide, T., Ildefonse, B., Jean, M.M., John, B.E., Koepke, J., Machi, S., Maeda, J., Marks, N.E., McCaig, A.M., Meyer, R., Morris, A., Nozaka, T., Python, M., Saha, A., and Wintsch, R.P., 2014. Primitive layered gabbros from fast-spreading lower oceanic crust. Nature, 505(7482):204–207. http://dx.doi.org/10.1038/nature12778 Hosford, A., Tivey, M., Matsumoto, T., Dick, H., Schouten, H., and Kinoshita, H., 2003. Crustal magnetization and accretion at the Southwest Indian Ridge near the Atlantis II Fracture Zone, 0–25 Ma. Journal of Geophysical Research: Solid Earth, 108(B3):2169. http:// dx.doi.org/10.1029/2001JB000604 Ildefonse, B., Blackman, D.K., John, B.E., Ohara, Y., Miller, D.J., MacLeod, C.J., and Integrated Ocean Drilling Program Expeditions 304/305 Science Party, 2007. Oceanic core com plexes and crustal accretion at slow-spreading ridges. Geology, 35(7):623–626. http:// dx.doi.org/10.1130/G23531A.1 Kikawa, E., and Pariso, J.E., 1991. Magnetic properties of gabbros from Hole 735B, Southwest Indian Ridge. References In Von Herzen, R.P., Robinson, P.T., et al., Proceedings of the Ocean Drilling Program, Scientific Results, 118: College Station, TX (Ocean Drilling Program), 285–307. http://dx.doi.org/10.2973/odp.proc.sr.118.148.1991 Kinoshita, J., Dick, H., and Shipboard Party JAMSTEC/WHOI, 1999. Deep sea diving expedi tion in SW Indian Ridge (paper presented in honor of Don Heinrichs for his dedication in Marine Geosciences of NSF), Eos, Transactions of the American Geophysical Union, 80(46):F526. Lavier, L., Buck, W.R., and Poliakov, A.N.B., 1999. Self-consistent rolling-hinge model for the evolution of large-offset low-angle normal faults. Geology, 27(12):1127–1130. http:// dx.doi.org/10.1130/0091-7613(1999)027<1127:SCRHMF>2.3.CO;2 Lissenberg, C.J., MacLeod, C.J., Howard, K.A., and Godard, M., 2013. Pervasive reactive melt migration through fast-spreading lower oceanic crust (Hess Deep, equatorial Pacific Ocean). Earth and Planetary Science Letters, 361:436–447. http://dx.doi.org/10.1016/ j.epsl.2012.11.012 MacLeod, C.J., Dick, H.J.B., Allerton, S., Robinson, P.T., Coogan, L.A., Edwards, S.J., Galley, A., Gillis, K.M., Hirth, G., Hunter, A.G., Hutchinson, D., Kvassnes, A.J., Natland, J.H., Salis bury, M., Schandl, E.S., Stakes, D.S., Thompson, G.M., and Tivey, M.A., 1998. Geological mapping of slow-spread lower ocean crust: a deep-towed video and wireline rock drilling survey of Atlantis Bank (ODP Site 735, SW Indian Ridge). InterRidge News, 7(2):39–43. MacLeod, C.J., Escartin, J., Banerji, D., Banks, G.J., Gleeson, M., Irving, D.H.B., Lilly, R.M., McCaig, A.M., Niu, Y., Allerton, S., and Smith, D.K., 2002. Direct geological evidence for oceanic detachment faulting: the Mid-Atlantic Ridge, 15°45′N. Geology, 30(10):879–882. http://dx.doi.org/10.1130/0091-7613(2002)030<0879:DGEFOD>2.0.CO;2 MacLeod, C.J., Wright, V.P., Perry, C.T., and Dick, H.J., 2000. Tectonic evolution and uplift/ subsidence history of Atlantis Bank, a transverse ridge near the Atlantis II Fracture Zone, SW Indian Ridge. Eos, Transactions of the American Geophysical Union, 81:1129. http:// abstractsearch.agu.org/meetings/2000/FM/sections/T/sessions/T62B/abstracts/ T62B-08.html 23 Expedition 360 Scientific Prospectus Matsumoto, T., Dick, H.J.B., and Scientific Party, 2002. Investigation of Atlantis Bank and the SW Indian Ridge from 568E to 588E: Preliminary Report: Tokyo (Japanese Agency for Marine-Earth Science and Technology). Miller, D.J., and Christensen, N.I., 1997. Seismic velocities of lower crustal and upper mantle rocks from the slow-spreading Mid-Atlantic Ridge, south of the Kane Transform Zone (MARK). In Karson, J.A., Cannat, M., Miller, D.J., and Elthon, D. (Eds.), Proceedings of the Ocean Drilling Program, Scientific Results, 153: College Station, TX (Ocean Drilling Pro gram), 437–454. http://dx.doi.org/10.2973/odp.proc.sr.153.043.1997 Minshull, T.A., Muller, M.R., Robinson, C.J., White, R.S., and Bickle, M.J., 1998. Is the oceanic Moho a serpentinization front? In Mills, R.A., and Harrison, K. (Eds.), Modern Ocean Floor Processes and the Geological Record. Geological Society Special Publications, 148(1):71–80. References http://dx.doi.org/10.1144/GSL.SP.1998.148.01.05 Miranda, E.A., and John, B.E., 2010. Strain localization along the Atlantis Bank oceanic detachment fault system, Southwest Indian Ridge. Geochemistry, Geophysics, Geosystems, 11(4):Q04002. http://dx.doi.org/10.1029/2009GC002646 Muller, M.R., Minshull, T.A., and White, R.S., 2000. Crustal structure of the Southwest Indian Ridge at the Atlantis II Fracture Zone. Journal of Geophysical Research: Solid Earth, 105(B11):25809–25828. http://dx.doi.org/10.1029/2000JB900262 Muller, M.R., Robinson, C.J., Minshull, T.A., White, R.S., and Bickle, M.J., 1997. Thin crust beneath Ocean Drilling Program Borehole 735B at the Southwest Indian Ridge? Earth and Planetary Science Letters, 148(1–2):93–107. http://dx.doi.org/10.1016/ S0012-821X(97)00030-7 Natland, J.H., and Dick, H.J.B., 2001. Formation of the lower ocean crust and the crystalliza tion of gabbroic cumulates at a very slowly spreading ridge. Journal of Volcanology and Geothermal Research, 110(3–4):191–233. http://dx.doi.org/10.1016/S0377- 0273(01)00211-6 Natland, J.H., and Dick, H.J.B., 2002. Stratigraphy and composition of gabbros drilled in Ocean Drilling Program Hole 735B, Southwest Indian Ridge: a synthesis of geochemical data. In Natland, J.H., Dick, H.J.B., Miller, D.J., and Von Herzen, R.P. (Eds.), Proceedings of the Ocean Drilling Program, Scientific Results, 176: College Station, TX (Ocean Drilling Pro gram), 1–69. http://dx.doi.org/10.2973/odp.proc.sr.176.002.2002 Palmiotto, C., Corda, L., Ligi, M., Cipriani, A., Dick, H.J.B., Douville, E., Gasperini, L., Mon tagna, P., Thil, F., Borsetti, A.M., Balestra, B., and Bonatti, E., 2013. Nonvolcanic tectonic islands in ancient and modern oceans. Geochemistry, Geophysics, Geosystems, 14(10):4698– 4717. http://dx.doi.org/10.1002/ggge.20279 Pettigrew, T.L., Casey, J.F., Miller, D.J., et al., 1999. Proceedings of the Ocean Drilling Program, Initial Reports, 179: College Station, TX (Ocean Drilling Program). http://dx.doi.org/ 10.2973/odp.proc.ir.179.1999 Robinson, P.T., Von Herzen, R., et al., 1989. Proceedings of the Ocean Drilling Program, Initial Reports, 118: College Station, TX (Ocean Drilling Program). http://dx.doi.org/10.2973/ odp.proc.ir.118.1989 Sauter, D., Cannat, M., Rouméjon, S., Andreani, M., Birot, D., Bronner, A., Brunelli, D., Car lut, J., Delacour, A., Guyader, V., MacLeod, C.J., Manatschal, G., Mendel, V., Ménez, B., Pasini, V., Ruellan, E., and Searle, R., 2013. Continuous exhumation of mantle-derived rocks at the Southwest Indian Ridge for 11 million years. Nature Geoscience, 6(4):314–320. http://dx.doi.org/10.1038/ngeo1771 24 Expedition 360 Scientific Prospectus Schroeder, T., and John, B.E., 2004. Strain localization on an oceanic detachment fault sys tem, Atlantis Massif, 30°N, Mid-Atlantic Ridge. Geochemistry, Geophysics, Geosystems, 5:Q11007. http://dx.doi.org/10.1029/2004GC000728 Schroeder, T., and John, B.E., 2004. Strain localization on an oceanic detachment fault sys tem, Atlantis Massif, 30°N, Mid-Atlantic Ridge. Geochemistry, Geophysics, Geosystems, 5:Q11007. http://dx.doi.org/10.1029/2004GC000728 Sinton, J.M., and Detrick, R.S., 1992. Mid-ocean ridge magma chambers. Journal of Geophysi cal Research: Solid Earth, 97(B1):197–216. Schroeder, T., and John, B.E., 2004. Strain localization on an oceanic detachment fault sys tem, Atlantis Massif, 30°N, Mid-Atlantic Ridge. Geochemistry, Geophysics, Geosystems, 5:Q11007. http://dx.doi.org/10.1029/2004GC000728 References http://dx.doi.org/10.1029/91JB02508 Sinton, J.M., and Detrick, R.S., 1992. Mid-ocean ridge magma chambers. Journal of Geophysi cal Research: Solid Earth, 97(B1):197–216. http://dx.doi.org/10.1029/91JB02508 Tucholke, B.E., and Lin, J., 1994. A geological model for the structure of ridge segments in slow spreading ocean crust. Journal of Geophysical Research: Solid Earth, 99(B6):11937– 11958. http://dx.doi.org/10.1029/94JB00338 Tucholke, B.E., and Lin, J., 1994. A geological model for the structure of ridge segments in slow spreading ocean crust. Journal of Geophysical Research: Solid Earth, 99(B6):11937– 11958. http://dx.doi.org/10.1029/94JB00338 Tucholke, B.E., Lin, J., and Kleinrock, M.C., 1998. Megamullions and mullion structure defin ing oceanic metamorphic core complexes on the Mid-Atlantic Ridge. Journal of Geophysi cal Research: Solid Earth, 103(B5):9857–9866. http://dx.doi.org/10.1029/98JB00167 Tucholke, B.E., Lin, J., and Kleinrock, M.C., 1998. Megamullions and mullion structure defin ing oceanic metamorphic core complexes on the Mid-Atlantic Ridge. Journal of Geophysi cal Research: Solid Earth, 103(B5):9857–9866. http://dx.doi.org/10.1029/98JB00167 25 Expedition 360 Scientific Prospectus Table T1. Operations plan for proposed sites, Expedition 360. RCB = rotary core barrel, UBI = Ultrasonic Borehole Imager, FMS = Formation MicroScanner, MSS = magnetic susceptibility sonde, VSI = Versatile Sonic Imager. Begin Expedition 5.0 10.8 AtBk-6 32° 42.3402' S 706 2.6 EPSP 57° 16.6910' E approved to 5.6 TBD Wireline log pilot hole with Triple Combo w/ UBI, FMS-Sonic 0.8 Open hole to 14" diameter and run 10 3/4" casing string to ~200 mbsf 3.6 23.6 Wireline log pilot hole with Triple Combo w/ UBI, FMS-Sonic, MSS 2.0 4.0 Sub-Total Days On-Site: 42.2 3.0 End Expedition 13.8 39.4 2.8 5.0 42.2 Weather Contingency and cement casing. RCB core from ~220 mbsf to ~1300 mbsf & VSI Tool Strings from 200 mbsf to ~1300 mbsf. Hole A: Drill in reentry cone with ~15 m of 13 3/8" 54.5 lb/ft conductor casing and cement reentry system in place. Drill out cement and RCB core to ~220 mbsf and MSS Tool Strings from 30 mbsf to ~220 mbsf. Transit 2710 nmi from Colombo to Site AtBk-1a @ 10.5 knots Exp-360 Indian Ridge Lower Crust and Moho (Pro800) Operations Plan Summary S. Midgley, 16 October 2014 Site No. Location (Latitude Longitude) Seafloor Depth (mbrf) Operations Description Transit (days) Drilling Coring (days) WL Log (days)2 Colombo, Sri Lanka Port Call Days 56.0 Sub-Total On-Site: Total Expedition: 61.0 Transit 762 nmi from AtBk-1a to Port Louis @ 10.5 knots Port Louis, Mauritius Port Call: Total Operating Days: Expedition 360 Scientific Prospectus Expedition 360 Scientific Prospectus Table T1. Figure F1. 3-D perspective view of the Atlantis II Transform, looking north-northeast. Data com- piled from multibeam data collected during Conrad Cruise C2709, James Clark Ross Cruise JR31, and Yokosuka and Kairei site survey cruises, combined with satellite gravity seafloor data and the Global Multi Resolution Bathymetry database. References Operations plan for proposed sites, Expedition 360. RCB = rotary core barrel, UBI = Ultrasonic Borehole Imager, FMS = Formation MicroScanner, MSS = magnetic susceptibility sonde, VSI = Versatile Sonic Imager. Begin Expedition 5.0 10.8 AtBk-6 32° 42.3402' S 706 2.6 EPSP 57° 16.6910' E approved to 5.6 TBD Wireline log pilot hole with Triple Combo w/ UBI, FMS-Sonic 0.8 Open hole to 14" diameter and run 10 3/4" casing string to ~200 mbsf 3.6 23.6 Wireline log pilot hole with Triple Combo w/ UBI, FMS-Sonic, MSS 2.0 4.0 Sub-Total Days On-Site: 42.2 3.0 End Expedition 13.8 39.4 2.8 5.0 42.2 Weather Contingency and cement casing. RCB core from ~220 mbsf to ~1300 mbsf & VSI Tool Strings from 200 mbsf to ~1300 mbsf. Hole A: Drill in reentry cone with ~15 m of 13 3/8" 54.5 lb/ft conductor casing and cement reentry system in place. Drill out cement and RCB core to ~220 mbsf and MSS Tool Strings from 30 mbsf to ~220 mbsf. Transit 2710 nmi from Colombo to Site AtBk-1a @ 10.5 knots Exp-360 Indian Ridge Lower Crust and Moho (Pro800) Operations Plan Summary S. Midgley, 16 October 2014 Site No. Location (Latitude Longitude) Seafloor Depth (mbrf) Operations Description Transit (days) Drilling Coring (days) WL Log (days)2 Colombo, Sri Lanka Port Call Days 56.0 Sub-Total On-Site: Total Expedition: 61.0 Transit 762 nmi from AtBk-1a to Port Louis @ 10.5 knots Port Louis, Mauritius Port Call: Total Operating Days: able T1. Operations plan for proposed sites, Expedition 360. RCB = rotary core barrel, UBI = Ultrasonic Borehole Imager, FMS = Formation MicroScanner, MSS = magnetic susceptibility sonde, VSI = Versatile Sonic Imager. RCB = rotary core barrel, UBI = Ultrasonic Borehole Imager, FMS = Formation MicroScanner, MSS = magnetic susceptibility sonde, VSI = Versatile Sonic Imager. 26 Expedition 360 Scientific Prospectus Figure F1. 3-D perspective view of the Atlantis II Transform, looking north-northeast. Data com- piled from multibeam data collected during Conrad Cruise C2709, James Clark Ross Cruise JR31, and Yokosuka and Kairei site survey cruises, combined with satellite gravity seafloor data and the Global Multi Resolution Bathymetry database. u t eso ut o at y et y database. AN-1 AN-2 Novara fault zone Atlantis II Transform Atlantis Bank 33°S, 57°E 32°S, 57°E 34°S, 57°E 32°S, 58°E 33°S, 58°E N 27 27 Expedition 360 Scientific Prospectus Figure F2. A. References Bathymetry of the Atlantis Bank area, with past and proposed drill sites marked. B. Summary of geological mapping of the Atlantis Bank area, with geological interpretation. Figure F2. A. Bathymetry of the Atlantis Bank area, with past and proposed drill sites marked. B. Summary of geological mapping of the Atlantis Bank area, with geological interpretation. -7500 -6750 -6000 -5250 -4500 -3750 -3000 -2250 -1500 -750 0 Depth (m) 655 652 652 650 646 466 467 458 459 648 462 461 460 644 651 653 647 645 173 174 643 649 1105A 735B 172 AtBk6 21’E 57°10’ 57°20’ 34’ 19’ 29’ 22’ 53’ 37’ 0 5 10 km ? ? ? ? ? ? ? ? ? Core complex margin Normal fault Detachment fault ROV/DSRV track Pillow basalt Dikes/Gabbro Gabbro Metaperidotite Peridotite Dikes/Peridotite Dredge haul Diabase Talc-serpentine schist patch Sediment/ clueless Slump deposit 57°10’E 32°40’ 32°50’ 32°30’ S 57°20’ 735B AtBk6 1105A AtBk5 AtBk2 AtBk4 B A -7500 -6750 -6000 -5250 -4500 -3750 -3000 -2250 -1500 -750 0 57°10’E 32°40’ 32°50’ 32°30’ S 57°20’ 735B AtBk6 1105A AtBk5 AtBk2 AtBk4 A B 655 652 652 650 646 466 467 458 459 648 462 461 460 644 651 653 647 645 173 174 643 649 1105A 735B 172 AtBk6 21’E 57°10’ 57°20’ 34’ 19’ 29’ 22’ 53’ 37’ 0 5 10 km ? ? ? ? ? ? ? ? ? Core complex margin Normal fault Detachment fault ROV/DSRV track Pillow basalt Dikes/Gabbro Gabbro Metaperidotite Peridotite Dikes/Peridotite Dredge haul Diabase Talc-serpentine schist patch Sediment/ clueless Slump deposit B 655 652 652 650 646 466 467 458 459 648 462 461 460 644 651 653 647 645 173 174 643 649 1105A 735B 172 AtBk6 21’E 57°10’ 57°20’ 34’ 19’ 29’ 22’ 53’ 37’ 0 5 10 km ? ? ? ? ? ? ? ? ? Core complex margin Normal fault Detachment fault ROV/DSRV track Pillow basalt Dikes/Gabbro Gabbro Metaperidotite Peridotite Dikes/Peridotite Dredge haul Diabase Talc-serpentine schist patch Sediment/ clueless Slump deposit B Depth (m) 28 Expedition 360 Scientific Prospectus Figure F3. High-resolution contoured narrow-beam bathymetry map of Atlantis Bank wave-cut plat- form, showing remotely operated vehicle (ROV) and submersible dive tracks plus locations of shal- low and deep drill cores. Primary proposed drill site is AtBk6; contingency Sites AtBk4 and AtBk5 are also marked. also marked. Figure F4. Bottom photo of primary deep drill proposed Site AtBk6, showing flat bare-rock pave- ment (with echinoid) and thin carbonate in distance. The leg of the BRIDGE seabed rock drill is in the foreground; drilling here (Site JR31-BR-8) yielded olivine gabbro with mylonitic shear bands. Field of view is ~4–5 m. References 57°17’ 57°18’ 32°40’ S 32°44’ 32°45’ 57°15’E 57°16’ 32°43’ 32°42’ 32°41’ 16 53 24 35 20 34 17 735B 1105A AtBk6 AtBk5 AtBk4 JR31 dredges 53 Shinkai 6500 462 461 Limestone Pillow basalt Diabase Gabbro Serpentine-talc JR31 drill cores Hydrothermal Last cap on gabbro ODP/IODP drill sites 430 435 437 438 425 434 426 429 433 436 Off-bottom N Atlantis Bank Cruise JR31 narrow-beam bathymetry Contour interval: 5 m Previous Proposed 1 km -1280 -1260 -1240 -1220 -1200 -1180 -1160 -1140 -1120 -1100 -1080 -1060 -1040 -1020 -1000 -980 -960 -940 -920 -900 -880 -860 -840 -820 -800 -780 -760 -740 -720 -700 -680 -660 -640 meters ROPOS ROV 24 29 Expedition 360 Scientific Prospectus Figure F4. Bottom photo of primary deep drill proposed Site AtBk6, showing flat bare-rock pave- ment (with echinoid) and thin carbonate in distance. The leg of the BRIDGE seabed rock drill is in the foreground; drilling here (Site JR31-BR-8) yielded olivine gabbro with mylonitic shear bands. Field of view is ~4–5 m. Figure F4. Bottom photo of primary deep drill proposed Site AtBk6, showing flat bare-rock pave- ment (with echinoid) and thin carbonate in distance. The leg of the BRIDGE seabed rock drill is in the foreground; drilling here (Site JR31-BR-8) yielded olivine gabbro with mylonitic shear bands. Field of view is ~4–5 m. 30 Expedition 360 Scientific Prospectus Figure F5. Velocity models for north–south seismic Line CAM101 over Atlantis Bank, together with principal proposed drill sites, modified after Muller et al. (2000). Green line = target depth of pro- posed Site AtBk6 drilled during Expedition 360, orange line = projected penetration during SloMo- Leg 2, red line = projected penetration during SloMo-Leg 2 (riser drilling). Velocity contours are 0.4 km/s. Upper panel shows an undifferentiated Layer 3; lower panel shows a lower serpentinized mantle Layer 3 and an upper gabbroic Layer 3. The models fit the data equally well. Triangles mark ocean-bottom hydrophones. Proposed Site AtBk6 lies on the line, and Hole 735B is projected from a kilometer to the west. Moho gaps are due to ocean bottom seismometer placement and are not real. Figure F5. Velocity models for north–south seismic Line CAM101 over Atlantis Bank, together with principal proposed drill sites, modified after Muller et al. (2000). References Green line = target depth of pro- posed Site AtBk6 drilled during Expedition 360, orange line = projected penetration during SloMo- Leg 2, red line = projected penetration during SloMo-Leg 2 (riser drilling). Velocity contours are 0.4 km/s. Upper panel shows an undifferentiated Layer 3; lower panel shows a lower serpentinized mantle Layer 3 and an upper gabbroic Layer 3. The models fit the data equally well. Triangles mark ocean-bottom hydrophones. Proposed Site AtBk6 lies on the line, and Hole 735B is projected from a kilometer to the west. Moho gaps are due to ocean bottom seismometer placement and are not real. 0 15 14 13 12 11 10 9 8 7 2 4 6 8 0 0 20 40 60 2 4 6 8 Depth (km) Distance (km) Approximate Age (Ma) Layer 2 Layer 3 Layer 2 Layer 3 Upper Lower South North Volcanic Ridge Normal Faults 6 6 6.8 6.8 7.8 7.8 Mo ho R e fle ctor Moho 735B AtBk6 0 15 14 13 12 11 10 9 8 7 2 4 6 8 0 0 20 40 60 2 4 6 8 Depth (km) Distance (km) Approximate Age (Ma) Layer 2 Layer 3 Layer 2 Layer 3 Upper Lower South North Volcanic Ridge Normal Faults 6 6 6.8 6.8 7.8 7.8 Mo ho R e fle ctor Moho 735B AtBk6 Depth (km) 31 Expedition 360 Scientific Prospectus Figure F6. Lithostratigraphy of ODP Hole 735B and comparison with IODP Hole U1309D from the Mid-Atlantic Ridge at 30°N, modified from Dick et al. (2000) and Blackman et al. (2006, 2011). Lith- ologies are running 20 m downhole averages of the rock proportions, with those for Hole 735B nor- malized to 100% recovery, whereas those for Hole U1309D are shown as a proportion of the intervals drilled. Different symbols for the points in the whole rock Mg# diagram are for different lithologies based on modal mineralogy and can be found in the ODP Legs 118 and 176 Initial Reports volumes and IODP Expedition 304/305 reports (Robinson, Von Herzen, et al., 1989; Dick, Natland, Miller, et al., 1999; Blackman, Ildefonse, John, Ohara, Miller, MacLeod, and Expedition. 304/305 Scientists, 2006). These sections represent the descriptions of two different scientific parties, and whereas both followed AGI conventions on nomenclature, the exact definitions for each lithology described may vary. References The red line at Mg# 80 is to emphasize the overall chemical differences in the sections. Primary melts emerging from the mantle would be in equilibrium with gabbroic cumulates and dunites with an Mg# ≥90, which are largely missing in both sections. and dunites with an Mg# ≥90, which are largely missing in both sections. 30 50 70 90 Depth (mbsf) 0 200 400 600 800 1000 1200 1400 Oxide gabbro Gabbro, gabbronorite Basalt/Diabase Olivine gabbro, troctolitic gabbro Olivine-rich troctolite, dunite, wehrlite, harzburgite Troctolite Troctolitic gabbro Olivine gabbro Gabbro, gabbronorite Disseminated oxide gabbro Oxide gabbro 0 100% Hole 735B, Atlantis Bank, Southwest Indian Ridge Hole U1309D, Atlantis Massif, Mid-Atlantic Ridge No Hole 5 0 Whole-rock Mg/(Mg + Fe) Lithology Plastic deformation Whole-rock Mg/(Mg + Fe) Lithology Plastic deformation 30 50 70 90 0 100% 5 0 30 50 70 90 Depth (mbsf) 0 200 400 600 800 1000 1200 1400 Oxide gabbro Gabbro, gabbronorite Basalt/Diabase Olivine gabbro, troctolitic gabbro Olivine-rich troctolite, dunite, wehrlite, harzburgite Troctolite Troctolitic gabbro Olivine gabbro Gabbro, gabbronorite Disseminated oxide gabbro Oxide gabbro 0 100% Hole 735B, Atlantis Bank, Southwest Indian Ridge Hole U1309D, Atlantis Massif, Mid-Atlantic Ridge No Hole 5 0 Whole-rock Mg/(Mg + Fe) Lithology Plastic deformation Whole-rock Mg/(Mg + Fe) Lithology Plastic deformation 30 50 70 90 0 100% 5 0 Hole 735B, Atlantis Bank, Southwest Indian Ridge Hole U1309D, Atlantis Massif, Mid-Atlantic Ridge Depth (mbsf) 32 Expedition 360 Scientific Prospectus Figure F7. A. Location and magnetization of Atlantis Bank platform drill cores. B. Contoured deep- towed magnetization. C. References Magnetic anomaly models with positions of Hole 735B and proposed Site AtBk6 700 800 800 800 900 900 900 1000 1000 1000 1100 1100 1100 1200 1200 1200 1300 1300 1400 735B 1105A AtBk6 Drill Cores BR reversed BGS reversed BR normal BGS normal 32°40’S 32°44’ 32°42’ 57°14’E 57°16’ 57°18’ 57°16’E 57°18’ 1105A AtBk6 735B −5 0 5 10 C5n.2n C5r.1n C5r.2n C5An Magnetization (A/m) −500 0 500 C5n.2n C5r.1n C5r.2n C5An Magnetic field (nT) C5n.2n C5r.2n C5An North South 735B AtBk6 C5r.1n 38 40 41 42 43 44 45 46 47 48 49 −2 −1.5 −1 −0.5 0 Latitude 32°S (minutes) Depth (km) Sea-surface magnetization Near-bottom magnetic field Bathymetry C A B 39 57°16’E 57°18 1105A AtBk6 735B B B −5 0 5 10 C5n.2n C5r.1n C5r.2n C5An Magnetization (A/m) −500 0 500 C5n.2n C5r.1n C5r.2n C5An Magnetic field (nT) C5n.2n C5r.2n C5An North South 735B AtBk6 C5r.1n 38 40 41 42 43 44 45 46 47 48 49 −2 −1.5 −1 −0.5 0 Latitude 32°S (minutes) Depth (km) Sea-surface magnetization Near-bottom magnetic field Bathymetry C 39 0C A 700 800 800 800 900 900 900 1000 1000 1000 1100 1100 1100 1200 1200 1200 1300 1300 1400 735B 1105A AtBk6 Drill Cores BR reversed BGS reversed BR normal BGS normal 32°40 32°44 32°42 57°14’E 57°16’ 57°18’ A Magnetization (A/m) 33 Expedition 360 Scientific Prospectus Site summaries Site AtBk6 Site AtBk4 Priority: 1 Position: 32°42.3402’S; 57°16.6910’E North-central part of Atlantis Bank wave-cut platform Water depth (m): 695 Target drilling depth (mbsf): As deep as possible (>3 km) Approved maximum penetration (mbsf): To be agreed; but >3 km Survey coverage (track map, seismic profile): Seabed drill core Site JR31-BR-8 Objective(s): Drill deep section through gabbro to crust–mantle transition Drilling program: Re-entry system: Plan A: 15-m long 13.375-inch drill-in casing Plan B conventional 15-m long 16-inch casing. RCB core to 220 mbsf; set 200- m long 10.75 inch casing (after logging). RCB core as deep as time permits. Log. Logging/downhole measurements program: Three runs with triple combo, FMS-sonic, and UBI before setting 200 m long 10.75 inch casing; same three runs plus VSI from 200 mbsf to maximum depth achieved. References Nature of rock anticipated: Gabbro, possible rare diabase; ultimately serpentinized and thence fresh peridotite Priority: 2 (weather contingency) Position: 32°44.6643’S; 57°17.0384’E Southern end of Atlantis Bank wave-cut platform Water depth (m): 839 Target drilling depth (mbsf): ~150 (single bit) Approved maximum penetration (mbsf): To be agreed Survey coverage (track map; seismic profile): Seabed drill core Site JR31-BGS-12 Objective(s): Drill short section of serpentinized peridotite Drilling program: Single-bit bare-rock spud-in Logging/downhole measurements program: None Nature of rock anticipated: Serpentinized peridotite Expedition 360 Scientific Prospectus Expedition 360 Scientific Prospectus Site summaries Site AtBk6 Priority: 1 Position: 32°42.3402’S; 57°16.6910’E North-central part of Atlantis Bank wave-cut platform Water depth (m): 695 Target drilling depth (mbsf): As deep as possible (>3 km) Approved maximum penetration (mbsf): To be agreed; but >3 km Survey coverage (track map, seismic profile): Seabed drill core Site JR31-BR-8 Objective(s): Drill deep section through gabbro to crust–mantle transition Drilling program: Re-entry system: Plan A: 15-m long 13.375-inch drill-in casing Plan B conventional 15-m long 16-inch casing. RCB core to 220 mbsf; set 200- m long 10.75 inch casing (after logging). RCB core as deep as time permits. Log. Logging/downhole measurements program: Three runs with triple combo, FMS-sonic, and UBI before setting 200 m long 10.75 inch casing; same three runs plus VSI from 200 mbsf to maximum depth achieved. References Nature of rock anticipated: Gabbro, possible rare diabase; ultimately serpentinized and thence fresh peridotite Site summaries Site AtBk4 Priority: 2 (weather contingency) Position: 32°44.6643’S; 57°17.0384’E Southern end of Atlantis Bank wave-cut platform Water depth (m): 839 Target drilling depth (mbsf): ~150 (single bit) Approved maximum penetration (mbsf): To be agreed Survey coverage (track map; seismic profile): Seabed drill core Site JR31-BGS-12 Objective(s): Drill short section of serpentinized peridotite Drilling program: Single-bit bare-rock spud-in Logging/downhole measurements program: None Nature of rock anticipated: Serpentinized peridotite 34 Expedition 360 Scientific Prospectus p p Site summaries (continued) Site AtBk5 Site AtBk2 Priority: 2 (weather contingency) Position: 32°40.8244’S; 57°17.5431’E Northernmost part of Atlantis Bank wave-cut platform Water depth (m): 709 Target drilling depth (mbsf): ~150 (single bit) Approved maximum penetration (mbsf): To be agreed Survey coverage (track map; seismic profile): Seabed drill core Site JR31-BGS-20 Objective(s): Drill short section of gabbro Drilling program: Single-bit bare-rock spud-in Logging/downhole measurements program: None Nature of rock anticipated: Gabbro Priority: 2 (weather contingency) Position: 32°41.00’S; 57°20.35’E Water depth (m): 1700 Target drilling depth (mbsf): ~150 (single bit) Approved maximum penetration (mbsf): To be agreed Survey coverage (track map; seismic profile): Objective(s): Core the dike–gabbro transition Drilling program: Single-bit bare-rock spud-in Logging/downhole measurements program: None Nature of rock anticipated: Gabbro, diabase p p Site summaries (continued) Site AtBk5 Priority: 2 (weather contingency) Position: 32°40.8244’S; 57°17.5431’E Northernmost part of Atlantis Bank wave-cut platform Water depth (m): 709 Target drilling depth (mbsf): ~150 (single bit) Approved maximum penetration (mbsf): To be agreed Survey coverage (track map; seismic profile): Seabed drill core Site JR31-BGS-20 Objective(s): Drill short section of gabbro Drilling program: Single-bit bare-rock spud-in Logging/downhole measurements program: None Nature of rock anticipated: Gabbro Site summaries (continued) Site AtBk2 Priority: 2 (weather contingency) Position: 32°41.00’S; 57°20.35’E Water depth (m): 1700 Target drilling depth (mbsf): ~150 (single bit) Approved maximum penetration (mbsf): To be agreed Survey coverage (track map; seismic profile): Objective(s): Core the dike–gabbro transition Drilling program: Single-bit bare-rock spud-in Logging/downhole measurements program: None Nature of rock anticipated: Gabbro, diabase 35 Expedition 360 Scientific Prospectus Expedition scientists and scientific participants Expedition scientists and scientific participants The current list of participants for Expedition 360 can be found at iodp.tamu.edu/science- ops/precruise/indianridge/participants.html. 36
https://openalex.org/W2053906245	https://scielo.isciii.es/pdf/azoo/v58n221/art5.pdf	Portuguese	null	Influência da queima e aditivos químicos e bacterianos na composição química de silagens de cana-de-açúcar	Archivos de zootecnia	2,009	cc-by-sa	6,813	Siqueira, G.R.1,2, R.A. Reis2, R.P. Schocken-Iturrino2, T.F. Bernardes2, A.J.V. Pires3, M.T.P. Roth2 e R.C. Amaral2 1Agência Paulista de Tecnologia dos Agronegócios (APTA). Pólo Regional de Desenvolvimento dos Agronegócios da Alta Mogiana. Av. Rui Barbosa, s/n. C.P. 35. CEP 14770-000 Colina-SP. Brasil. siqueiragr@aptaregional.sp.gov.br 2Faculdade de Ciências Agrárias e Veterinárias. UNESP. Jaboticabal-SP. Brasil. 3Universidade Estadual do Sudoeste da Bahia. Itapetinga-BA. Brasil. RESUMO Objetivou-se avaliar o efeito da queima e do tratamento com aditivos químicos (uréia, benzoato de sódio e hidróxido de sódio) e inoculantes microbiológicos (Propionibacterium acidipro- pionici + Lactobacillus plantarum e Lactobacillus buchneri) na ensilagem da cana-de-açúcar, utili- zando o esquema fatorial 2 (cana-de-açúcar crua e queimada) x 6 (cinco aditivos uréia, benzoato de sódio, hidróxido de sódio, Propionibacterium acidipropionici + Lactobacillus plantarum e Lactobacillus buchneri mais o grupo controle). Avaliou-se a composição química da forragem antes de ensilar e após a abertura dos silos. Observou-se concentração dos teores de FDN em todas as silagens, de 51,3% (antes da ensilagem) para 67,8% após a abertura dos silos. Maiores recuperações da matéria seca digestível verdadeira, 83,6 e 79,8% foram observadas nas silagens de cana-de-açúcar queimada e tratadas com NaOH ou com L. buchneri, respectivamente. Observou-se elevação da recuperação da matéria seca digestível de 45% nas silagens de cana crua controle para 74,3% nas silagens tratadas com L. buchneri. Nas silagens de cana queimada as melhores recuperações dessa fração foram ob- servadas nas silagens tratadas com NaOH (83,6%) e nas com L. buchneri (79,8%). Os aditivos NaOH e L. buchneri foram os mais eficientes em contro- lar as perdas qualitativas durante o processo fermentativo da ensilagem da cana-de-açúcar crua ou queimada. Additives. Fermentation. Inoculants. Silage. Additives. Fermentation. Inoculants. Silage. Aditivos. Fermentação. Inoculantes. Silagem. Additives. Fermentation. Inoculants. Silage. Arch. Zootec. 58 (221): 43-54. 2009. Recibido: 4-6-06. Aceptado: 5-11-07. INTRODUÇÃO comparada à silagem controle, não foi veri- ficada diferença estatística em relação às perdas de matéria seca, sendo os resultados encontrados de 31,6 e 30,2% nas silagens controle e tratadas com 0,5% de uréia, res- pectivamente. Na década de 70 vários autores realizaram pesquisas com uso de aditivos químicos na ensilagem da cana-de-açúcar, objetivando melhorar o perfil de fermentação e controlar a população de leveduras (Nussio e Schmidt, 2004). Recentemente, alguns aditivos voltaram a serem utilizados em pesquisas no Brasil, mostrando a retomada do interesse da comunidade científica pela pesquisa com silagem de cana-de-açúcar. Nussio e Schmidt (2004) mencionam que o interesse da pes- quisa brasileira sobre a ensilagem de cana- de-açúcar vem crescendo ano a ano. Na ensilagem da cana-de-açúcar a utilização do hidróxido de sódio foi avaliada por Castrillón et al. (1978), os autores observaram redução no conteúdo de etanol de 5,1 para 0,85% e aumento do ácido lático de 2,1 para 10,2%, quando procederam-se ao tratamento da cana-de-açúcar com 4% de NaOH na matéria seca. A utilização de inoculantes bacterianos na ensilagem sempre foi baseada em microrganismos homoláticos, apresentando como principais características: rápida redução do pH e alta produção de ácido lático (Costa et al., 2001). No entanto, pro- blemas relacionados à estabilidade aeróbia das silagens geraram buscas por micror- ganismos com características distintas daquelas antes desejadas (Filya et al., 2004). Resultados de pesquisa têm apontado que o principal problema na ensilagem da cana-de-açúcar é a fermentação alcoólica realizada por leveduras durante o período de armazenamento, com conseqüente redução do conteúdo de carboidratos solúveis (Bernardes et al., 2007; Pedroso et al., 2007 e Siqueira et al., 2007). Esses mesmos autores afirmam em seus trabalhos a importância da busca por aditivos com potencial para o controle desses micror- ganismos. Neste cenário, a bactéria Lactobacillus buchneri começou a ser utilizada por pesquisadores dos EUA e da Europa a partir do final dos anos 90. O intuito de sua utilização foi o controle da instabilidade aeróbia causada por leveduras e fungos em silagens de alto valor nutritivo (Weinberg e Muck, 1996). Weinberg et al. (2002) avaliando silagens de trigo, observaram elevação no teor de ácido acético, redução no teor de ácido lático, sendo que esses acontecimentos reduziram a população de leveduras de 3,7 (silagem controle) para <2,0 log ufc/g de matéria seca de silagem. Nesse sentido, pesquisa pioneira na utilização do L. SUMMARY The objective of this trial was to evaluate the effect of the chemical (urea, sodium benzoate, and sodium hydroxide) and microbiological (Propionibacterium acidipropionici + Lactoba- cillus plantarum, and Lactobacillus buchneri) additives on the sugarcane nutritive value, ensiled crude or after burned, using a factorial scheme 2 (burned or crude sugar cane) x 6 (five additives urea, sodium benzoate, sodium hydroxide, Propionibacterium acidipropionici + Lactobaci- llus plantarum, and Lactobacillus buchneri plus control). It was evaluated the sugar cane chemical composition, before and after ensilage. The sugar cane NDF contents increased (51.3%, before ensilage) to 67.8% after fermentation period. The highest true digestible dry matter recovery values, 83.6 and 79.8% were observed on the burned sugar cane silage treated with NaOH or L. buchneri, respectively. The NaOH, and L. buchneri showed more efficiency in reducing nutritive looses during the fermentation phase of the crude or burned sugar cane silage. Arch. Zootec. 58 (221): 43-54. 2009. Arch. Zootec. 58 (221): 43-54. 2009. Recibido: 4-6-06. Aceptado: 5-11-07. Archivos de zootecnia vol. 58, núm. 221, p. 44. MATERIAL E MÉTODOS A cana-de-açúcar (Saccharum offici- narum L.) utilizada foi o cultivar SP70-1143, proveniente da Usina Andrade Açúcar e Álcool. A cultura foi adubada com 400 kg/ ha do formulado 20-05-20, após o terceiro corte e o controle de invasoras foi realizado com o herbicida Platô. A colheita manual procedeu-se no mês de Outubro de 2003, quando a cana-de-açúcar apresentava-se apta para o quarto corte, com produção de 80 t MV/ha aos 15 meses de crescimento vegetativo e com 16% de pol. Segundo Weinberg e Muck (1996), o uso de microrganismos alternativos, que são produtores de ácidos orgânicos consi- derados fracos (acético ou propiônico), no que se refere à eficiência em reduzir o pH da massa ensilada, podem ter a ação sobre o metabolismo de leveduras e fungos fila- mentosos (Moon, 1983 e McDonald et al., 1991). A forragem foi cortada e transportada até as dependências da Unesp-Jaboticabal. No dia posterior ao corte parte da cana-de- açúcar foi queimada, e colocada em forma de pilhas. A forragem foi processada em pica- dora estacionária em partículas de 1 a 3 cm. ) As bactérias Propionibacterium acidi- propionici e o Lactobacillus buchneri en- tre outros compostos produzem principal- mente ácido propiônico e ácido acético, respectivamente (Dawson et al., 1998 e Danner et al., 2003). Esses ácidos em pH inferior ao seu pKa (4,87 e 4,73 dos ácidos propiônico e acético, respectivamente), ficam na forma não dissociada, sendo a membrana dos microrganismos permeável a eles, conseqüentemente a entrada do ácido é realizada via transporte passivo. Dentro das células, eles são dissociados (RCOO- e H+) devido ao pH interno do microrganismo ser por volta de 7,0 (superior ao pKa), libe- rando íons H+, conseqüentemente ocorre rápida redução do pH intracelular. Para ele- var novamente o pH, o microrganismo tem que expulsar os íons H+, implicando em gasto de energia, por se tratar de um processo de transporte ativo, retardando o cresci- mento e podendo causar a morte celular (McDonald et al., 1991). Utilizaram-se um esquema fatorial 2 (for- mas: cana-de-açúcar crua e queimada) x 6 aditivos (cinco aditivos uréia, benzoato de sódio, hidróxido de sódio, Propionibac- terium acidipropionici + Lactobacillus plantarum e Lactobacillus buchneri mais o grupo controle) num total de 12 tratamentos com três repetições. QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR produção de ácido propiônico. Filya et al. (2004) avaliaram a inoculação do P. acidipropionici, L. plantarum e a associa- ção dos dois microrganismos na ensilagem de trigo, sorgo e milho. A concentração de ácido propiônico foi superior em todas as silagens inoculadas apenas com P. acidipro- pionici e a população de leveduras foi redu- zida da faixa de 5,5 a 5,8 para <2,0 log ufc/g de silagem e a população de mofos de 4,1 a 5,0 também para <2,0 log ufc/g de silagem. propionici + Lactobacillus plantarum e Lactobacillus buchneri) sobre a composição química na ensilagem da cana-de-açúcar. propionici + Lactobacillus plantarum e Lactobacillus buchneri) sobre a composição química na ensilagem da cana-de-açúcar. INTRODUÇÃO buchneri na ensilagem da cana-de-açúcar foi desenvolvida por Pedroso et al. (2007). Neste estudo o autor observou redução nas perdas por gases e melhores recuperações de matéria seca. A utilização de aditivos químicos ou bacterianos visa o controle de leveduras durante o período fermentativo das silagens. Lättemäe e Lingvall (1996) avaliaram o efeito do benzoato de sódio na fermentação de silagens de gramíneas (azevém perene e festuca) e observaram redução na população de leveduras quando se adicionou benzoato de sódio na ensilagem. O uso da uréia na ensilagem da cana-de- açúcar foi estudado por Schmidt et al. (2004) e Pedroso et al. (2007), não sendo observa- da tendência definida da utilização desse aditivo, quanto à redução de perdas ocasio- nadas por leveduras. Pedroso et al. (2007) observou redução das perdas de matéria seca de 18,2% (controle) e de até 6,56% nas silagens com 1,5% de uréia. No entanto, não houve efeito sobre o teor de etanol das silagens. Todavia, no estudo de Schmidt et al. (2004) usando a dose de 0,5% de uréia Outro grupo de microrganismos que vêm sendo estudado com finalidade semelhante ao L. buchneri é o gênero Propionibac- terium que têm como característica a Archivos de zootecnia vol. 58, núm. 221, p. 44. Archivos de zootecnia vol. 58, núm. 221, p. 45. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL hidróxido de sódio (NaOH) foi utilizado em solução de 33,3%. Imediatamente após a aplicação dos aditivos a cana-de-açúcar foi ensilada. seca foi determinada pelo método de Tilley e Terry (Silva e Queiroz, 2002). Determinaram-se as recuperações dos CNF e da matéria seca digestível verdadeira, pela equação descrita abaixo: Utilizou-se como silos experimentais foram utilizados canos de PVC de 50 cm de altura e 10 cm de diâmetro, com tampas com válvulas de Bunsen para permitir o escape do gás. A ensilagem foi realizada com auxílio de bastões de ferro, objetivando alcançar a densidade de 650 kg de forragem/m3. Foi determinado o volume de cada silo experi- mental e pesou-se a quantidade de forragem necessária para obter a densidade desejada. Após a compactação da forragem com bastões de cano ferro e concreto na ponta, os silos foram vedados com fita adesiva, pesados e armazenados à temperatura am- biente. Decorridos 60 dias de fermentação os silos foram novamente pesados. Rec= (MSF * CFf) / (MSI * CFi) * 100 sendo: Rec= (MSF * CFf) / (MSI * CFi) * 100 sendo: Rec: recuperação da fração X (% da fração X), MSF: matéria seca no momento da abertura (quantidade de forragem (kg) * teor de matéria seca (%)), MSF: matéria seca no momento da abertura (quantidade de forragem (kg) * teor de matéria seca (%)), CFf: concentração da fração X, no momento da abertura, CFf: concentração da fração X, no momento da abertura, MSI: matéria seca ensilada (quantidade de forragem (kg) * teor de matéria seca (%)), MSI: matéria seca ensilada (quantidade de forragem (kg) * teor de matéria seca (%)), CFi: concentração da fração X, no momento da ensilagem. CFi: concentração da fração X, no momento da ensilagem. CFi: concentração da fração X, no momento da ensilagem. O delineamento experimental utilizado foi inteiramente casualizado em esquema fatorial (2 x 6), com três repetições. Os fatores estudados são descritos a seguir: queima (cana-de-açúcar crua, cana-de-açúcar queimda) e aditivos (controle, benzoato de sódio, uréia, hidróxido de sódio, P. acidipro- pionici + L.plantarum e L. buchneri). Antes da ensilagem e após a aplicação dos referidos inoculantes e/ou aditivos a forragem foi amostrada três vezes em cada tratamento. Sendo que de cada amostra foi fragmentada ainda em duas sub-amostras. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL Uma sub-amostra foi destinada à determi- nação do pH segundo Silva e Queiroz (2002), a outra sub-amostra foi pesada e levada para estufa de ventilação forçada a 55ºC durante 72 horas (Silva e Queiroz, 2002). p p ) Os dados foram submetidos à análise de variância pelo procedimento de Modelos Lineares Gerais (Proc GLM - General Linear Models Procedures). As médias foram ajus- tadas pelo método dos quadrados mínimos (LSMEANS, least squares means). As análises estatísticas foram efetuadas utili- zando o programa SAS (1999). Determinaram-se os teores de matéria seca (MS), matéria orgânica (MO), matéria mineral (MM), proteína bruta (PB), nitro- gênio ligado à fibra em detergente neutro (N-FDN) e extrato etéreo (EE) segundo os métodos descritos por Silva e Queiroz (2002). Os teores de fibra em detergente neutro (FDN), fibra em detergente ácido (FDA) foram avaliados pelo método seqüencial segundo as técnicas descritas por Van Soest et al. (1991). Para determinação da celulose foi utilizado o ácido sulfúrico a 72% (Van Soest, 1994), enquanto os teores de lignina foram calculados por diferença entre FDA e celulose. O teor de carboidratos não fibrosos (CNF) foi calculado pela expressão (CNF = 100 – (FDN + MM + PB + EE)). A digestibilidade verdadeira in vitro da matéria MATERIAL E MÉTODOS Os microrganismos Propionibacterium acidipropionici (Cepa MS 01) e Lacto- bacillus plantarum (Cepa MA 18/50) utili- zados são encontrados no inoculante co- mercial Propiolact® (PROP), o micror- ganismo Lactobacillus buchneri (Cepa NCIMB 40788) é encontrado no inoculante comercial LalsilCana® (BUCH). Os inocu- lantes bacterianos foram aplicados confor- me recomendações dos fabricantes obser- vando as doses e diluições. A aplicação dos aditivos químicos foi calculada com base na matéria natural da forragem. A uréia foi diluída em água e aplicada em solução de 35 l/t de forragem, o benzoato de sódio foi aplicado através de solução de 15 l/t e o No presente trabalho objetivou-se avaliar o efeito da queima e do tratamento com aditivos químicos (uréia, benzoato de sódio e hidróxido de sódio) e inoculantes microbiológicos (Propionibacterium acidi- Archivos de zootecnia vol. 58, núm. 221, p. 46. QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR doses de uréia (0, 2 e 4%) observaram elevação do pH de 3,87 para 4,07 e 4,28 em função das doses. produzidos. Castrillón et al. (1978) trataram a cana-de-açúcar com 4% de NaOH na matéria seca e observaram que as silagens controle tiveram 1,6% enquanto as tratadas apresentaram 12,2% de ácido lático, mesmo com essa alta produção de ácido lático as silagens tratadas apresentaram pH de 4,41, enquanto as não tratadas 4,12. O NaOH tem grande capacidade de ele- var o pH, haja visto, os valores obtidos na cana-de-açúcar antes de ensilar. Aliado a esse fato tem-se a elevação do poder tampão, propiciando assim os maiores valores de pH após a abertura. No entanto, ao se comparar a redução do pH das silagens controle (2,1 unidades de pH) com a redução das silagens tratadas com NaOH (7 unidades de pH), infere-se que nas silagens tratadas com NaOH ocorreu maior extensão de fermen- tação, pois o pH após a abertura é fruto do pH antes de ensilar associado ao poder tampão e modificado pelos ácidos orgânicos Observa-se pequena diferença entre a cana-de-açúcar crua e queimada antes da ensilagem (tabela I). O processo de queima elimina a palhada da cana-de-açúcar, mate- rial esse que possui alto teor de MS, conseqüentemente há redução do teor de MS na cana-de-açúcar queimada. Entre os aditivos, não houve diferença estatística no teor MS (p>0,05). Após a abertura dos silos Tabela I. Valores de pH e teores de matéria seca da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Values of pH, and dry matter of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Archivos de zootecnia vol. 58, núm. 221, p. 47. RESULTADOS E DISCUSSÃO A queima antes da ensilagem, propiciou valores de pH estatisticamente inferiores (p<0,05), no entanto, diferenças de pequena magnitude (tabela I). Diferenças estatísticas em relação à queima também foram observa- das após a abertura, todas inferiores a 0,2 unidades de pH. Em relação aos aditivos obteve-se os maiores valores nas silagens tratadas com NaOH seguidas das aditivadas com uréia. Hill e Leaver (2002) estudando a ensilagem da planta de trigo tratada com Archivos de zootecnia vol. 58, núm. 221, p. 46. Médias seguidas da mesma letra, maiúscula na linha e minúscula na coluna, não diferiram estatisticamente pelo teste Tukey (p<0,05). QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR Tratamentos Antes da ensilagem Após a ensilagem Crua Queimada Média Crua Queimada Média Valores de pH Controle 5,9 5,7 5,8b 3,75Ac 3,72Ac 3,73c Benzoato (0,1%) 6,0 5,7 5,8b 3,65Acd 3,72Ac 3,69cd Uréia (1,5%) 5,8 5,7 5,8b 4,24Ab 4,05Bb 4,1b NaOH (1,0%) 11,7 11,7 11,7a 4,64Ba 4,86Aa 4,75a PROP1 5,6 5,7 5,7b 3,55Bce 3,66Acd 3,60d BUCH2 5,7 5,6 5,7b 3,43Ae 3,52Ad 3,48e Média 6,8A 6,7B 6,7 3,88B 3,92A 3,9 CV (%) 1,88 1,47 Teores de MS (%) Controle 35,2 34,1 34,6a 27,4Bc 29,2Abc 28,3d Benzoato (0,1%) 35,2 34,4 34,8a 29,4Bb 30,7Ab 30,0bc Uréia (1,5%) 35,8 33,1 34,5a 29,1Ab 29,1Abc 29,1cd NaOH (1,0%) 36,8 34,5 35,6a 29,1Bb 32,7Aa 30,9b PROP1 35,6 34,4 35,0a 28,0Abc 28,0Ac 28,0d BUCH2 35,1 34,4 34,7a 31,9Ba 33,4Aa 32,7a Média 35,6A 34,1B 34,9 29,1B 30,5A 29,8 CV (%) 2,69 2,21 Médias seguidas da mesma letra, maiúscula na linha e minúscula na coluna, não diferiram estatisticamente pelo teste Tukey (p<0,05). 1Propionibacterium acidipropionici + Lactobacillus plantarum; 2Lactobacillus buchneri. Archivos de zootecnia vol. 58, núm. 221, p. 48. Médias seguidas da mesma letra, maiúscula na linha e minúscula na coluna, não diferiram estatisticamente pelo teste Tukey (p<0,05). p y (p , ) 1Propionibacterium acidipropionici + Lactobacillus plantarum; 2 Lactobacillus buchneri. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL observou-se redução em todos os teores de MS. Excetuando as silagens tratadas com PROP e uréia, todas as demais apresentaram maiores teores de MS quando a cana-de- açúcar foi queimada, devido a interação ocorrida entre aditivos e forma de manejo (crua ou queimada). Destacam-se os teores de MS das silagens tratadas com BUCH (crua e queimada) e das silagens de cana- de-açúcar queimada tratadas com NaOH. Na ensilagem da cana-de-açúcar ocorre gran- de redução dos teores de MS durante a armazenagem (Siqueira et al., 2007). Aditi- vos que impedem este fato propiciam dimi- nuição das perdas ocorridas na armazenagem. mada, tratadas com uréia foram superiores aos demais (p<0,05) (tabela II). A uréia é um ingrediente rico em nitrogênio não protéico (45% de N na MS), sendo assim já era espe- rado essa superioridade em relação aos teores de PB. observou-se redução em todos os teores de MS. Excetuando as silagens tratadas com PROP e uréia, todas as demais apresentaram maiores teores de MS quando a cana-de- açúcar foi queimada, devido a interação ocorrida entre aditivos e forma de manejo (crua ou queimada). Destacam-se os teores de MS das silagens tratadas com BUCH (crua e queimada) e das silagens de cana- de-açúcar queimada tratadas com NaOH. Na ensilagem da cana-de-açúcar ocorre gran- de redução dos teores de MS durante a armazenagem (Siqueira et al., 2007). Aditi- vos que impedem este fato propiciam dimi- nuição das perdas ocorridas na armazenagem. Observa-se que após a abertura houve elevação numérica em todos os teores de PB, comparado aos teores de antes da ensilagem. Esse aumento está associado à concentração do nutriente devido às perdas de MS. A uréia aplicada na ensilagem pode ser perdida no decorrer do processo fermentativo. Nussio e Schmidt (2004) em revisão sobre ensilagem da cana de açúcar reportaram que a recuperação do nitrogênio aplicado na forma de uréia é da ordem de p g Os teores de proteína bruta (PB) das silagens e da cana-de-açúcar crua e quei- Tabela II. Teores de proteína bruta (PB), nitrogênio ligado à fibra em relação ao nitrogênio total (N-FDN ) da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Crude protein (PB), neutral detergent insoluble nitrogen related to the total nitrogen (N-FDN) of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Tabela II. Archivos de zootecnia vol. 58, núm. 221, p. 49. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL Teores de proteína bruta (PB), nitrogênio ligado à fibra em relação ao nitrogênio total (N-FDN ) da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Crude protein (PB), neutral detergent insoluble nitrogen related to the total nitrogen (N-FDN) of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Tabela II. Teores de proteína bruta (PB), nitrogênio ligado à fibra em relação ao nitrogênio total (N-FDN ) da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Crude protein (PB), neutral detergent insoluble nitrogen related to the total nitrogen (N-FDN) of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Tratamentos Antes da ensilagem Após a ensilagem Crua Queimada Média Crua Queimada Média PB (%) Controle 1,5 1,3 1,4b 1,7 2,3 2,0b Benzoato (0,1%) 1,3 1,2 1,3b 1,8 2,1 2,0b Uréia (1,5%) 12,4 12,5 12,5a 13,9 14,1 14,0a NaOH (1,0%) 1,4 1,3 1,4b 2,0 1,9 2,0b PROP1 1,4 1,4 1,4b 2,0 2,8 2,4b BUCH2 1,5 1,4 1,4b 1,9 2,1 2,0b Média 3,3A 3,2A 3,2 3,9B 4,2A 4,1 CV (%) 12,07 8,35 N-FDN (% NT) Controle 45,4Bc 51,1Ab 48,2b 55,8Aa 47,7Ba 51,8a Benzoato (0,1%) 47,3Abc 48,6Ab 48,0b 37,5Abc 40,1Ab 38,8b Uréia (1,5%) 5,3Ad 6,2Ac 5,8c 8,7Ad 7,9Ad 8,3d NaOH (1,0%) 51,6Bbc 59,1Aa 55,3a 58,4Aa 39,5Bb 48,9a PROP1 50,0Abc 53,0Ab 51,5ab 42,4Ab 37,5Bbc 39,9b BUCH2 55,7Aa 50,9Bb 53,3a 35,1Ac 33,4Ac 34,3c Média 42,6b 44,8A 43,7 39,6A 34,4B 37,0 CV (%) 5,00 5,28 Médias seguidas da mesma letra, maiúscula na linha e minúscula na coluna, não diferiram estatisticamente pelo teste Tukey (p<0,05). 1Propionibacterium acidipropionici + Lactobacillus plantarum; 2 Lactobacillus buchneri. QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR 70%, no presente estudo observou-se recuperação do nitrogênio total nas silagens tratadas com uréia de 82%. nitrogênio solúvel, sendo assim, mesmo se a cana-de-açúcar apresenta-se teores de N- FDN semelhantes, ocorreria diluição desse devido à inclusão de nitrogênio solúvel. Os teores de nitrogênio ligado a FDN em relação ao N total (N-FDN) foram apresen- tados na tabela II. Observa-se que as forragens tratadas com uréia tiveram os menores valores, pois a uréia é fonte de Antes da ensilagem os valores de fibra em detergente neutro (FDN) na cana-de- açúcar queimada foram inferiores aos teores na cana-de-açúcar crua (tabela III). Ao Tabela III. Teores de fibra em detergente neutro (FDN), fibra em detergente ácido (FDA) e lignina (LIG) da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Values of the neutral detergent fiber (FDN), acid detergent fiber (FDA) and lignin (LIG) of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Tratamentos Antes da ensilagem Após a ensilagem Crua Queimada Média Crua Queimada Média FDN (%) Controle 54,7 49,8 52,3bc 75,1Aa 74,4Aab 74,8a Benzoato (0,1%) 53,0 48,3 50,7c 68,4Ab 68,1Ac 68,2b Uréia (1,5%) 53,6 49,3 51,5bc 70,8Aab 71,6Abc 71,2b NaOH (1,0%) 49,3 45,1 47,2d 59,5Ac 47,7Be 53,6d PROP1 53,8 51,1 52,5ab 74,1Aa 76,2Aa 75,1a BUCH2 56,5 51,5 54,0a 66,7Ab 61,4Bd 64,1c Média 53,5A 49,2B 51,3 69,1A 66,6B 67,8 CV (%) 1,86 2,63 FDA (%) Controle 34,8 31,0 32,9a 48,7Ba 52,2Ab 50,5b Benzoato (0,1%) 35,0 30,4 32,7a 44,6Ac 46,3Ac 45,5c Uréia (1,5%) 35,2 29,4 32,3a 45,8Aab 47,9Ac 46,8c NaOH (1,0%) 35,8 30,8 33,3a 41,1Ac 37,6Bc 39,3d PROP1 35,6 30,8 33,2a 48,2Ba 57,5Aa 52,9a BUCH2 35,0 31,6 33,3a 45,6Aab 47,7Ac 46,6c Média 35,2A 30,7B 33,0 45,7B 48,2A 46,9 CV (%) 3,07 2,77 LIG (%) Controle 6,9Bbc 8,7Aa 7,8ab 8,1Bb 9,9Aa 9,0b Benzoato (0,1%) 9,6Aa 8,1Ba 8,9a 7,8Ab 8,1Ab 8,0bc Uréia (1,5%) 6,6Bc 8,0Aa 7,3b 7,3Ab 7,9Ab 7,6c NaOH (1,0%) 8,0Aabc 5,9Bb 7,0b 7,1Ab 5,8Bc 6,4d PROP1 8,7Aab 8,0Aa 8,4ab 9,8Ba 10,9Aa 10,4a BUCH2 7,7Aabc 7,2Aab 7,4b 8,2Ab 8,0Ab 8,1bc Média 7,9A 7,6A 7,8 8,0A 8,4A 8,2 CV (%) 10,22 7,38 Médias seguidas da mesma letra, maiúscula na linha e minúscula na coluna, não diferiram estatisticamente pelo teste Tukey (p<0,05). 1Propionibacterium acidipropionici + Lactobacillus plantarum; 2Lactobacillus buchneri. Tabela III. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL queimar a cana-de-açúcar ocorre a elimi- nação da palha, que é uma porção da planta rica em FDN. Outro resultado, que vale ser salientado, é a redução da FDN pelo uso de NaOH mesmo antes da ensilagem. Balieiro Neto et al. (2007) avaliando a ensilagem de cana-de-açúcar tratadas com aditivos alcalinos, observou redução da FDN de 55,48% para 45,36% quando se adicionou 2% de CaO na forragem antes da ensilagem. Resultados semelhantes foram observados por Pedroso et al. (2007) na ensilagem da cana-de-açúcar, onde observou redução da FDN de 64,5% para 55,4% nas forragens tratadas com 1% de NaOH, antes da ensilagem. pode ter sido perdido, além da intensa perda na forma de CO2 durante a fermentação por leveduras, elevando assim a concentração da FDN. Em relação à fibra em detergente ácido (FDA) antes da ensilagem, verificou-se diferença estatística apenas no efeito da queima (p<0,05). Como foi relatado na FDN, o processo de queima eliminou a presença de palhas que por sua vez é uma parte da planta rica em FDA. Os teores de FDA após a abertura foram numericamente superiores aos observados antes da ensilagem. A se- melhança do ocorrido com a FDN pode atri- buir a elevação da FDA ao consumo de carboidratos solúveis durante a fermen- tação. g Após a abertura observou-se elevação nos teores de FDN em todas as silagens. Esse fato é conseqüência do consumo de conteúdo celular, notadamente carboidratos solúveis pelos microrganismos durante a ensilagem, principalmente leveduras. Pode- se associar a elevação da FDN às perdas de matéria seca, destaca-se nesse sentido, os teores de FDN das silagens de cana-de- açúcar queimada e tratada com NaOH, que além de sofrerem o processo de hidrólise alcalina da fibra (Tetlow e Mason, 1987), pode ter havido maior fermentação homo- lática, e conseqüentemente houve maior produção de ácido lático que não é perdido por volatilização e redução na produção de CO2, diminuindo assim o efeito de concen- tração da FDN. Os teores de lignina (LIG) não seguiram a mesma tendência da FDN e da FDA, pois não tiveram comportamento consistente durante o processo de ensilagem, ocorren- do, elevação, redução e manutenção dos teores. A porção de carboidratos representa cerca de 90% da planta da cana-de-açúcar, sendo fragmentado em dois grandes gru- pos: carboidratos fibrosos, representados pela FDN e carboidratos não fibrosos (CNF). QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR Teores de fibra em detergente neutro (FDN), fibra em detergente ácido (FDA) e lignina (LIG) da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Values of the neutral detergent fiber (FDN), acid detergent fiber (FDA) and lignin (LIG) of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Tabela III. Teores de fibra em detergente neutro (FDN), fibra em detergente ácido (FDA) e lignina (LIG) da cana-de-açúcar antes da ensilagem e após a abertura dos silos em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Values of the neutral detergent fiber (FDN), acid detergent fiber (FDA) and lignin (LIG) of the sugarcane and sugarcane ensiled after burning or bacterial inoculants addition). Archivos de zootecnia vol. 58, núm. 221, p. 50. QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR IV). Os maiores teores de CNF foram obtidos nas silagens tratadas com NaOH, seguidas pelas silagens inoculadas com BUCH. Os teores de CNF estão intimamente relaciona- dos com os teores de FDN, pois são calcu- lados pela subtração da FDN do teor de carboidratos totais. Conseqüentemente, quanto mais alto o teor de FDN, elevado pela perda de carboidratos solúveis, menor será o teor de CNF, constatação essa que pode ser comprovada pela análise conjunta das tabelas III e IV. Tabela IV. Teores de carboidratos não fibrosos (CNF %) e valores de digestibilidade verdadeira in vitro da matéria seca (DVIVMS %) da cana-de-açúcar antes da ensilagem e após a abertura dos silos e recuperações dos carboidratos não fibrosos (Rec CNF) e da matéria seca digestível verdadeira (Rec MSDV), em percentagem da fração ensilada,em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Values of the no fiber carbohydrate (CNF %), and dry matter true in vitro digestibility (DVIVMS %) of the sugarcane and sugarcane silage, no fiber carbohydrates recovery (Rec. CNF) and digestible dry matter recovery (Rec. MSDV) related to the ensiled portion in function of the burning or bacterial inoculants addition). Tabela IV. Teores de carboidratos não fibrosos (CNF %) e valores de digestibilidade verdadeira in vitro da matéria seca (DVIVMS %) da cana-de-açúcar antes da ensilagem e após a abertura dos silos e recuperações dos carboidratos não fibrosos (Rec CNF) e da matéria seca digestível verdadeira (Rec MSDV), em percentagem da fração ensilada,em função do efeito da queima e do tratamento com aditivos químicos e bacterianos. (Values of the no fiber carbohydrate (CNF %), and dry matter true in vitro digestibility (DVIVMS %) of the sugarcane and sugarcane silage, no fiber carbohydrates recovery (Rec. CNF) and digestible dry matter recovery (Rec. MSDV) related to the ensiled portion in function of the burning or bacterial inoculants addition). SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL Os CNF possuem alta digestibilidade, acima de 90% (Costa, 2002) e podem ser conside- rados, uma porção de grande importância no valor nutritivo da cana-de-açúcar. Na tabela IV estão apresentados os teores de CNF antes e após a ensilagem. Antes da ensilagem vê-se que o efeito da queima elevou os teores de CNF, conseqüência da redução dos teores da FDN. Entre as silagens aditivadas, as tratadas com uréia apre- sentaram os menores teores de CNF, pois no cálculo deste parâmetro desconta-se o teor de proteína, sendo a uréia uma fonte de nitrogênio não protéico, o teor de CNF fica subestimado nestas silagens, pois 1 g de nitrogênio não protéico advindo da uréia representa em desconto na formula de CNF o valor de 6,25 g. Outro valor relevante foi observado na silagem de cana-de-açúcar queimada e ino- culada com BUCH. No processo de ensila- gem da cana-de-açúcar e ou de outras forra- gens com presença do BUCH ocorre efetivo consumo de carboidratos solúveis (Pedroso et al., 2007 e Nishino et al., 2003). Entretan- to, a concentração da FDN nessas silagens de cana-de-açúcar foi menor que naquelas tratadas com uréia, PROP e não tratadas, pois possivelmente ocorreu predominância de produção de ácido acético com conse- qüente redução da produção de etanol. Sendo o etanol um produto volátil esse Após a ensilagem houve redução do teor de CNF em todas as silagens (tabela Archivos de zootecnia vol. 58, núm. 221, p. 50. Archivos de zootecnia vol. 58, núm. 221, p. 51. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL Pedroso et al. (2007) avaliando o teor de carboidratos solúveis no perfil de fer- mentação na ensilagem da cana-de-açúcar observou redução de 23,3% para 9,13 e 5,69% nas silagens controle e inoculadas com L. buchneri, respectivamente. Wein- berg et al. (2002) também observaram maiores consumos de carboidratos solúveis em silagem de milho inoculadas com L. buchneri, quando comparado às silagens controle ou silagens inoculadas com L. plantarum. Ressalva-se que a interpretação dos resultados de CNF em termos relativos pode ser associada aos carboidratos solúveis, porém no que diz respeito aos valores absolutos deve ser diferenciada daquela realizada pelos valores de car- boidratos solúveis, pois na fração de CNF, inserem-se produtos da fermentação pre- sentes na matéria seca, enquanto carboi- dratos solúveis representam um grupo es- pecífico de carboidratos. CNF, todos esses baseados na inibição da fermentação por leveduras e no caso do NaOH outro fator contribuinte na elevação da DVIVMS foi a solubilização da fração fibrosa. A recuperação de CNF (tabela IV) fornece um indicativo de preservação da constituição original da forragem estudada, no entanto além da conservação do material original faz-se necessário conhecer ou mesmo inferir sobre os produtos oriundos da fermentação. Destacam-se os valores de recuperação de CNF das silagens tratadas com NaOH e inoculadas com BUCH, princi- palmente nas silagens de cana-de-açúcar queimada. Segundo McDonald et al. (1991) na fermentação por leveduras ocorre perda de 48% da matéria seca fermentada, consi- derando que as leveduras fermentam carboidratos solúveis que estão contidos na fração de CNF, infere-se que, ocorreram nessas silagens inibição da atuação de leveduras mais pronunciadas que nas demais silagens. A digestibilidade verdadeira in vitro da matéria seca (DVIVMS) da cana-de-açúcar antes de ensilar foi superior na cana-de- açúcar queimada, provavelmente devido à eliminação da palha. Destaca-se ainda, a superioridade dos valores de DVIVMS na cana-de-açúcar tratada com NaOH antes da ensilagem. No estudo de Tetlow e Mason (1987) foi observada elevação da diges- tibilidade in vitro das culturas de centeio, cevada e trigo antes de ensilar quando essas foram tratadas com NaOH. Resultados semelhantes à recuperação de CNF foram observados nas recuperações da matéria seca digestível verdadeira (tabela IV). A análise desse parâmetro traduz-se em indicativo de perdas quantitativas e qualitativas. A explicação para esses resul- tados baseia-se nas perdas quantitativas, bem como pelas alterações químicas como a elevação da FDN. QUEIMA E ADITIVOS E COMPOSIÇÃO DE SILAGENS DE CANA-DE-AÇÚCAR Tratamentos Antes da ensilagem Após a ensilagem Crua Queimada Média Crua Queimada Média CNF (%) Controle 37,3 45,1 41,2bc 15,1Ac 18,1Ad 16,6d Benzoato (0,1%) 40,9 46,5 43,7a 23,4Aa 24,6Ac 24,0c Uréia (1,5%) 27,7 34,2 30,9d 6,5Ac 9,2Ae 7,8e NaOH (1,0%) 39,0 45,0 42,0ab 27,4Ba 40,7Aa 34,1a PROP1 39,8 43,4 41,6bc 17,2Ab 15,7Ad 16,4d BUCH2 36,6 43,0 39,8c 24,9Ba 31,8Ab 28,3b Média 36,9B 42,9A 39,9 19,1B 23,4A 21,2 CV (%) 2,79 9,2 DVIVMS (%) Controle 52,6Bc 60,8Ab 56,7bc 35,1 39,7 37,4d Benzoato (0,1%) 55,8Bbc 60,4Abc 58,1b 39,7 43,0 41,4c Uréia (1,5%) 53,2Bc 58,7Abcd 55,9bc 37,7 44,1 40,9c NaOH (1,0%) 66,2Ba 69,3Aa 67,8a 59,6 63,0 61,3a PROP1 57,8Ab 57,0Acd 57,4b 34,6 39,8 37,2d BUCH2 52,7Bc 56,1Ad 54,4c 48,4 51,7 50,1b Média 56,4B 60,4A 58,4 42,5B 46,9A 44,7 CV (%) 2,60 3,98 Rec CNF Rec MSDV Controle 27,3Ac 28,3Ad 27,8d 45,0Acd 46,0Ac 45,5c Benzoato (0,1%) 42,7Ab 39,0Ac 40,8c 53,2Ab 52,4Abc 52,8b Uréia (1,5%) 16,9Ad 19,5Ad 18,2e 51,6Abc 54,4Ab 53,0b NaOH (1,0%) 53,6Ba 83,1Aa 68,3a 68,5Ba 83,6Aa 76,1a PROP1 28,7Ac 24,9Ad 26,8d 39,8Ad 48,3Abc 44,0c BUCH2 55,0Ba 64,1Ab 59,6b 74,3Aa 79,8Aa 77,1a Média 37,4B 43,1A 40,2 55,4B 60,7A 58,1 CV (%) 10,08 5,26 Médias seguidas da mesma letra, maiúscula na linha e minúscula na coluna, não diferiram estatisticamente pelo teste Tukey (p<0,05). 1Propionibacterium acidipropionici + Lactobacillus plantarum; 2Lactobacillus buchneri. SIQUEIRA, REIS, SCHOCKEN-ITURRINO, BERNARDES, PIRES, ROTH E AMARAL Vale ainda ressaltar, que a recuperação das frações supracitadas foram calculadas em relação ao material no momento da ensilagem, no entanto no processo de queima ocorrem perdas no cam- po pela ação do fogo, que no presente estudo não foram mensuradas. Após a ensilagem ocorreu redução acen- tuada na DVIVMS das silagens. A digesti- bilidade da cana-de-açúcar é conseqüência da relação entre CNF e FDN. Nesse sentido, observa-se que a queima da cana-de-açúcar propiciou maiores DVIVMS independente do aditivo utilizado, devido à redução da FDN e maiores teores de CNF. Com relação ao efeito dos aditivos as silagens tratadas com NaOH apresentaram os maiores valo- res de DVIVMS, seguidas pelas silagens inoculadas com BUCH. A explicação para esses resultados pode ser baseada, nos menores teores de FDN e maiores teores de Archivos de zootecnia vol. 58, núm. 221, p. 52. BIBLIOGRAFIA Hill, J. and J.D. Leaver. 2002. 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https://openalex.org/W3115845394	https://strathprints.strath.ac.uk/79271/7/Kolln_etal_JCS_2022_training_a_neural_network_to_selectively_restore_cellular_structures.pdf	English	null	Label2label: Training a neural network to selectively restore cellular structures in fluorescence microscopy	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	19,071	ABSTRACT Fluorescence microscopy is a commonly used technique in cell biology for determining the spatial distribution and abundance of target proteins in cells. It relies on the use of highly specific labels to visualise the cellular components of interest. Fluorescent labels can be chemical stains, antibody labelling or molecular labelling, whereby cells are genetically altered to express fluorescent proteins (Suzuki et al., 2007). In this work, we mainly focus on immunofluorescence labelling in fixed cells. Immunofluorescence microscopy is routinely used to visualise the spatial distribution of proteins that dictates their cellular function. However, unspecific antibody binding often results in high cytosolic background signals, decreasing the image contrast of a target structure. Recently, convolutional neural networks (CNNs) were successfully employed for image restoration in immunofluorescence microscopy, but current methods cannot correct for those background signals. We report a new method that trains a CNN to reduce unspecific signals in immunofluorescence images; we name this method label2label (L2L). In L2L, a CNN is trained with image pairs of two non-identical labels that target the same cellular structure. We show that after L2L training a network predicts images with significantly increased contrast of a target structure, which is further improved after implementing a multiscale structural similarity loss function. Here, our results suggest that sample differences in the training data decrease hallucination effects that are observed with other methods. We further assess the performance of a cycle generative adversarial network, and show that a CNN can be trained to separate structures in superposed immunofluorescence images of two targets. Recently, CNNs were employed for content-aware image restoration (CARE) of corrupted fluorescence images. By training a U-Net with image pairs that were acquired with, for instance, different laser powers or exposure times, it was shown that a network was able to successfully restore denoised images of cell structures, such as the plasma membrane or the nucleus (Ronneberger et al., 2015; Weigert et al., 2018). CNNs were further used for the upsampling of images that were detected below the Nyquist sampling frequency (Weigert et al., 2018). Moreover, generative adversarial networks (GANs) were trained to enhance image resolution in immunofluorescence images, using, for example, stimulated emission depletion (STED) microscopy to acquire the training benchmarks (Goodfellow et al., 2014; Wang et al., 2019). Both mentioned methods rely on clean benchmark images for the network training, which can be challenging to acquire in practice. © 2022. Published by The Company of Biologists Ltd \| Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE ABSTRACT Hence, semi-supervised/unsupervised deep learning-based restoration methods have emerged. For instance, networks based on the architecture of a cycle generative adversarial network (CycleGAN) (Zhu et al., 2017) were employed to deconvolve fluorescence images of microtubules, using unpaired high- resolution images as reference (von Chamier et al., 2021) or simulated low- and high-resolution images (Lim et al., 2020) for the training. The CycleGAN architecture addresses the ‘hallucination problem’, which is the introduction of artificial features in generated images that is often observed when training a classical GAN (Zhu et al., 2017). Here, a GAN is first trained to generate a higher quality image based on a corrupted input. Then, the generated image is fed into a second GAN that translates it back into the original image (back translation), giving the network less freedom to make changes to an input (Zhu et al., 2017; Lim et al., 2020). Other examples include noise2void, an unsupervised method that removes camera shot noise (Krull et al., 2019), and noise2noise (N2N) (Lehtinen et al., 2018). For N2N, a CNN is trained with corrupted image pairs of the same sample. Owing to the statistical nature of how loss is minimised by the network during training with a deviation- minimising loss function, it was shown that uncorrelated image signals that follow, for example, Gaussian, Poisson or Bernoulli noise are rejected, whereas correlating fluorescence signals in the training data are recovered by this method (Lehtinen et al., 2018). Although these restoration methods enhance the contrast in KEY WORDS: Convolutional neural networks, Content-aware image restoration, Antibody labelling, Noise2noise, Cellular structures, Fluorescence microscopy Label2label: training a neural network to selectively restore cellular structures in fluorescence microscopy n1,2,3,, Omar Salem2,3, Jessica Valli4, Carsten Gram Hansen2,3 and Gail McConnell1 Lisa Sophie Kö lln1,2,3,, Omar Salem2,3, Jessica Valli4, Carsten Gram Hansen2,3 and Gail McConnell1 Handling Editor: Jennifer Lippincott-Schwartz Received 24 June 2021; Accepted 17 December 2021 1University of Strathclyde, Department of Physics, Glasgow G4 0NG, UK. 2University of Edinburgh, Centre for Inflammation Research, Edinburgh EH16 4TJ, UK. 3University of Edinburgh, Institute for Regeneration and Repair, Edinburgh EH16 4UU, UK. 4Edinburgh Super Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh EH14 4AS, UK. 1University of Strathclyde, Department of Physics, Glasgow G4 0NG, UK. 2University of Edinburgh, Centre for Inflammation Research, Edinburgh EH16 4TJ, UK. 3University of Edinburgh, Institute for Regeneration and Repair, Edinburgh EH16 4UU, UK. 4Edinburgh Super Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh EH14 4AS, UK. INTRODUCTION In recent years, deep learning has increasingly been used for image processing in cell biology (Belthangady and Royer, 2019). Specifically, convolutional neural networks (CNNs) with a U-Net architecture (Ronneberger et al., 2015) are employed for various tasks, from protein detection in transmission microscopy images (Christiansen et al., 2018; Ounkomol et al., 2018) to image segmentation of single cells (Falk et al., 2019) or specific cellular structures (Caicedo et al., 2019). Journal of Cell Science L.S.K., 0000-0001-9562-7292; O.S., 0000-0002-3072-9228; J.V., 0000-0003- 4346-0317; C.G.H., 0000-0003-0746-7482; G.M., 0000-0002-7213-0686 Loss functions for training a convolutional neural network Loss functions for training a convolutional neural network We trained a CNN with different loss functions with the aim of restoring cell images with enhanced structural contrast. A CNN, which can be described as a function gθ with its model parameters θ, is trained to minimise the error between two images based on a loss function L: argmin q X T t¼1 LðgqðxtÞ; ytÞ; ð1Þ ð1Þ with gθ(xt) as the predicted image for the input xt, yt as the benchmark and T as the number of input-benchmark image pairs that are used for the training (Lehtinen et al., 2018). Common loss functions are the least absolute deviation loss function L1 and the least square deviation loss function L2 (Zhao et al., 2016; Lehtinen et al., 2018): L1ð^yt; ytÞ ¼ 1 N X N n¼1 j^yt;n yt;nj; ð2Þ L2ð ^yt; ytÞ ¼ 1 N X N n¼1 ð^yt;n yt;nÞ2; ð3Þ ð2Þ g pp p g L2L is also different to restoration methods with a noise2clean approach in which a clean benchmark is required to train a network (Goodfellow et al., 2014; Weigert et al., 2018; Wang et al., 2019). For noise2clean, the training data are generated by acquiring two images of the same label with, for example, different exposure time, sampling density or frame averaging. Notably, training a network to restore immunofluorescence images that are acquired with low exposure time or sampling density is rarely feasible, as cell specimens are fixed, and commercial antibodies are comparably efficient and photostable, allowing the image acquisition with both parameters optimised right away. Generating image pairs to train a network to restore images acquired with low frame averaging, however, is time-consuming, and further complicated by stage drift and photobleaching, overall resulting in a low benefit-cost ratio. More importantly, as in N2N, background signals originating from the label are still present in the benchmark, and as such they also cannot be corrected by this method. ð3Þ where ^yt (=gθ(xt)) is the predicted image and N the total pixel number. Because L2 is minimal if it equals the mean value of the observations, it was used for N2N in cases in which the image corruption resembles, for example, Gaussian noise whose mean is zero (Lehtinen et al., 2018). L1 is the loss function of the CSBDeep framework in default configuration (for non-probabilistic training). Author for correspondence (lisa.kolln@strath.ac.uk) Author for correspondence (lisa.kolln@strath.ac.uk) L.S.K., 0000-0001-9562-7292; O.S., 0000-0002-3072-9228; J.V., 0000-0003- 4346-0317; C.G.H., 0000-0003-0746-7482; G.M., 0000-0002-7213-0686 L.S.K., 0000-0001-9562-7292; O.S., 0000-0002-3072-9228; J.V., 0000-0003- 4346-0317; C.G.H., 0000-0003-0746-7482; G.M., 0000-0002-7213-0686 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 that the original work is properly attributed. Although these restoration methods enhance the contrast in immunofluorescence images by upsampling or denoising, they do not correct for inherent cytosolic background signals in the specimen itself, which, in immunofluorescence microscopy, 1 Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE for the training that is also known as CARE network (Weigert et al., 2018). Moreover, we trained a CycleGAN with unpaired images to assess whether, in principle, a network can also be trained with immunofluorescence images that stem from two different datasets (Zhu et al., 2017). originate from the cell label or labelling protocol. Unspecific labelling by a stain or antibody binding, as well as internalisation or residue dyes after the specimen preparation, can significantly limit the image contrast of a target (Saper, 2009). The performance of antibodies or stains vary for a number of reasons. Epitopes can be altered in the target protein by the fixation step, effectively changing the location or accessibility to the antibody (Miller and Shakes, 1995). Also, unspecific antibody binding can be caused by attractive intermolecular interaction, such as van der Waals or hydrogen bonding interactions, or by binding to proteins with similar epitopes, which overall results in an underlying cytosolic background signal in images of cells (Wagner et al., 2011). To establish and evaluate our method, we generated image data across four different distinct cellular structures: the actin cytoskeleton (Dugina et al., 2009; Suarez and Kovar, 2016); the microtubule network (Nogales, 2000; Pellegrini and Budman, 2005); and discrete plasma membrane structures, namely caveolae (Rausch and Hansen, 2020) and focal adhesions (Martino et al., 2018). The ability of a CNN to selectively restore distinct cellular structures in immunofluorescence images after training with carefully selected data was further assessed by training a CNN as separator of two markers in superposed immunofluorescence images. Here, images were acquired of fixed cells that were dual- labelled with a nuclear marker and an antibody against the plasma membrane protein CD44 (Ilangumaran et al., 2010). Label2label We propose a new application of deep learning in fluorescence microscopy in which a neural network is trained to significantly reduce label-induced unspecific cytosolic signals in fluorescence images of cellular structures. We call this method label2label (L2L). For L2L, a CNN is trained with image pairs of cells that are dual- labelled for the same distinct cellular structure of interest. L2L utilises the varying performance of antibodies and stains in immunofluorescence microscopy. We hypothesized that a CNN trained with two images of a cellular structure that originate from two non-identical labels would act content filter-like, whereby fluorescence signals that systematically vary in the images are rejected, whereas correlating structural signal is restored. Here, the underlying principle of L2L is a so-called style transfer in which a neural network is trained to merge the content in input images with the style of reference images (Jing et al., 2020). As input and benchmark images highly correlate, L2L is also comparable to N2N. In both methods, a network is trained without clean benchmark images; however, in L2L, differences between the training images are not only originating from dynamic image corruptions like noise, but also inherent sample (=label) differences. Consequently, fluorescence signals from cytosolic protein and unspecific binding that, in practice, lower structural contrast in immunofluorescence images are retrieved in restorations after N2N training, whereas a network reduces such signals after L2L training when selecting appropriate training data. Label2label for reducing depolymerised β-actin in images of HeLa cells Image pairs were acquired of fixed HeLa cells (N=68) that were dual labelled with the monoclonal anti-β-actin antibody AC-15 and a phalloidin stain (see Materials and Methods). Fig. 1A shows an example confocal image pair of a fixed cell. Phalloidin almost exclusively labelled the actin filaments, but images of the antibody (AC-15) showed an additional high background signal in the cell body. This background signal likely originates from unspecific binding and/or binding to cytosolic protein by the antibodies, resulting in high intensity punctate regions, as observed in the cell cytoplasm. Notably, a 20-frame average image of AC-15 exhibited less image noise, but cytosolic background signal was still present, significantly lowering the contrast of the actin filaments (Fig. 1C). The difference in image contrast between both labels is quantifiable by calculating the mean Michelson and root mean square (RMS) contrast for each label (Peli, 1990). For that, we applied a Gaussian filter (sigma=2) to the images, normalised them to their 1st/99th percentile and derived the sample intensities assuming that the 10% brightest image pixels represented the sample. For the cell images of LMSSSIMð^yt; ytÞ ¼ 1 1 N X N n¼1 MS SSIMð^yt;n; yt;nÞ: ð5Þ ð5Þ We hypothesized that a CNN would restore images with higher structural contrast after L2L training with a LMS-SSIM instead of a ‘classical’ L1. To test this hypothesis, we trained a CNN with different loss functions [L1, LSSIM (M=1), L3S-SSIM (M=3), L5S-SSIM (M=5)]. Weights of the MS-SSIM loss function were selected such that they follow the contrast sensitivity function of the human visual system (Wang et al., 2003). The results were compared to the denoising method N2N. For N2N training, the same settings were applied, but the network was trained with two noise realisations of the same label instead. The aim of this work is to compare two deep learning-based restoration methods that improve the contrast of cellular structures in fluorescence images, do not require clean benchmark data and have requirements for the training data generation that are feasible in standard immunofluorescence ig. 1. Qualitative L2L and N2N results for images of actin. (A) Confocal image pair of a fixed HeLa cell that was dual labelled with the anti-β-actin antibody C-15 and a phalloidin stain, which was excluded from the CNN training. Scale bar: 20 µm. Loss functions for training a convolutional neural network As for both, L1 and L2, loss is calculated on a pixel-to-pixel basis during the network training, predicted images are often of low quality for a human observer (Larsen et al., 2016; Zhao et al., 2016). Journal of Cell Science To better take into account the properties of the human visual system, a multiscale structural similarity (MS-SSIM) index was proposed as an alternative to compare the similarity between two images (Wang et al., 2003, 2004). It follows: MS SSIMð pÞ ¼ ðlMð pÞÞgM Y M j¼1 ðcjð pÞÞgj ðsjð pÞÞgj; ð4Þ For L2L, the image pairs for the network training can be acquired simultaneously, under near-identical imaging conditions (see the imaging sections in Materials and Methods). The images of one fluorescent cell label are selected as training input, and the images of the label that yields a higher contrast of the cellular structure are used as a training benchmark. We selected the CSBDeep framework ð4Þ with the exponent γj as the weight for the individual scale j ðP M j¼1 gj ¼ 1Þ, and measures that compare the luminance (lM), with the exponent γj as the weight for the individual scale M j ðP j 1 gj ¼ 1Þ, and measures that compare the luminance (lM), 2 2 Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE microscopy. We show proof-of-principle that by introducing systematic sample differences in the training data a CNN can be successfully trained to reject not only image noise but also diffuse label-dependent cytosolic signals in immunofluorescence images. Both can decrease the contrast of a target structure significantly in practice. contrast (cj) and structure (sj) between two images that are functions of the local statistics of a pixel p (for a detailed explanation see Wang et al., 2003, 2004). For the calculation, a low-pass filter is applied to the image patches after each iteration (if M>1), followed by downsampling by a factor of 2, making the MS-SSIM index sensitive to differing viewing conditions. The MS-SSIM index exhibits values between (−1, 1) where −1/0/1 imply a negative/no/positive correlation between the images. To satisfy Eqn 1, for an MS-SSIM loss function (LMS-SSIM) follows (Zhao et al., 2016): Label2label for reducing depolymerised β-actin in images of HeLa cells (B) Reconstructed image of AC-15 by a CNN after L2L training with mages of AC-15/phalloidin as training input/benchmark, using an L3S-SSIM loss function. (C) Original and processed images of AC-15 for two ROIs (6 µm×6 µm). rom left to right: raw image, restored images after N2N and L2L training with an L1 and L3S-SSIM loss function, respectively, and a 20-frame average. (D) The orresponding image of phalloidin and the RMS map between the raw and the predicted image of AC-15 by the network after L2L training. J l f C ll S i Journal of Cell Science Fig. 1. Qualitative L2L and N2N results for images of actin. (A) Confocal image pair of a fixed HeLa cell that was dual labelled with the anti-β-actin antibody AC-15 and a phalloidin stain, which was excluded from the CNN training. Scale bar: 20 µm. (B) Reconstructed image of AC-15 by a CNN after L2L training with images of AC-15/phalloidin as training input/benchmark, using an L3S-SSIM loss function. (C) Original and processed images of AC-15 for two ROIs (6 µm×6 µm). From left to right: raw image, restored images after N2N and L2L training with an L1 and L3S-SSIM loss function, respectively, and a 20-frame average. (D) The corresponding image of phalloidin and the RMS map between the raw and the predicted image of AC-15 by the network after L2L training. 3 Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE RESEARCH ARTICLE Table 1. Label2label for reducing depolymerised β-actin in images of HeLa cells In the L2L results, however, not only image noise was removed, but also the contrast of filamentous signal was clearly enhanced, even compared to the training benchmark (phalloidin) (Fig. 1D, left). The RMS maps between the raw images of AC-15 and the L2L results revealed a selective removal of high intensity punctate regions [Fig. 1D (right); for further maps see Fig. S1]. AC-15/phalloidin, mean Michelson contrast values of 0.44±0.08/ 0.96±0.04 and mean±s.d. RMS contrast values of 0.15±0.03/0.19 ±0.01 were calculated. Consequently, images of AC-15 were used as input and images of the phalloidin stain were used as a benchmark for L2L training. For N2N, two noise realisations of AC- 15, acquired through sequential imaging, were used as training data. training with a LMS-SSIM showed cell structures with increased sharpness, and erroneous predictions by the network occurred (with lower M) (see annotated ROIs in Fig. S2A-C). Hallucination effects were substantially more pronounced in N2N results. Here, punctuate regions in the cell cytoplasm appeared with artificial contrast in restored images of AC-15, and actin filaments with low contrast in the input image were restored with intensity fluctuations along the structure. For L2L, these artefacts were barely observed when using an LMS-SSIM for the training. q g q g g g Fig. 1B shows the restoration of an image of AC-15 in a HeLa cell (Fig. 1A, left) by a CNN after L2L training with a L3S-SSIM (see ‘Training the CNN’ section in Materials and Methods). A trained CNN reduced cytoplasmic signal throughout the cell body in the restoration, and the relative signal of filamentous actin labelled with AC-15 increased. In Fig. 1C, for two regions of interest (ROIs), the original cell image of AC-15 and the prediction by a CNN after N2N and L2L training with a L1 and L3S-SSIM, respectively, are shown. Although noise was reduced in the N2N result, making it optically similar to a 20-frame average, the contrast of the actin filaments was still low due to the high background signal. In the L2L results, however, not only image noise was removed, but also the contrast of filamentous signal was clearly enhanced, even compared to the training benchmark (phalloidin) (Fig. 1D, left). The RMS maps between the raw images of AC-15 and the L2L results revealed a selective removal of high intensity punctate regions [Fig. 1D (right); for further maps see Fig. S1]. Label2label for reducing depolymerised β-actin in images of HeLa cells To further evaluate the network performance after L2L training, the average peak signal-to-noise ratio (PSNR), normalised root- mean square error (NRMSE) and MS-SSIM indices (M=1, 3 and 5) were calculated for the raw or predicted images of AC-15, and the corresponding images of phalloidin, dependent on the training loss function. For that, validation image patches that were excluded from the training data were used (see the ‘Training the CNN’ section in Materials and Methods). All calculated metrics indicated an increased correlation between the restorations and the benchmark (phalloidin) compared to the original image (see Table 1). Notably, using an L5S-SSIM for the training narrowly yielded the best PSNR and NRMSE. Journal of Cell Science Journal of Cell Science Label2label for reducing depolymerised β-actin in images of HeLa cells Loss function-dependent evaluation of L2L for images of different cellular structures PSNR (dB) NRMSE MS-SSIM PSNR (dB) NRMSE MS-SSIM M=5 M=3 M=1 M=5 M=3 M=1 Actin cytoskeleton Microtubule network Input 16.67 0.150 0.434 0.331 0.150 17.20 0.140 0.540 0.359 0.135 L1 19.76 0.107 0.635 0.531 0.314 16.88 0.146 0.415 0.271 0.103 L5S-SSIM 20.08 0.103 0.673 0.565 0.339 18.37 0.123 0.660 0.475 0.203 L3S-SSIM 20.07 0.103 0.672 0.567 0.343 18.23 0.125 0.652 0.469 0.200 LSIM 19.91 0.105 0.662 0.561 0.340 18.03 0.128 0.648 0.468 0.199 Caveolae PXN Input 14.91 0.181 0.156 0.110 0.049 18.73 0.118 0.455 0.337 0.152 L1 14.79 0.184 0.136 0.114 0.052 19.78 0.107 0.465 0.339 0.135 L5S-SSIM 15.26 0.175 0.216 0.207 0.131 21.27 0.091 0.605 0.497 0.329 L3S-SSIM 15.19 0.176 0.200 0.196 0.129 21.20 0.092 0.600 0.496 0.330 LSIM 14.88 0.182 0.175 0.186 0.111 21.13 0.093 0.592 0.489 0.328 SYTOX CD44 Input 15.03 0.192 0.553 0.499 0.309 20.99 0.114 0.816 0.825 0.715 L1 36.15 0.019 0.943 0.932 0.898 30.05 0.034 0.942 0.916 0.841 L5S-SSIM 36.34 0.019 0.958 0.945 0.904 27.80 0.046 0.950 0.921 0.838 L3S-SSIM 36.27 0.019 0.958 0.947 0.910 28.23 0.044 0.950 0.924 0.845 LSIM 36.56 0.019 0.954 0.945 0.914 28.83 0.040 0.944 0.921 0.849 Evaluation after training a CNN with image pairs of HeLa cells that were dual labelled with an anti-actin antibody (input) and a phalloidin stain (benchmark) (see ‘Actin cytoskeleton’), of MeT5A cells that were labelled with the anti-α-tubulin antibodies DM1A (input) and YOL1/34 (benchmark) (see ‘Microtubule network’), of MeT5A cells that were labelled with antibodies against CAVIN-1 (D1P6W; input) and caveolin-1 (CAV1) (4H312; benchmark) (see ‘Caveolae’), and of MeT5A, HeLa and U2OS cells that were dual labelled with the anti-PXN antibodies 5H11 (input) and Y113 (benchmark) (see ‘PXN’). Calculated metrics are also shown after training a CNN to separate markers in superposed images of cells that were labelled with the nuclear marker SYTOX and an anti-CD44 antibody. Average metrics were calculated between the respective training inputs or restored images of the input after training the network with an L1, L5S-SSIM, L3S-SSIM or LSSIM loss function, and the corresponding training benchmarks. To achieve this, the validation image patches were used that were created from non-background areas in the raw images (see Table 2). Metrics for the caveolae dataset were calculated after applying a Gaussian filter (sigma=1.5) due to high image noise in the dataset. The best value for each column is shown in bold. Label2label for reducing depolymerised β-actin in images of HeLa cells Table 1. Loss function-dependent evaluation of L2L for images of different cellular structures Evaluation after training a CNN with image pairs of HeLa cells that were dual labelled with an anti-actin antibody (input) and a phalloidin stain (benchmark) (see ‘Actin cytoskeleton’), of MeT5A cells that were labelled with the anti-α-tubulin antibodies DM1A (input) and YOL1/34 (benchmark) (see ‘Microtubule network’), of MeT5A cells that were labelled with antibodies against CAVIN-1 (D1P6W; input) and caveolin-1 (CAV1) (4H312; benchmark) (see ‘Caveolae’), and of MeT5A, HeLa and U2OS cells that were dual labelled with the anti-PXN antibodies 5H11 (input) and Y113 (benchmark) (see ‘PXN’). Calculated metrics are also shown after training a CNN to separate markers in superposed images of cells that were labelled with the nuclear marker SYTOX and an anti-CD44 antibody. Average metrics were calculated between the respective training inputs or restored images of the input after training the network with an L1, L5S-SSIM, L3S-SSIM or LSSIM loss function, and the corresponding training benchmarks. To achieve this, the validation image patches were used that were created from non-background areas in the raw images (see Table 2). Metrics for the caveolae dataset were calculated after applying a Gaussian filter (sigma=1.5) due to high image noise in the dataset. The best value for each column is shown in bold. AC-15/phalloidin, mean Michelson contrast values of 0.44±0.08/ 0.96±0.04 and mean±s.d. RMS contrast values of 0.15±0.03/0.19 ±0.01 were calculated. Consequently, images of AC-15 were used as input and images of the phalloidin stain were used as a benchmark for L2L training. For N2N, two noise realisations of AC- 15, acquired through sequential imaging, were used as training data. Fig. 1B shows the restoration of an image of AC-15 in a HeLa cell (Fig. 1A, left) by a CNN after L2L training with a L3S-SSIM (see ‘Training the CNN’ section in Materials and Methods). A trained CNN reduced cytoplasmic signal throughout the cell body in the restoration, and the relative signal of filamentous actin labelled with AC-15 increased. In Fig. 1C, for two regions of interest (ROIs), the original cell image of AC-15 and the prediction by a CNN after N2N and L2L training with a L1 and L3S-SSIM, respectively, are shown. Although noise was reduced in the N2N result, making it optically similar to a 20-frame average, the contrast of the actin filaments was still low due to the high background signal. L2L to enhance the structural contrast in images of the microtubule network and caveolae inhomogeneous binding or selective binding to specific epitopes of polymerised tubulin; this was not observable for the clone DM1A (compare 20-frame average images in Fig. S2D,F). Microtubules appear overall sharper in images of YOL1/34 compared to DM1A, with a lower ‘haze’ in the cytoplasm. This haze might be caused by unspecific binding, binding to cytosolic tubulin and/or out-of-focus signal. Notably, the optical resolution in images of DM1A labelled with the secondary antibody Alexa Fluor 633 was lower than in images of YOL1/34, which was conjugated to Alexa Fluor 488. inhomogeneous binding or selective binding to specific epitopes of polymerised tubulin; this was not observable for the clone DM1A (compare 20-frame average images in Fig. S2D,F). Microtubules appear overall sharper in images of YOL1/34 compared to DM1A, with a lower ‘haze’ in the cytoplasm. This haze might be caused by unspecific binding, binding to cytosolic tubulin and/or out-of-focus signal. Notably, the optical resolution in images of DM1A labelled with the secondary antibody Alexa Fluor 633 was lower than in images of YOL1/34, which was conjugated to Alexa Fluor 488. were generated. To train a CNN as a content filter for caveolae, STED image pairs (N=60) were acquired of fixed MeT5A cells that were dual labelled with antibodies against the two essential caveolae components CAVIN-1 (D1P6W) and caveolin-1 (CAV1; 4H312) (Hansen and Nichols, 2010). For the tubulin dataset, we calculated mean±s.d. Michelson contrast values of 0.45±0.14/0.9±0.05 and mean±s.d. RMS contrast values of 0.13±0.03/0.17±0.02 for DM1A/YOL1/34; for the caveolae dataset, these values were 0.27 ±0.05/0.98±0.02 and 0.13±0.02/0.22±0.02 for D1P6W/4H312 (see previous section; for the caveolae dataset, the 1% brightest pixels in the image were regarded as a sample). Hence, for L2L training, images of DM1A and D1P6W were used as input, and images of YOL1/34 and 4H312 were used as a benchmark to train the CNN for the respective target structure. In Fig. 2A, the results after N2N and L2L training with the tubulin dataset are displayed for two ROIs and loss functions (L1 and L3S-SSIM). Restorations are shown of a representative training input (top) and an image of the same cell specimen, which was acquired with a different microscope that allowed additional STED imaging (bottom) (see the imaging section in Materials and Methods). STED images exhibit a circa 3× enhanced resolution In images of cells labelled with YOL1/34, intensity fluctuations along the microtubules were observed that likely originate from Fig. 2. L2L to enhance the structural contrast in images of the microtubule network and caveolae p g ] In Fig. S2A-C, the qualitative N2N and L2L results are shown for images of AC-15 dependent on the training loss function (L1, L5S-SSIM, L3S-SSIM or LSSIM), including the corresponding benchmarks (phalloidin) for L2L and 20-frame average images of both labels to better assess the network performance. For both methods, using an L1 for the training leads in comparison to more conservative predictions, in which, with L2L, non-filamentous signal was reduced by the network but actin filaments appeared relatively blurry. On the other hand, predictions by a CNN after In Fig. S2A-C, the qualitative N2N and L2L results are shown for images of AC-15 dependent on the training loss function (L1, L5S-SSIM, L3S-SSIM or LSSIM), including the corresponding benchmarks (phalloidin) for L2L and 20-frame average images of both labels to better assess the network performance. For both methods, using an L1 for the training leads in comparison to more conservative predictions, in which, with L2L, non-filamentous signal was reduced by the network but actin filaments appeared relatively blurry. On the other hand, predictions by a CNN after To further study L2L as a method to increase image contrast of distinct cellular structures, fluorescence image pairs were acquired of the microtubule network that has a distinct branched spatial distribution in cells, and caveolae that are 60-100 nm large invaginations in the plasma membrane (Bates et al., 2007; Khater et al., 2018). For the former, fixed MeT5A cells were dual labelled with two monoclonal antibodies against α-tubulin (DM1A raised in mouse and YOL1/34 raised in rat), and confocal image pairs (N=51) 4 4 Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE Table 2. Training settings Dataset Actin Tubulin Caveolae PXN SYTOX plus CD44 Input marker AC-15 DM1A D1P6W 5H11 Superposed‡ Benchmark marker Phalloidin YOL1/34 4H312 Y113 SYTOX/CD44‡ Number of raw image pairs 68+272* 51+204* 60+240* 77+308* 58 Size of raw images (4096 pixels)2 (4096 pixels)2 (7730 pixels)2 (4096 pixels)2 (2608 pixels)2 Percentiles for normalisation 1/99.9 1/99.9 2/99.5 1/99.9 2/99.8 Number of validation/training patches (128 pixels)2 13,926/125,338 10,445/94,003 18,432/165,888 15,770/141,926 11,878/106,906 Learning rate 2×10−4 2×10−4 1×10−5 2×10−4 2×10−5 Batch size/steps per epoch/epoch number 32/100/400 32/100/500 16/500/100 32/100/500 32/1000/250 *augmented images, ‡ two channel images. Overview of the training data and settings in the CSBDeep framework for L2L training. Training networks to reduce cytosolic content in images of PXN with paired and unpaired images p p g We also trained a CNN with image pairs of two non-identical labels against the focal adhesion protein paxillin (PXN) (Martino et al., 2018), with the aim to reduce immunofluorescence signal in the images that stems from cytosolic protein. Image pairs of fixed MeT5A (N=47), HeLa (N=17) and U2OS (N=13) cells, dual labelled with the monoclonal anti-PXN antibodies 5H11 and Y113, were acquired. As expected, the raw immunofluorescence images of both antibodies showed correlating focal adhesion structures in the cells but also a diffuse signal throughout the cell body that differed in relative intensity to the focal adhesion signal between the two clones (compare Fig. 3A, left, and Fig. 3B). For all cell lines, we observed the same trend; the images of antibody Y113 showed a higher focal adhesion-to-cytosolic signal ratio than images of 5H11. To determine whether the cytoplasmic signal originated from clone-dependent binding to protein in the cytosol or unspecific binding, short hairpin (sh)RNA-mediated PXN knockdown MeT5A cells were generated (see ‘Generation and verification of knockdown PXN MeT5A cells’ in Materials and Methods). The knockdown was confirmed via a qPCR analysis that showed a knockdown efficiency of >70% on mRNA level. Additionally, PXN protein levels were substantially lower in the shRNA PXN cells, as analysed by western blotting (Fig. S6A,B). For both antibodies, the immunofluorescence signal of PXN was reduced in images of fixed shPXN cells in both the focal adhesions and in the cytoplasm (Fig. S6C). Hence, the cytoplasmic signal that is observed in images of both clones is largely not caused by unspecific binding. Instead, the different relative cytosolic content in cell images of 5H11 and Y113 indicates a differing accessibility of the respective binding sites for both clones for protein that is cytosolic or bound to focal adhesions, and that cytosolic PXN likely functions as a readily available replenishable buffer for focal adhesion-localised PXN. py ( g ) The STED image pairs that we acquired of cells that were dual- labelled for CAVIN-1 (D1P6W) and CAV1 (4H312) showed a low signal-to-noise ratio, further amplified by the pre-processing step that was undertaken to prevent artefacts that we observed when training with the nearly binary raw data (see ‘Data augmentation and pre-processing’ in Materials and Methods; Fig. 2C,D). Moreover, the correlation of the fluorescence signal originating from caveolae structure was relatively low between the training input (D1P6W) and benchmark (4H312) for some image pairs (Fig. L2L to enhance the structural contrast in images of the microtubule network and caveolae The restoration of the cell image of DM1A that was acquired with a different imaging setup than the training input exhibited a slightly less homogeneous intensity distribution along the microtubules, likely due to the changed image noise (compare Fig. 2A, left). For N2N and L2L, the restoration success is dependent on the tubule density in the image. The closer microtubules are packed in the cell, the less likely the successful recovery of separate structure by the CNN, as evident by comparing the results of both methods with the corresponding STED image (see also Fig. S4A,B). Notably, the intensity fluctuations along the cellular structure in the benchmark (YOL1/34) for L2L do not result in artefacts in restorations of the input (DM1A). Further N2N and L2L results for representative training inputs are shown in Fig. S2E, after using an L1, L5S-SSIM, L3S-SSIM or LSSIM for the training. A loss function-dependent trend was observable for both methods: using an LMS-SSIM instead of an L1, the CNN learned (with decreasing M) to restore microtubules with increased sharpness, especially when trained with images of two non-identical labels (Fig. S2E). This effect was quantifiable; extracted full width at half maxima (FWHMs) of line profiles across single microtubules in the images showed that results obtained with L2L were closest to the microtubule diameters detected with STED microscopy (Fig. S4C). more pronounced when using the loss function with a small number of iterations (M) (compare N2N results for LSSIM and L5S-SSIM in Fig. S5B). Moreover, weak stripe-like artefacts were introduced by the network after N2N training (Fig. 2C). Although caveolae structures appeared slightly sharper than in the L2L results, the level of sharpness that was observed in the N2N result was not verifiable in the 20-frame average image (Fig. 2C, bottom). Again, the network performance was evaluated after L2L training with different loss functions by calculating the average PSNR, NRMSE and MS-SSIM indices (Table 1). For both datasets, the calculated metrics indicated that the correlation between reconstructed and benchmark image was highest after training with an L5S-SSIM. For the tubulin dataset, a decrease in the correlation between the restored image of DM1A and the benchmark (YOL1/ 34) after training with a L1 was observed. L2L to enhance the structural contrast in images of the microtubule network and caveolae For N2N and L2L, the restoration success is dependent on the tubule density in the image. The closer microtubules are packed in the cell, the less likely the successful recovery of separate structure by the CNN, as evident by comparing the results of both methods with the corresponding STED image (see also Fig. S4A,B). Notably, the intensity fluctuations along the cellular structure in the benchmark (YOL1/34) for L2L do not result in artefacts in restorations of the input (DM1A). Further N2N and L2L results for representative training inputs are shown in Fig. S2E, after using an L1, L5S-SSIM, L3S-SSIM or LSSIM for the training. A loss function-dependent trend was observable for both methods: using an LMS-SSIM instead of an L1, the CNN learned (with decreasing M) to restore microtubules with increased sharpness, especially when trained with images of two non-identical labels (Fig. S2E). This effect was quantifiable; extracted full width at half maxima (FWHMs) of line profiles across single microtubules in the images showed that results obtained with L2L were closest to the microtubule diameters detected with STED microscopy (Fig. S4C). The STED image pairs that we acquired of cells that were dual- labelled for CAVIN-1 (D1P6W) and CAV1 (4H312) showed a low signal-to-noise ratio, further amplified by the pre-processing step that was undertaken to prevent artefacts that we observed when training with the nearly binary raw data (see ‘Data augmentation and pre-processing’ in Materials and Methods; Fig. 2C,D). Moreover, the correlation of the fluorescence signal originating from caveolae structure was relatively low between the training input (D1P6W) and benchmark (4H312) for some image pairs (Fig. S5A,C). This was also indicated by the calculated mean PSNR, NRMSE and MS- SSIM indices for both images, which suggested the lowest correlation among the L2L datasets that were generated in this work (compare Table 1). It posed the question as to whether a CNN would be prone to introduce artificial structure after L2L training with this dataset. RESEARCH ARTICLE Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE compared to the standard confocal image and are therefore a better reference for assessing the restoration performance of a CNN for this sample in ROIs in which microtubules are densely packed (Bates et al., 2007). L2L to enhance the structural contrast in images of the microtubule network and caveolae Comparing the restorations by a CNN after ‘classical’ N2N training with an L1 with the L2L result after employing an L3S−SSIM for the training, a clear improvement of the contrast of the microtubule network with L2L was observed (Fig. 2A). Here, both methods outperformed the corresponding 20- frame average image (Fig. 2A, top; Fig. S2D,F) and classical image processing methods, such as a Gaussian or top-hat filter (Fig. S3, Table S1). The restoration of the cell image of DM1A that was acquired with a different imaging setup than the training input exhibited a slightly less homogeneous intensity distribution along the microtubules, likely due to the changed image noise (compare Fig. 2A, left). For N2N and L2L, the restoration success is dependent on the tubule density in the image. The closer microtubules are packed in the cell, the less likely the successful recovery of separate structure by the CNN, as evident by comparing the results of both methods with the corresponding STED image (see also Fig. S4A,B). Notably, the intensity fluctuations along the cellular structure in the benchmark (YOL1/34) for L2L do not result in artefacts in restorations of the input (DM1A). Further N2N and L2L results for representative training inputs are shown in Fig. S2E, after using an L1, L5S-SSIM, L3S-SSIM or LSSIM for the training. A loss function-dependent trend was observable for both methods: using an LMS-SSIM instead of an L1, the CNN learned (with decreasing M) to restore microtubules with increased sharpness, especially when trained with images of two non-identical labels (Fig. S2E). This effect was quantifiable; extracted full width at half maxima (FWHMs) of line profiles across single microtubules in the images showed that results obtained with L2L were closest to the microtubule diameters detected with STED microscopy (Fig. S4C). compared to the standard confocal image and are therefore a better reference for assessing the restoration performance of a CNN for this sample in ROIs in which microtubules are densely packed (Bates et al., 2007). Comparing the restorations by a CNN after ‘classical’ N2N training with an L1 with the L2L result after employing an L3S−SSIM for the training, a clear improvement of the contrast of the microtubule network with L2L was observed (Fig. 2A). Here, both methods outperformed the corresponding 20- frame average image (Fig. 2A, top; Fig. S2D,F) and classical image processing methods, such as a Gaussian or top-hat filter (Fig. S3, Table S1). L2L to enhance the structural contrast in images of the microtubule network and caveolae Loss function-dependent L2L and N2N results for images of the microtubule network and caveolae. (A-D) Confocal images of MeT5A cells that were dual labelled with the anti-tubulin antibodies DM1A (A) and YOL1/34 (B), and STED images of MeT5A cells that were dual labelled with the anti-CAVIN-1 antibody D1P6W (C) and the anti-CAV1 antibody 4H312 (D). (A,C) From left to right: raw image of a representative training input, reconstructed images after N2N and L2L training with an L1 or L3S-SSIM loss function, and a corresponding 20-frame average or high resolution STED image. (B,D) Representative training benchmarks for L2L training are displayed. Images shown were excluded from the network training. Scale bars: 1 µm (A,B); 200 nm (C,D). Journal of Cell Science Fig. 2. Loss function-dependent L2L and N2N results for images of the microtubule network and caveolae. (A-D) Confocal images of MeT5A cells that were dual labelled with the anti-tubulin antibodies DM1A (A) and YOL1/34 (B), and STED images of MeT5A cells that were dual labelled with the anti-CAVIN-1 antibody D1P6W (C) and the anti-CAV1 antibody 4H312 (D). (A,C) From left to right: raw image of a representative training input, reconstructed images after N2N and L2L training with an L1 or L3S-SSIM loss function, and a corresponding 20-frame average or high resolution STED image. (B,D) Representative training benchmarks for L2L training are displayed. Images shown were excluded from the network training. Scale bars: 1 µm (A,B); 200 nm (C,D). 5 compared to the standard confocal image and are therefore a better reference for assessing the restoration performance of a CNN for this sample in ROIs in which microtubules are densely packed (Bates et al., 2007). Comparing the restorations by a CNN after ‘classical’ N2N training with an L1 with the L2L result after employing an L3S−SSIM for the training, a clear improvement of the contrast of the microtubule network with L2L was observed (Fig. 2A). Here, both methods outperformed the corresponding 20- frame average image (Fig. 2A, top; Fig. S2D,F) and classical image processing methods, such as a Gaussian or top-hat filter (Fig. S3, Table S1). The restoration of the cell image of DM1A that was acquired with a different imaging setup than the training input exhibited a slightly less homogeneous intensity distribution along the microtubules, likely due to the changed image noise (compare Fig. 2A, left). Training networks to reduce cytosolic content in images of PXN with paired and unpaired images (A) From left to right: raw image of 5H11, the restored images by a CNN/CycleGAN er L2L training with paired/unpaired images, and the corresponding image of Y113 (B). Scale bar: 20 µm. (C) Training results for two ROIs (6 µm×6 µm). From t to right: input (5H11), the restored images by a CNN after N2N and L2L training with an L1 loss function, the restored image by a CycleGAN, and a 20-frame erage. (D) Corresponding benchmark images (Y113) for L2L training and its predictions by a CNN after L2L training as outlined above. A CNN that was trained h L2L data in-paints focal adhesions (see white arrowheads) and reduces cytosolic protein (see ROI number 2) for both the training input and benchmark. ages shown were excluded from the network training. Fig. 3. Network architecture-dependent L2L results for images of PXN. Confocal images of a HeLa cell dual labelled with the two anti-PXN antibodies 5H11 and Y113, which were used as training input and benchmark for L2L training. (A) From left to right: raw image of 5H11, the restored images by a CNN/CycleGAN after L2L training with paired/unpaired images, and the corresponding image of Y113 (B). Scale bar: 20 µm. (C) Training results for two ROIs (6 µm×6 µm). From left to right: input (5H11), the restored images by a CNN after N2N and L2L training with an L1 loss function, the restored image by a CycleGAN, and a 20-frame average. (D) Corresponding benchmark images (Y113) for L2L training and its predictions by a CNN after L2L training as outlined above. A CNN that was trained with L2L data in-paints focal adhesions (see white arrowheads) and reduces cytosolic protein (see ROI number 2) for both the training input and benchmark. Images shown were excluded from the network training. background signal in the training benchmark (Y113), although these images were not used as input for L2L training (Fig. 3D). In Fig. 3A, the restorations of a representative training input (5H11) by a CNN (using an L1) and a CycleGAN are shown after L2L training with aligned and unaligned images, respectively. Both networks learned to reduce cytosolic content in images of 5H11 and selectively recovered focal adhesion structure, but the CNN outperformed the CycleGAN for this task. Training networks to reduce cytosolic content in images of PXN with paired and unpaired images S5A,C). This was also indicated by the calculated mean PSNR, NRMSE and MS- SSIM indices for both images, which suggested the lowest correlation among the L2L datasets that were generated in this work (compare Table 1). It posed the question as to whether a CNN would be prone to introduce artificial structure after L2L training with this dataset. We found that the CNN performance after N2N and L2L training was highly dependent on the training loss function (Fig. 2C). Using an L1, in both methods, a trained CNN only learned to restore structures in high signal-to-noise regions in the input image (compare Fig. 2C, top and bottom). However, when the training loss function was replaced with an L3S-SSIM, the recovery of structural signal by the CNN from the noisy input (DM1A) was much enhanced for N2N and L2L. Here, cytosolic background signal in the image was clearly reduced by the network after L2L training, resulting in restorations with higher sample-to-background ratios than in corresponding 20-frame average STED images of both labels (Fig. 2; Fig. S5A-C). For N2N, on the other hand, significant hallucination effects were observed after using an L3S-SSIM for the training, with unspecific cytosolic signals often recovered as structure-like content (Fig. 2C, top). These artificial features got Journal of Cell Science All cell lines were prepared and imaged under the same conditions, and were used indistinguishably to generate the training data (see ‘Training the CNN’ section in Materials and Methods). For the cell images of 5H11/Y113 (N=77), mean Michelson contrast values of 0.57±0.07/0.99±0.01, and mean±s.d. RMS contrast values of 0.15 ±0.01/0.18±0.02 were calculated. Cell images of 5H11/Y113 were used as input/benchmark to train a network to decrease cytosolic signal in images of focal adhesions. To test whether an artificial network can also be trained with unpaired L2L data, a CycleGAN was trained and its performance was compared to the CNN (see ‘Training the CycleGAN’ in Materials and Methods). Further results for other datasets are shown in Fig. S7. 6 RESEARCH ARTICLE Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE g. 3. Network architecture-dependent L2L results for images of PXN. Confocal images of a HeLa cell dual labelled with the two anti-PXN antibodies 5H11 d Y113, which were used as training input and benchmark for L2L training. Training networks to reduce cytosolic content in images of PXN with paired and unpaired images In the generated image of the CycleGAN, high intensity areas in the cell cytoplasm that did not stem from focal adhesions were, in part, still present in the predicted images with a tendency to translate intensity fluctuations in the input as weak structure (Fig. 3C, bottom). A similar effect was observed after training a CNN with an LMS-SSIM with N2N data (Fig. S5E). Here, intensity fluctuations in the cytosol were, as observed with the caveolae dataset (see previous section), artificially accentuated in the restorations, which became more pronounced with a small M, leading to very different qualitative N2N results after using an L1 and an LSSIM. These artefacts were much less pronounced in the L2L results (Fig. S5E). Training a CNN to separate cellular structures in superposed images Scale bar: 10 µm. The image pairs were obtained via sequential imaging by changing the excitation wavelength. (B) Qualitative result for an ROI (5 µm×5 µm). Prediction success is dependent on the level of superposition of both labels. Structures appear slightly blurry in the restorations compared to the benchmark, but image noise and jitter are reduced. The images shown were excluded from the training. Fig. 4. Qualitative results after training a CNN to separate cellular structures in superposed images of a nuclear stain and an antibody against a plasma membrane protein. (A) Training input and benchmark images of a MeT5A cell that was dual labelled with the nuclear stain SYTOX Green and an anti-CD44 antibody, and corresponding reconstructions after training a CNN with an L3S-SSIM. Scale bar: 10 µm. The image pairs were obtained via sequential imaging by changing the excitation wavelength. (B) Qualitative result for an ROI (5 µm×5 µm). Prediction success is dependent on the level of superposition of both labels. Structures appear slightly blurry in the restorations compared to the benchmark, but image noise and jitter are reduced. The images shown were excluded from the training. (validation) and the training data, making it impossible to assess how much the network performance depends on whether a specific image pair is used for the training. in the restorations, and overlapping structures in the nuclear area were mostly, but not fully, recovered in the images of CD44. A loss function-dependent evaluation of the network performance showed no clear trend as to which loss function was best suited to train the CNN as a separator of the two structures (Table 1). The mean relative change of the NRMSE and 5S-SSIM indices between the input and the restored image (both versus the training benchmark) is displayed in Fig. 5. Each data point represents the mean value of a cross validation dependent on N. A Gaussian filter (sigma=1.5) was applied to images of caveolae prior to the calculations, as noise levels were so high in the raw input images that no trend was observable for unprocessed data. The results for nearly all datasets showed a similar trend: even when using a small N, the structural similarity to the benchmark increased for restored images compared to the input. Training a CNN to separate cellular structures in superposed images g Lastly, the ability of a CNN to transfer style between immunofluorescence images with correlating structural signal was tested by training a network to separate superposed confocal images of two different cellular targets. For that, fixed MeT5A cells were stained with the nuclear marker SYTOX Green and labelled with an antibody against CD44, a plasma membrane protein. Then, image pairs (N=58) were acquired of both markers (superposed) and of each marker separately (see the imaging section in Materials and Methods). The spatial distribution in the cell of both targets partly overlapped in the images but the targets were structurally distinguishable, with the nucleus appearing with high intensity in the cell centre and CD44 distributed at the periphery of the cell (Fig. 4A). The CNN was trained with the superposed image of both the nucleus and the plasma membrane as training input in a two channel image, and the separate cell images of SYTOX and CD44, respectively, as training benchmark (see ‘Training the CNN’ in Materials and Methods). The qualitative results in Fig. 4 for an example image pair show that a CNN separated the nucleus and plasma membrane marker in the input image successfully after the training. Noticeably, similar to N2N, image jitter and noise that were present in the input image were removed by the network in the restorations (Fig. 4B). However, the CNN slightly blurred structure Journal of Cell Science Contrary to the results of the evaluation (Table 1) that, again, indicated the highest network performance after using an L5S-SSIM, subjectively, training the CNN with an L1 led to the best L2L results overall. Cytosolic signal was significantly lowered in the restorations after L2L training compared to the 20-frame average image or N2N result, and focal adhesions were selectively recovered and appeared with increased contrast (Fig. 3C). Also, the trained CNN in-painted focal adhesion structures that were inhomogeneously labelled (see white arrowheads in Fig. 3C). In addition, we found that the trained CNN reduced cytosolic 7 7 Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE Fig. 4. Qualitative results after training a CNN to separate cellular structures in superposed images of a nuclear stain and an antibody against a plasma membrane protein. (A) Training input and benchmark images of a MeT5A cell that was dual labelled with the nuclear stain SYTOX Green and an anti-CD44 antibody, and corresponding reconstructions after training a CNN with an L3S-SSIM. Training a CNN to separate cellular structures in superposed images However, the calculated ΔNRMSE and Δ5S-SSIM were dependent on the particular images that are used for the training – indicated by a wide distribution for small N. This could indicate that certain acquired image data are better suited for training the network, or easier to predict for the network. As expected, on average, the highest performance was achieved using a high N. Although the results for Δ5S-SSIM indicate that with N>30 or 32 the network performance was relatively consistent for all datasets, the ΔNRMSE improved continuously with increasing N. Here, the trend of ΔNRMSE deviated for the caveolae dataset for which, altogether, only slight changes were observed between different N, without a clear trend. Testing the robustness of L2L g The robustness of L2L training was evaluated for the actin (Ntot=68), tubulin (Ntot=51), caveolae (Ntot=60) and PXN (Ntot=77) datasets by analysing how much the network performance was impacted by using specific image pairs for the training data generation. To carry this out, repeated cross validations were conducted with randomly selected image pairs N from the total dataset (Ntot). For the actin and PXN dataset, we conducted 8-fold cross validations with N=8, 16, 32 and 64; for the tubulin and caveolae dataset, we conducted 10-fold cross validations with N=10, 30 and 50. We increased the number of repetitions for small N, and the epoch number for the training was linearly adapted to N to prevent overfitting (see ‘Training the CNN’ in Materials and Methods; Table 3). The difference between this approach and the default validation in the CSBDeep framework is that in the latter the validation is conducted via a train/test split of the training data, which is generated from all raw image pairs. This way, image patches generated from a specific image pair appear in both, the test Journal of Cell Science Table 3. Settings for repeated cross validations Dataset Actin (N=68)/PXN (N=77) 8-fold cross validations Tubulin (N=51)/Caveolae (N=60) 10-fold cross validations Number of repetitions 10 5 3 2 8 3 2 Total number of raw image pairs 8 16 32 64 10 30 50 Loss function L3S-SSIM/L1 L3S-SSIM Learning rate/batch size/steps per epoch 5×10−4/32/150 5×10−4/1×10−5/32/150 Epoch number 20 40 60 120 20 60 100 Overview of the settings in the CSBDeep framework that were used for repeated 8-fold or tenfold cross validations, training the network for L2L with the different datasets. Table 3. Settings for repeated cross validations Table 3. Settings for repeated cross validations 8 Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 RESEARCH ARTICLE Fig. 5. Repeated cross validation for L2L training. The mean relative change (input/benchmark versus restoration/benchmark) of the NRMSE and 5S-SSIM index after L2L training with image pairs of different cellular structures. Boxes represent 25th and 75th percentiles with median, whiskers represent standard deviations. The image pairs for the trainings were generated of cells that were dual labelled for the actin cytoskeleton (Ntot=68), tubulin (Ntot=51), caveolae (Ntot=60) or PXN (Ntot=77), dependent on the number of raw image pairs that were randomly selected from the total dataset for the cross validation. DISCUSSION L2L is different to restoration methods that use images of the same label as training data, e.g. N2N. After N2N training a CNN restores all signals originating from the label without distinction, whereas a CNN systematically learns to lower non-structural signals after L2L training. We found that a network trained with L2L data restores images with high contrast of the target structure, even compared to the training benchmark or its high frame average image (see, for example, Fig. 2A,C). Such a CNN further improved the images that were used as the benchmark for the training (Fig. 3D; Fig. S8). g ( g ; g ) The network performance is dependent on the level of correlation between the images of the two labels and the training loss functions. Using a single-scale SSIM loss function for the training increased the likelihood of hallucination effects in restored images after L2L training, especially for the PXN dataset in which cytosolic content in the cell body was not entirely without structure and present in both training input and output. However, an LMS-SSIM was better suited for training a network to restore sharp cellular structures. Notably, the restorations increasingly (with M) converged to results obtained with an L1 (Fig. S2, Fig. S5). For images of actin, tubulin and caveolae we determined that the best results were obtained after using an L3S-SSIM. Here, unspecific background signals differed sufficiently between the images of both labels. For the PXN dataset, an L1 was more suitable for training a CNN to recover focal adhesions only, likely due to the correlating cytosolic signal in the training images, albeit both at different relative intensity to the focal adhesion signal (Fig. 3A,B). ( ) ( g ) Although clean benchmark data are not required for L2L and N2N training, in images of structures that are not sufficiently resolved by the imaging technique, either a posteriori knowledge about the structure or clean reference data are required to assess the qualitative performance of a trained CNN. This was especially noticeable in cell images of tubulin, in which the fine microtubule network is not fully resolved with confocal microscopy (Fig. 2A,B). We observed erroneous predictions by a CNN in image regions where the microtubule network was densely packed after both N2N and L2L training. Testing the robustness of L2L Each data point is the mean value for an eightfold (actin and PXN) or tenfold (tubulin and caveolae) cross validation that was repeated for small image pair numbers. Fig. 5. Repeated cross validation for L2L training. The mean relative change (input/benchmark versus restoration/benchmark) of the NRMSE and 5S-SSIM index after L2L training with image pairs of different cellular structures. Boxes represent 25th and 75th percentiles with median, whiskers represent standard deviations. The image pairs for the trainings were generated of cells that were dual labelled for the actin cytoskeleton (Ntot=68), tubulin (Ntot=51), caveolae (Ntot=60) or PXN (Ntot=77), dependent on the number of raw image pairs that were randomly selected from the total dataset for the cross validation. Each data point is the mean value for an eightfold (actin and PXN) or tenfold (tubulin and caveolae) cross validation that was repeated for small image pair numbers. DISCUSSION powerful loss functions, such as the LMS-SSIM, are only of limited use (e.g. the N2N results in Fig. S5B,E). Using an LMS-SSIM for N2N training led to a significantly higher occurrence of artefacts compared to L2L, in which cytosolic non-structural content (as present in the training input for the actin, caveolae and PXN datasets) was restored with accentuated artificial contrast. However, the qualitative N2N and L2L results for the caveolae dataset indicate that a CNN picks up structure much more efficiently in very noisy images when using an L3S-SSIM instead of an L1 for the training (Fig. 2C). But using two STED images of the same label (D1P6W) as training data caused the network to hallucinate significantly when using an LMS-SSIM, with cytosolic background signals recovered as structure. Also, some N2N results exhibited artefacts that might originate from static image corruptions introduced by the imaging system itself, which then would be present in both noise realisations of a sample (Fig. 2C; Fig. S5B). A clear improvement between the restored images after N2N training with an L1 and an L3S-SSIM, respectively, is only observed for the tubulin dataset, in which the background signal appears very unspecific in images of the tubulin marker (DM1A) (Fig. 2A). We show that a CNN can be successfully trained to reduce unspecific label-induced fluorescence signals detected in the cell cytoplasm in immunofluorescence images of cellular structures, requiring images of two non-identical labels of a target for the training that exhibit systematic sample differences. L2L is different to restoration methods that use images of the same label as training data, e.g. N2N. After N2N training a CNN restores all signals originating from the label without distinction, whereas a CNN systematically learns to lower non-structural signals after L2L training. We found that a network trained with L2L data restores images with high contrast of the target structure, even compared to the training benchmark or its high frame average image (see, for example, Fig. 2A,C). Such a CNN further improved the images that were used as the benchmark for the training (Fig. 3D; Fig. S8). We show that a CNN can be successfully trained to reduce unspecific label-induced fluorescence signals detected in the cell cytoplasm in immunofluorescence images of cellular structures, requiring images of two non-identical labels of a target for the training that exhibit systematic sample differences. DISCUSSION A higher performance might be achieved with cleaner reference images. The ability of a CNN to selectively restore specific cell structure is also highlighted in this work by training a CNN to separate a nuclear marker and a plasma membrane protein label in superposed immunofluorescence images (Fig. 4). Our results show that CNNs could be used in the future to separate the fluorescence signals from multiple markers in microscopy images that were acquired with imaging setups that have a limited number of excitation sources or detectors. L2L could also be applied in multiplex imaging experiments if antibodies are not selected based on performance but compatibility issues between the species in which they are raised. Moreover, L2L could be considered for post-processing in live-cell imaging, in which high-performance labels are rare. Training data can be generated post image acquisition in vitro by fixing the cells and labelling with a higher performing antibody against the target structure. Our results for the caveolae dataset suggest that training a CNN with L2L data might be particularly advantageous to restore noisy images, as it allows the implementation of an MS-SSIM loss function without introducing artefacts that we otherwise observed after training with images of the same label (Fig. 2C; Fig. S5B). Here, L2L results exhibit a high structure-to-background signal ratio, clearly outperforming high- frame average images that were acquired with the STED microscope. As expected, the evaluation of repeated eightfold/tenfold cross validations (dependent on the number of raw image pairs that were used to generate the training data) showed that using a high number of image pairs to train a CNN for the style transfer between two labels is advisable (Fig. 5). The mean PSNR and 5S-SSIM index significantly increased even after L2L training with a small number of image pairs, but the results were dependent on the selected image pairs. Although the evaluation would be more meaningful if each cross validation had been conducted with unique image pairs instead of image pairs but that were randomly selected from a finite dataset, the results indicate that a network converges to the optimal result of a particular dataset during training (Fig. 5). p g g ( g ) Initially, it was unclear whether the difference in the training data would increase hallucinations by a CNN after L2L training. For instance, in the caveolae dataset, the sample differences between input and output were quite significant (compare Fig. DISCUSSION Further, the sharpening of structure with L2L after using an MS-SSIM loss function for the training – although advantageous for images of tubulin (Fig. S4C) for which we know that the true microtubule diameter is not resolved with confocal microscopy – may be less desirable in images of other cell structures. Journal of Cell Science Thus far, most deep learning-based image restoration methods have relied on image pairs of the same label that were acquired under different imaging conditions. Here, our N2N results suggest that 9 RESEARCH ARTICLE Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 labels were good indicators for assigning labels to ‘input’ and ‘benchmark’. Here, training a CNN with the reverse order or pairing the labels in both directions resulted in either worse or comparable prediction success. We found that metrics such as the PSNR and NRMSE are inadequate to forecast which loss function yields the best restorations by a CNN after L2L training, and this is exacerbated by the unavailability of clean reference images of the respective target structure. For instance, the calculated metrics for the L2L results for all datasets barely deviated for different LMS-SSIM, although, qualitatively, differences in the restorations were observed (Table 1). Additionally, although the calculated SSIM indices indicate scarcely any correlation between the input and benchmark, with values around 0.05-0.15 for all L2L datasets, the correlation was higher according to the 5S-SSIM indices (by a factor of ∼3). Therefore, a high-scale SSIM index might be more suitable for detecting correlation in fluorescence images of cellular structure than other metrics, likely because microscopy images are the convolved signal of a sample volume rather than the strict two- dimensional depiction of a sample at a specific section. This observation fits with the MS-SSIM index theory (Wang et al., 2003). We also trained a CycleGAN with unaligned label pairs of a target structure (Fig. 3A,C; Fig. S7). Although the generated images of a trained CycleGAN exhibited reduced unspecific cytosolic signals, it was outperformed by a trained CNN. As the generator in the CycleGAN is trained to fool a discriminator based on noisy benchmarks, either little to no change to the input image was observed (tubulin/caveolae) or artefacts were introduced (actin/ PXN) by the network to match the style of the reference image. Prior denoising of the images via Gaussian filtering led to slightly better results (Fig. S7). DISCUSSION S5A,C), and inhomogeneous labelling of the microtubule structure by the anti- tubulin antibody YOL1/34 (benchmark) was not visible in the training input (DM1A) (compare Fig. S2D,F). Yet, we could not observe that the CNN introduced artificial structure in the restored images after L2L training that resembled either trend. Instead, our results suggest that sample differences can be advantageous when training with an LMS-SSIM to lower the occurrence of hallucination effects observed after N2N training. However, an in-painting effect was observed for L2L, primarily in restorations of AC-15 (actin; Fig. 1C,D) and 5H11 (PXN; see Fig. 3C,D). Consequently, L2L can be used to correct images for inhomogeneous binding of a stain or antibody but restorations cannot be used to quantify the distribution of a target protein in a cell. Instead, L2L could serve as an image pre- processing step to extract the binary information about the location of a structure in the cell (see examples in Fig. S8). Notably, the systematic recovery of specific structure and the adaptability of L2L to images of a multitude of targets potentially makes L2L superior to classical image processing methods (as shown in Fig. S3 and Table S1). In conclusion, we present a new deep learning-based image restoration method for images of cellular structures that utilises the varying performance of labels in immunofluorescence microscopy; we call this method L2L. With L2L, we show that by training a CNN for a style transfer between two non-identical labels of a shared target, the network can be systematically trained to reduce unspecific cytosolic background signals and enhance structural contrast in immunofluorescence images. Like other methods, L2L relies on the convention of the network to underestimate inherently unpredictable signal. However, with L2L, not only image noise but also label-induced fluorescence signals in the cell specimen can be reduced in the images after selecting appropriate training data. The ability to significantly lower unspecific binding, inhomogeneous labelling of a structure or binding to cytosolic protein in immunofluorescence images makes L2L, to our knowledge, unique in comparison to other deep learning-based image restoration methods that are currently used in cell biology. Journal of Cell Science Training data for L2L can be generated with one imaging setup using two detectors simultaneously, which makes the images independent of stage drift and sample dynamics, and is time efficient. DISCUSSION However, contrary to N2N, L2L requires sample preparation with two markers that exhibit systematic differences in the respective images to allow training for a useful style transfer between labels. Therefore, not all label pairs of a target structure are suitable for generating the necessary training data. We found that the calculated RMS and Michelson contrast values for images of two Confocal microscopy py Image pairs of the immunofluorescence sample labelled for the actin cytoskeleton, tubulin and PXN, as well as SYTOX Green and CD44, were taken using a commercial confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems), using a HC PL Apo 63×/1.4 N.A. CS2 objective with no digital zoom (for image sizes see Table 2). To acquire data for L2L training, both markers in the individual sample were imaged simultaneously using the two in-built photomultiplier detectors, each equipped with a prism-based tunable spectral filter. Each field of view was imaged twice to acquire the two noise realisations of the samples that were used for N2N training of a CNN. The sample dual labelled for actin was excited with a 488 nm and 552 nm laser line, and the two spectral detectors were set to detect light between 495-540 and 560-700 nm. The samples dual labelled for tubulin and PXN were excited with a 488 nm and 638 nm laser line, and the two spectral detectors were set to detect light between 495-620 nm and 645-750 nm. The immunofluorescence sample labelled for SYTOX Green and CD44 was imaged by setting the spectral range of the detector to 560- 660 nm. Separate images of SYTOX and CD44 were acquired by exciting the sample with a 488 and 552 nm laser line, respectively. A corresponding superposed image of both markers was acquired by exciting the sample with both lasers simultaneously. Generation and verification of knockdown paxillin MeT5A cells Knockdown of MeT5A cells was achieved by a shRNA-mediated knockdown using pKLO.1 vectors coding for shRNAs targeting paxillin (TRC N0000123137) or control. HEK293T cells were used for virus generation, and the virus was harvested, filtered and added to polybrene- treated MeT5A cells. The retroviral transduction of MeT5A cells was followed by puromycin selection. Stable knockdown cell lines were verified by qPCR (mRNA abundance) and western blots (protein abundance). For western blotting, cell lysates were prepared in reducing lysis buffer, boiled for 10 min and separated on conventional homemade SDS gels, and developed onto Medical Blue Sensitive X-Ray Film (Scientific Laboratory Supplies). The PXN specific antibody Y113 was used (rabbit; ab32084, Abcam). Ponceau staining of the PVDF membrane was carried out to establish equal loading. For qPCR (RT-qPCR), mRNA was extracted from cells using an RNeasy Plus Mini kit (Qiagen). cDNA was generated using a High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems). Stimulated emission depletion microscopy Fixed MeT5A cells dual labelled for tubulin were further imaged with a Leica SP8 TCS 3X STED microscope, using a Leica HC PL APO 100×/1.40 Oil STED WHITE objective (15506378, Leica). The sample was excited with a supercontinuum white light laser at 488 nm and 633 nm for confocal imaging, and at 633 nm for STED imaging. For confocal imaging, two Leica photomultiplier tube detectors were set to detect light between 498- 600 nm and 643-743 nm. For STED imaging, a Leica HyD hybrid detector was set to detect light between 643-743 nm. STED depletion was performed using a Leica 775 nm depletion laser set to 50% with time gating from 0.3-8 ns. Pairs of confocal and STED images were acquired with a 15 nm pixel size. Sequential STED image pairs of the cell specimen labelled for caveolae were acquired by first exciting the sample at 646 nm and detecting light between 656-750 nm, then exciting at 591 nm and using a spectral range of 601-650 nm for the detection. The depletion laser power was set to 100% with time gating from 0.3-8 ns, and the pixel size was set to 10 nm. Sample preparation for immunofluorescence microscopy If not stated otherwise, cells were plated onto #1.5 coverslips a day prior to fixation, then fixed with 4% paraformaldehyde for 15 min at 37°C, followed by a permeabilisation step with 2.5% FBS and 0.3% Triton X-100 in PBS for 30 min at room temperature (Rausch and Hansen, 2019). HeLa cells that were labelled for actin were fixed 6 h after plating. The following antibodies or stains were used to generate cell specimens dual labelled for the same target structure: monoclonal anti-β-actin antibody AC-15 conjugated to Alexa Fluor 488 (1:250; mouse; ab6277, Abcam) and the phalloidin- Atto 565 stain (1:100; 94072, Sigma-Aldrich) to visualise the actin cytoskeleton in MeT5As; monoclonal antibodies YOL1/34 conjugated to Alexa Fluor 488 (1:500; rat; ab195883, Abcam) and DM1A (1:250; mouse; T6199, Sigma-Aldrich) for α-tubulin labelling in MeT5As; monoclonal antibodies 4H312 (CAV1) (1:200; mouse; sc-70516, Santa Cruz Biotechnology) and D1P6W (CAVIN-1) (1:200; rabbit; 69036, Cell Signaling Technology) for labelling caveolae in MeT5As; and monoclonal antibodies Y113 (1:250; rabbit; ab32084, Abcam) and 5H11 (1:500; mouse; MA5-13356, Life Technologies) for PXN labelling in MeT5A, U2OS and HeLa cells. Stimulated emission depletion microscopy Further, fixed MeT5A cells were labelled with a nuclear SYTOX Green marker (S7020, Life Technologies) and a monoclonal anti-CD44 antibody (1:100; rat; MA4400, Life Technologies). The following secondary antibodies were used: anti-rabbit IgG Alexa Fluor 488 (1:200; donkey; A-21206, Life Technologies) to conjugate Y113; anti- rat IgG Alexa Fluor 555 (1:100; goat; A-21434, Life Technologies) to conjugate the antibody against CD44; anti-mouse IgG Alexa Fluor 594 (1:200; goat; A32742, Life Technologies) to conjugate 4H312; anti-mouse IgG Alexa Fluor 633 (1:200; goat; A-11001, Life Technologies) to conjugate DM1A and 5H11; and anti-rabbit IgG Atto 647N (1:200; goat; 40839, Sigma-Aldrich) to conjugate D1P6W. All immunofluorescence samples were mounted in ProLong Glass Antifade Mountant (Life Technologies) onto microscope slides. Cell culture For imaging, the human mesothelial cell line MeT5A (ATCC CRL-9444), the adenocarcinoma cervical cancer cell line HeLa and the human osteosarcoma cell line U2OS were used. HeLa cells were a gift from Margaret Cunningham and U2OS cells were a gift from Kathryn McIntosh (both Strathclyde Institute 10 RESEARCH ARTICLE Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 of Pharmacy and Biomedical Sciences, Glasgow, UK). MeT5A cells were cultured in RPMI-1640 medium (Corning), supplemented with 10% (v/v) fetal bovine serum (FBS) (Labtech), 100 μg/ml penicillin-streptomycin (Gibco), 1 mM sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco) and 2 mM HEPES buffer solution (Gibco). HeLa and U2OS cells were cultured in Dulbecco’s modified Eagle medium (DMEM) plus GlutaMAX medium (Gibco) supplemented with 10% (v/v) FBS and 100 μg/ml penicillin-streptomycin. Human embryonic kidney cells HEK293T were grown in DMEM supplemented with 100 μg/ml penicillin-streptomycin and 2 mM L-glutamine. All cells were kept at 37°C/5% CO2 in a humidified atmosphere. of Pharmacy and Biomedical Sciences, Glasgow, UK). MeT5A cells were cultured in RPMI-1640 medium (Corning), supplemented with 10% (v/v) fetal bovine serum (FBS) (Labtech), 100 μg/ml penicillin-streptomycin (Gibco), 1 mM sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco) and 2 mM HEPES buffer solution (Gibco). HeLa and U2OS cells were cultured in Dulbecco’s modified Eagle medium (DMEM) plus GlutaMAX medium (Gibco) supplemented with 10% (v/v) FBS and 100 μg/ml penicillin-streptomycin. Human embryonic kidney cells HEK293T were grown in DMEM supplemented with 100 μg/ml penicillin-streptomycin and 2 mM L-glutamine. All cells were kept at 37°C/5% CO2 in a humidified atmosphere. Confocal microscopy qPCR was performed on 1 ng of cDNA using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) and an Applied Biosystems QuantStudio 5 Real-Time PCR System. Expression of paxillin was analysed and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT1) levels. qPCR data originate from five independent biological replicates, plotted in Prism (GraphPad) and analysed using Mann–Whitney. Image drift correction Fluorescence images that were taken sequentially were corrected for stage drift by estimating the extent of the displacement between the images. A total of 100 image pairs of size 60×60 pixels were generated for each image in a temporal image stack, after applying a Gaussian filter (sigma=2) and using the first detected image as benchmark. For that, the create_patches function from the CSBDeep framework was used, which cuts random image patch pairs in non-background areas of the image. For each created image patch, the SSIM index (filter size=11, sigma=1.5) was calculated by shuffling the uncorrected image patch relative to the benchmark patch by 5 pixels in all directions, effectively cutting both into 50×50-pixel patches. The shuffling vector that, on average, yielded the highest structural similarity between both images was selected as optimal position for the sequential image, and the images were cropped accordingly. Journal of Cell Science Author contributions Author contributions Conceptualization: L.S.K., C.G.H., G.M.; Methodology: L.S.K., O.S., C.G.H., G.M.; Software: L.S.K.; Validation: L.S.K., O.S.; Formal analysis: L.S.K., O.S.; Investigation: L.S.K., O.S., J.V.; Resources: C.G.H., G.M.; Data curation: L.S.K., O.S., J.V.; Writing - original draft: L.S.K., C.G.H., G.M.; Writing - review & editing: L.S.K., O.S., J.V., C.G.H., G.M.; Visualization: L.S.K., O.S.; Supervision: C.G.H., G.M.; Project administration: L.S.K., C.G.H., G.M.; Funding acquisition: C.G.H., G.M. Data augmentation and pre-processing Images were augmented to create more training data. To achieve this, the images were interpolated, using the zooming factors 0.5, 0.75, 1.25 and 1.5 (0.8, 0.9, 1.1 and 1.2 for the caveolae dataset), and then randomly rotated by 0°, 90°, 180° or 270°. STED images for the caveolae dataset were pre- processed by adding Poisson noise to broaden the image histogram that, due to being acquired with hybrid detectors, was nearly binary in the raw images, resulting in poor results for the network training. Prior to the training patch generation in the CSBDeep framework, a percentile normalisation was conducted (Table 2). 11 RESEARCH ARTICLE RESEARCH ARTICLE Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 References Bates, M., Huang, B., Dempsey, G. T. and Zhuang, X. (2007). Multicolor super- resolution imaging with photo-switchable fluorescent probes. Science 317, 1749-1753. doi:10.1126/science.1146598 Data availability D t d i i Data underpinning this publication are openly available from the University of Strathclyde KnowledgeBase at: https://doi.org/10.15129/ee2ff5ad-5bbd-459d- 953b-7d45b057fc05. Training the CNN To generate distance maps or binarised images (Fig. S8), the following pre-processing steps were undertaken using above mentioned functions: for images of actin, a rolling-ball background subtraction (radius=10), a top-hat filter (filter size=7) and CLAHE (tile size 11) were applied; for images of tubulin, a rolling-ball background subtraction (radius=10) and a top-hat filter (filter size=11) were applied; for images of caveolae, a Gaussian filter (sigma=0.75) and a rolling-ball background subtraction (radius=5) were applied; and for images of PXN, a rolling-ball background subtraction (radius=5) was applied. Lastly, objects below a size of 20 pixels (caveolae)/ 50 pixels (all else) were removed. Binary images were generated using the 75th/60th/93th/90th percentile as threshold for images of actin/tubulin/ caveolae/PXN. Distance maps were generated using scipy (Virtanen et al., 2020). For N2N and L2L training, the CSBDeep framework was used, which is a CNN with U-Net architecture (Ronneberger et al., 2015) that was developed for CARE in fluorescence microscopy (Weigert et al., 2018). The CSBDeep framework (version 0.6.0) was downloaded from GitHub and used with its default settings unless stated otherwise (https://github.com/CSBDeep/ CSBDeep). In the CSBDeep framework, training patches were generated randomly from the raw images after pre-processing (see previous section). Half of the training patches were generated from the raw images, and the other half from augmented images. Validation data were generated via a 90/10 train/test split, and the training and validation loss were monitored to rule out overfitting. A least absolute deviation loss function (L1) and different multiscale SSIM loss functions were used for the training [LSSIM (M=1), L3S-SSIM (M=3), L5S-SSIM (M=5)]. For an L3S-SSIM, the weights were set to 0.2096, 0.4659 and 0.3245, and for a L5S-SSIM, the size for the Gaussian filter was set to 7, which is the maximum possible filter size for the selected patch size; otherwise the suggested settings by Wang et al. (2003) were used. Training the CycleGAN The implementation of a CycleGAN in Pytorch was downloaded from GitHub and, if not stated otherwise, trained with the default parameters (https://github.com/junyanz/pytorch-CycleGAN-and-pix2pix) (Zhu et al., 2017). The CycleGAN was trained with unaligned images of the actin, tubulin, caveolae and PXN datasets that were pre-processed as outlined in the previous sections (see also Table 2), using a least squares GAN with a ResNet-9 generator architecture and a 70×70 PatchGAN discriminator architecture. Training was conducted with a batch size of 4, an epoch number of 4/10 (3/9 with linear decay of the learning rate) for the PXN/other dataset(s) and a scaling factor of 0.0005 for the network initialization. Funding To further evaluate L2L, repeated eightfold or tenfold cross validations were conducted on the datasets using the settings outlined in Table 3. The selection of the raw image pairs from the total dataset for each cross validation and the fold allocation were conducted randomly in Python. The image pairs for each training were generated as described above, including the pre-processing, and disabling the train/test split in the CSBDeep Framework. The relative change of the NRMSE and 5S-SSIM indices were calculated between input/benchmark and prediction/benchmark for the test images of each fold, deriving an average for each cross validation (Fig. 5). L.S.K. was supported by the Engineering and Physical Sciences Research Council and the Medical Research Council (MRC) for Doctoral Training in Optical Medical Imaging (EP/L016559/1), and J.V. was supported by the Wellcome Trust (208345/Z/ 17/Z). STED imaging was performed at the Edinburgh Super-Resolution Imaging Consortium, which is supported by the MRC and the Wellcome Trust. Work ongoing in the Gram Hansen lab is supported by a University of Edinburgh Chancellor’s Fellowship, Worldwide Cancer Research (19-0238) and the June Hancock Mesothelioma Research Fund. G.M. was supported by the Biotechnology and Biological Sciences Research Council (BB/P02565X/1 and BB/T011602/1) and the MRC (MR/K015583/1). Open Access funding was provided by University of Edinburgh. Deposited in PMC for immediate release. Peer review history Th i hi t The peer review history is available online at https://journals.biologists.com/jcs/ article-lookup/doi/10.1242/jcs.258994 Competing interests Competing interests The authors declare no competing or financial interests. The authors declare no competing or financial interests. The authors declare no competing or financial interests. The settings that were applied for the L2L training are shown in Table 2. The same settings were used to train the network for N2N, but the training data were generated from two sequential images of the respective antibody. To train the CNN as a separator of two cellular markers (the SYTOX Green stain and a CD44 antibody) in superposed immunofluorescence images, the CSBDeep Framework was trained with two channel images, where the input consisted of the superposed image in both channels, and the separately acquired images of SYTOX and CD44 as the output channels (see Table 2, right). 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(2018). Content-aware image restoration: pushing the limits of fluorescence microscopy. Nat. Methods 15, 1090-1097. doi:10.1038/s41592-018-0216-7 Peli, E. (1990). Contrast in complex images. J. Opt. Soc. Am. Image processing and analysis Belthangady, C. and Royer, L. A. (2019). Applications, promises, and pitfalls of deep learning for fluorescence image reconstruction. Nat. Methods 16, 1215-1225. doi:10.1038/s41592-019-0458-z Image processing and analysis was conducted in Python using the following functions/libraries in default, if not stated otherwise. To compare L2L with classical image processing methods (Fig. S3, Table S1), the following steps were undertaken for images of actin/tubulin/caveolae/PXN: Gaussian filters were applied with a sigma of 2/2/3/2 using ndimage.gaussian_filter in scipy (Virtanen et al., 2020); for rolling-ball background subtraction, subtract_background_rolling_ball from https://github.com/mbalatsko/ opencv-rolling-ball was used with a radius of 20/10/5/5; top-hat filters were applied with a filter size of 11/25/13/17; and contrast limited adaptive histogram equalization (CLAHE) was conducted with a grid size of 11/7/7/ 7, using getStructuringElement(cv2.MORPH_RECT) or createCLAHE, respectively, from OpenCV (Bradski, 2000). Bradski, G. (2000). The OpenCV library. Dr. Dobb’s J. Software Tools 120, 122-125. Caicedo, J. C., Roth, J., Goodman, A., Becker, T., Karhohs, K. W., Broisin, M., Molnar, C., Mcquin, C., Singh, S., Theis, F. J. et al. (2019). Evaluation of deep learning strategies for nucleus segmentation in fluorescence images. Cytometry Part A 95, 952-965. doi:10.1002/cyto.a.23863 Journal of Cell Science Christiansen, E. M., Yang, S. J., Ando, D. M., Javaherian, A., Skibinski, G., Lipnick, S., Mount, E., O’Neil, A., Shah, K., Lee, A. K. et al. (2018). In silico labeling: predicting fluorescent labels in unlabeled images. Cell 173, 792-803. doi:10.1016/j.cell.2018.03.040 Dugina, V., Zwaenepoel, I., Gabbiani, G., Cleì Ment, S. and Chaponnier, C. (2009). Beta and gamma-cytoplasmic actins display distinct distribution and functional diversity. J. Cell Sci. 122, 2980-2988. doi:10.1242/jcs.041970 ̈ The FWHM was derived from 20 randomly selected line profiles across single microtubules in images of tubulin, by averaging the line profile across 20 pixels and determining the Gaussian fit with scipy (Virtanen et al., 2020) (Fig. S4C). Falk, T., Mai, D., Bensch, R., Çiçek, Ö ., Abdulkadir, A., Marrakchi, Y., Bö hm, A., Deubner, J., Jä ckel, Z., Seiwald, K. et al. (2019). U-Net: deep learning for cell counting, detection, and morphometry. Nat. Methods 16, 67-70. doi:10.1038/ s41592-018-0261-2 12 RESEARCH ARTICLE Journal of Cell Science (2022) 135, jcs258994. doi:10.1242/jcs.258994 Rausch, V. and Hansen, C. G. (2020). The hippo pathway, YAP/TAZ, and the plasma membrane. Trends Cell Biol. 30, 32-48. doi:10.1016/j.tcb.2019.10.005 Rausch, V. and Hansen, C. G. (2020). The hippo pathway, YAP/TAZ, and the plasma membrane. Trends Cell Biol. 30, 32-48. doi:10.1016/j.tcb.2019.10.005 Goodfellow, I. Image processing and analysis A 7, 2032-2040. doi:10.1364/JOSAA.7.002032 Zhao, H., Gallo, O., Frosio, I. and Kautz, J. (2016). Loss functions for image restoration with neural networks. IEEE Trans. Comput. Imaging 3, 47-57. doi:10. 1109/TCI.2016.2644865 Pellegrini, F. and Budman, D. R. (2005). Review: tubulin function, action of antitubulin drugs, and new drug development. Cancer Investig. 23, 264-273. doi:10.1081/CNV-200055970 Zhu, J. Y., Park, T., Isola, P. and Efros, A. A. (2017). Unpaired image-to-image translation using cycle-consistent adversarial networks. In Proceedings of the IEEE International Conference on Computer Vision. Institute of Electrical and Electronics Engineers Inc., pp. 2242-2251. Rausch, V. and Hansen, C. G. (2019). Immunofluorescence study of endogenous YAP in mammalian cells. Methods Mol. Biol. 1893, 97-106. doi:10.1007/978-1- 4939-8910-2_8 Journal of Cell Science Journal of Cell Science 13 J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S1. RMS and SSIM maps between the input and predicted images after L2L training. (A) Two representative inputs for L2L training, corresponding 20-frame average images and L2L results for four different cellular structures (top-to-bottom): AC-15 (actin), DM1A (α- tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). The CNN was trained with a L1 (PXN)/L3S-SSIM (all else) loss function. RMS and SSIM maps between (B) the input and predicted images, and (C) the 20- frame average images and the predicted images. The LUT of the SSIM maps is selected such that 'blue', 'white' and 'red' indicate a negative, no and a positive correlation between the images, respectively. The trained network reduces cytosolic background signals in the images (RMS maps) and sharpens the cellular structure (SSIM maps). The shown image pairs were excluded from the trainings (scale bar (top-to-bottom) = 1/1/0.2/1 µm). Journal of Cell Science • Supplementary in Fig. S1. RMS and SSIM maps between the input and predicted images after L2L training. (A) Two representative inputs for L2L training, corresponding 20-frame average images and L2L results for four different cellular structures (top-to-bottom): AC-15 (actin), DM1A (α- tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). The CNN was trained with a L1 (PXN)/L3S-SSIM (all else) loss function. RMS and SSIM maps between (B) the input and predicted images, and (C) the 20- frame average images and the predicted images. The LUT of the SSIM maps is selected such that 'blue', 'white' and 'red' indicate a negative, no and a positive correlation between the images, respectively. Image processing and analysis The trained network reduces cytosolic background signals in the images (RMS maps) and sharpens the cellular structure (SSIM maps). The shown image pairs were excluded from the trainings (scale bar (top-to-bottom) = 1/1/0.2/1 µm). J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S2. Loss function-dependent label2label and noise2noise results for confocal images of the actin cytoskeleton and the microtubule network. (Left) Training inputs and corresponding 20-frame average images of (A) HeLa cells labell with AC-15 (actin) and (D) MeT5A cells labelled with DM1A (tubulin). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (B) Example of hallucination effects are highlighted in green boxes. (Right) The benchmarks for L2L training and 20-frame average images for the (C) actin (phalloidin) or (F) tubulin (YOL1/34) dataset respectively The shown image pairs were excluded from the trainings Fig. S2. Loss function-dependent label2label and noise2noise results for confocal images of the actin cytoskeleton and the microtubule network. (Left) Training inputs and corresponding 20-frame average images of (A) HeLa cells labe with AC-15 (actin) and (D) MeT5A cells labelled with DM1A (tubulin). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (B) Example of hallucination effects are highlighted in green boxes. (Right) The benchmarks for L2L training and 20-frame average images for the (C) actin (phalloidin) or (F) tubulin (YOL1/34) dataset, respectively. The shown image pairs were excluded from the trainings, and contrast adjusted to the 2nd and 99.8th percentile (image dimensions: 4.5 µm x 4.5 µ Journal of Cell Science • Suppleme Fig. S2. Loss function-dependent label2label and noise2noise results for confocal images of the actin cytoskeleton and the microtubule network. (Left) Training inputs and corresponding 20-frame average images of (A) HeLa cells labelled with AC-15 (actin) and (D) MeT5A cells labelled with DM1A (tubulin). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (B) Examples of hallucination effects are highlighted in green boxes. (Right) The benchmarks for L2L training and 20-frame average images for the (C) actin (phalloidin) or (F) tubulin (YOL1/34) dataset, respectively. The shown image pairs were excluded from the trainings, and contrast adjusted to the 2nd and 99.8th percentile (image dimensions: 4.5 µm x 4.5 µm). Fig. S2. Loss function-dependent label2label and noise2noise results for confocal images of the actin cytoskeleton and the microtubule network. images of the actin cytoskeleton and the microtubule network. Image processing and analysis (Left) Training inputs and corresponding 20-frame average images of (A) HeLa cells labelled with AC-15 (actin) and (D) MeT5A cells labelled with DM1A (tubulin). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (B) Examples of hallucination effects are highlighted in green boxes. (Right) The benchmarks for L2L training and 20-frame average images for the (C) actin (phalloidin) or (F) tubulin (YOL1/34) dataset, respectively. The shown image pairs were excluded from the trainings, and contrast adjusted to the 2nd and 99.8th percentile (image dimensions: 4.5 µm x 4.5 µm). J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S3. Classical methods to enhance image contrast versus label2label. (A) Two representative inputs for L2L training for four different cellular structures (top-to- bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). (B) Images after applying a Gaussian filter, after applying a Gaussian filter and rolling-ball background subtraction, a Gaussian filter and top-hat filter, a Gaussian filter and contrast limited adaptive histogram equalization (CLAHE), and the predicted images of a CNN after L2L Fig. S3. Classical methods to enhance image contrast versus label2label. (A) Two representative inputs for L2L training for four different cellular structures (top-to- bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). (B) Images after applying a Gaussian filter, after applying a Gaussian filter and rolling-ball background subtraction, a Gaussian filter and top-hat filter, a Gaussian filter and contrast limited adaptive histogram equalization (CLAHE), and the predicted images of a CNN after L2L training with a L1 (PXN)/L3S-SSIM (all else) loss function. (C) Corresponding 20-frame average images. The shown images were excluded from the network trainings (scale bar (top-to-bottom) = 1/1/0.2/1 µm). For a quantitative evaluation see Table S1. Journal of Cell Science • Supplementary information Fig. S3. Classical methods to enhance image contrast versus label2label. (A) Two representative inputs for L2L training for four different cellular structures (top-to- bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). Image processing and analysis Image resolution decreases after using (grey) classical image processing methods, while DL-based methods decrease the average extracted width of single microtubules increasingly, using a MS-SSIM loss function with decreasing scale (M). Here, (pink) L2L outperforms (green) N2N. The extracted FWHMs in predictions of DM1A after L2L training with a LSSIM are closest to FWHMs obtained with STED microscopy (see also (blue) the mean FWHMs in nm for each category). (A) Confocal image of DM1A (tubulin), the prediction by a CNN after training with confocal images of DM1A/YOL1/34 as input/benchmark (see Fig. S2) using a L3S-SSIM loss function, and the corresponding STED image (scale bar = 1 μm). (B) Region of interest of the L2L result and STED image in (A). Close structures that are not resolved with confocal microscopy collapse into one microtubule in the prediction of a network. Journal of Cell Science • Supplementary information (C) Extracted full width at half maxima (FWHMs) for 20 line profiles across single microtubules in (from-left-to-right) confocal images of DM1A which are used as training input for L2L/N2N training, images of DM1A after applying classical image processing methods (see Fig. S3), restored images of DM1A by a CNN after N2N and L2L training with different loss functions, STED images of DM1A, and confocal images of YOL1/34 which were used as benchmark for L2L. Image resolution decreases after using (grey) classical image processing methods, while DL-based methods decrease the average extracted width of single microtubules increasingly, using a MS-SSIM loss function with decreasing scale (M). Here, (pink) L2L outperforms (green) N2N. The extracted FWHMs in predictions of DM1A after L2L training with a LSSIM are closest to FWHMs obtained with STED microscopy (see also (blue) the mean FWHMs in nm for each category). (C) Extracted full width at half maxima (FWHMs) for 20 line profiles across single microtubules in (from-left-to-right) confocal images of DM1A which are used as training input for L2L/N2N training, images of DM1A after applying classical image processing methods (see Fig. S3), restored images of DM1A by a CNN after N2N and L2L training with different loss functions, STED images of DM1A, and confocal images of YOL1/34 which were used as benchmark for L2L. Image resolution decreases after using (grey) classical image processing methods, while DL-based methods decrease the average extracted width of single microtubules increasingly, using a MS-SSIM loss function with decreasing scale (M). Here, (pink) L2L outperforms (green) N2N. Image processing and analysis (B) Images after applying a Gaussian filter, after applying a Gaussian filter and rolling-ball background subtraction, a Gaussian filter and top-hat filter, a Gaussian filter and contrast limited adaptive histogram equalization (CLAHE), and the predicted images of a CNN after L2L training with a L1 (PXN)/L3S-SSIM (all else) loss function. (C) Corresponding 20-frame average images. The shown images were excluded from the network trainings (scale bar (top-to-bottom) = 1/1/0.2/1 µm). For a quantitative evaluation see Table S1. (A) Two representative inputs for L2L training for four different cellular structures (top-to- bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). (B) Images after applying a Gaussian filter, after applying a Gaussian filter and rolling-ball background subtraction, a Gaussian filter and top-hat filter, a Gaussian filter and contrast limited adaptive histogram equalization (CLAHE), and the predicted images of a CNN after L2L training with a L1 (PXN)/L3S-SSIM (all else) loss function. (C) Corresponding 20-frame average images. The shown images were excluded from the network trainings (scale bar (top-to-bottom) = 1/1/0.2/1 µm). For a quantitative evaluation see Table S1. J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S4. Measured resolution in images of tubulin dependent on the image processing method. Fig. S4. Measured resolution in images of tubulin dependent on the image processing h d Fig. S4. Measured resolution in images of tubulin dependent on the image processing method. method. (A) Confocal image of DM1A (tubulin), the prediction by a CNN after training with confocal images of DM1A/YOL1/34 as input/benchmark (see Fig. S2) using a L3S-SSIM loss function, and the corresponding STED image (scale bar = 1 μm). (B) Region of interest of the L2L result and STED image in (A). Close structures that are not resolved with confocal microscopy collapse into one microtubule in the prediction of a network. (C) Extracted full width at half maxima (FWHMs) for 20 line profiles across single microtubules in (from-left-to-right) confocal images of DM1A which are used as training input for L2L/N2N training, images of DM1A after applying classical image processing methods (see Fig. S3), restored images of DM1A by a CNN after N2N and L2L training with different loss functions, STED images of DM1A, and confocal images of YOL1/34 which were used as benchmark for L2L. Image processing and analysis The extracted FWHMs in predictions of DM1A after L2L training with a LSSIM are closest to FWHMs obtained with STED microscopy (see also (blue) the mean FWHMs in nm for each category). Journal of Cell Science • Supplementary in J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S5. Loss function-dependent label2label and noise2noise results for STED images of caveolae and confocal images of PXN. (Left) Training inputs and corresponding 20-frame average images of (A) MeT5A cells labelled with D1P6W (CAVIN-1, an essential caveolae component) and (D) MeT5A cells labelled with 5H11 (PXN, an essential focal adhesion component). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (Right) The benchmarks for L2L training and 20-frame average images for the (C) caveolae (4H312) or (F) PXN (Y113) dataset, respectively. The shown image pairs were excluded from the trainings. Each image is contrast adjusted to (A,C) their 2nd and 100th percentile, or (D,F) contrast Fig. S5. Loss function-dependent label2label and noise2noise results for STED images of caveolae and confocal images of PXN. Fig. S5. Loss function-dependent label2label and noise2noise results for STED images of caveolae and confocal images of PXN. (Left) Training inputs and corresponding 20-frame average images of (A) MeT5A cells labelled with D1P6W (CAVIN-1, an essential caveolae component) and (D) MeT5A cells labelled with 5H11 (PXN, an essential focal adhesion component). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (Right) The benchmarks for L2L training and 20-frame average images for the (C) caveolae (4H312) or (F) PXN (Y113) dataset, respectively. The shown image pairs were excluded from the trainings. Each image is contrast adjusted to (A,C) their 2nd and 100th percentile, or (D,F) contrast adjusted to the 2nd and 99.8th percentile of the respective input image (image dimensions: 1 1 ( l )/ 9 1 9 1 (PXN)) (Left) Training inputs and corresponding 20-frame average images of (A) MeT5A cells labelled with D1P6W (CAVIN-1, an essential caveolae component) and (D) MeT5A cells labelled with 5H11 (PXN, an essential focal adhesion component). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (Right) The benchmarks for L2L training and 20-frame average images for the (C) caveolae (4H312) or (F) PXN (Y113) dataset, respectively. The shown image pairs were excluded from the trainings. Image processing and analysis (Left) Training inputs and corresponding 20-frame average images of (A) MeT5A cells labelled with D1P6W (CAVIN-1, an essential caveolae component) and (D) MeT5A cells labelled with 5H11 (PXN, an essential focal adhesion component). (B,E) Corresponding restorations by a CNN after L2L and N2N training with different loss functions. (Right) The benchmarks for L2L training and 20-frame average images for the (C) caveolae (4H312) or (F) PXN (Y113) dataset, respectively. The shown image pairs were excluded from the trainings. Each image is contrast adjusted to (A,C) their 2nd and 100th percentile, or (D,F) contrast adjusted to the 2nd and 99.8th percentile of the respective input image (image dimensions: 1 µm x 1 µm (caveolae)/ 9 1 µm x 9 1 µm (PXN)) Each image is contrast adjusted to (A,C) their 2nd and 100th percentile, or (D,F) contrast adjusted to the 2nd and 99.8th percentile of the respective input image (image dimensions: 1 µm x 1 µm (caveolae)/ 9.1 µm x 9.1 µm (PXN)). J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S6. PAXILLIN (PXN) knockdown in MeT5A cells. PXN is a focal adhesion molecule. (A) Results of a RT-qPCR analysis, including the standard error of the mean: the relative expression of the PXN knockdown cells is 26.14±0.04%. (B) Western analysis of cell lysates of MeT5A cells visualising the PXN protein knockdown, with Ponceau stain ensuring equal loading. The monoclonal anti-PXN antibody Y113 was used. The PXN expression is reduced by around a half in the knockdown cells compared to the control. (C) Immunofluorescence images of MeT5A cells that were dual-labelled with the anti-PXN antibodies (left) 5H11 and (right) Y113 after (top) mock-infection and (bottom) shRNA mediated PXN-knockdown. The low signal of cytosolic protein in the knockdown cells for both antibodies indicate that the difference in cytosolic signal between the two, as observed in the control cells, originates from clone-dependent antibody binding to cytosolic PXN rather than unspecific binding (scale bar = 20 µm). Fig. S6. PAXILLIN (PXN) knockdown in MeT5A cells. PXN is a focal adhesion molecule. (A) Results of a RT-qPCR analysis, including the standard error of the mean: the relative expression of the PXN knockdown cells is 26.14±0.04%. (B Fig. S6. PAXILLIN (PXN) knockdown in MeT5A cells. Fig. S6. PAXILLIN (PXN) knockdown in MeT5A cells. PXN is a focal adhesion molecule. Image processing and analysis (A) Results of a RT-qPCR analysis, including the standard error of the mean: the relative expression of the PXN knockdown cells is 26.14±0.04%. (B) Western analysis of cell lysates of MeT5A cells visualising the PXN protein knockdown, with Ponceau stain ensuring equal loading. The monoclonal anti-PXN antibody Y113 was used. The PXN expression is reduced by around a half in the knockdown cells compared to the control. (C) Immunofluorescence images of MeT5A cells that were dual-labelled with the anti-PXN antibodies (left) 5H11 and (right) Y113 after (top) mock-infection and (bottom) shRNA mediated PXN-knockdown. The low signal of cytosolic protein in the knockdown cells for both antibodies indicate that the difference in cytosolic signal between the two, as observed in the control cells, originates from clone-dependent antibody binding to cytosolic PXN rather than unspecific binding (scale bar = 20 µm). Journal of Cell Science • Supplementary information J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information ig. S7. Predictions by a CycleGAN and CNN after label2label training. A) Two representative inputs for L2L training and corresponding 20-frame average images of fo ifferent cellular structures (top-to-bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, a ssential caveolae component), and 5H11 (PXN, an essential focal adhesion component). ( redicted images after training (left-to-right) a CycleGAN with unaligned image pairs without/wi pplying a Gaussian filter (σ=2) to the image patches prior to the network training, and predictio y a CNN after using a L1 (PXN)/L3S-SSIM (all else) for the training. (C) Corresponding benchmar or L2L (top-to-bottom): phalloidin (actin), YOL1/34 (α-tubulin), 4H312 (CAVEOLIN-1, an essenti aveolae component), and Y113 (PXN). The CNN outperforms the CycleGAN in restoring images wi nhanced contrast of the target structure (scale bar (top to bottom) 1/1/0 2/1 µm) Fig. S7. Predictions by a CycleGAN and CNN after label2label training. (A) Two representative inputs for L2L training and corresponding 20-frame average images of four different cellular structures (top-to-bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). (B) Predicted images after training (left-to-right) a CycleGAN with unaligned image pairs without/with applying a Gaussian filter (σ=2) to the image patches prior to the network training, and predictions by a CNN after using a L1 (PXN)/L3S-SSIM (all else) for the training. (C) Corresponding benchmarks for L2L (top-to-bottom): phalloidin (actin), YOL1/34 (α-tubulin), 4H312 (CAVEOLIN-1, an essential caveolae component), and Y113 (PXN). Image processing and analysis The CNN outperforms the CycleGAN in restoring images with enhanced contrast of the target structure (scale bar (top-to-bottom) = 1/1/0.2/1 µm). (A) Two representative inputs for L2L training and corresponding 20-frame average images of four different cellular structures (top-to-bottom): AC-15 (actin), DM1A (α-tubulin), D1P6W (CAVIN-1, an essential caveolae component), and 5H11 (PXN, an essential focal adhesion component). (B) Predicted images after training (left-to-right) a CycleGAN with unaligned image pairs without/with applying a Gaussian filter (σ=2) to the image patches prior to the network training, and predictions by a CNN after using a L1 (PXN)/L3S-SSIM (all else) for the training. (C) Corresponding benchmarks for L2L (top-to-bottom): phalloidin (actin), YOL1/34 (α-tubulin), 4H312 (CAVEOLIN-1, an essential caveolae component), and Y113 (PXN). The CNN outperforms the CycleGAN in restoring images with enhanced contrast of the target structure (scale bar (top-to-bottom) = 1/1/0.2/1 µm). J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Fig. S8. Distance maps or binarisation of images dependent on pre-processing. Top-to-bottom) Images of dual-labelled (A,B) HeLa cells targeting the actin cytoskeleto and MeT5A cells targeting the (C,D) microtubule network, (E,F) caveolae and (G,H) focal adhesions (PXN), respectively. For N2N, a CNN was trained with two noise realisations of th respective label. For L2L, images of (A,B) AC-15/Phalloidin, (C,D) DM1A/YOL1/34, (E,F) D1P6W/4H312, (G,H) 5H11/Y113 are used as input/benchmark for the training. For the network trainings, a L1 (PXN)/L3S-SSIM (all else) loss function was used. (A-D) Distance ma E-H) binarised images are shown for the respective pre-processed images (see also Fig. S7 corresponding raw images). L2L facilitates post-processing due to reduced non-structural si n the cell cytoplasm in the predictions (scale bar (top-to-bottom) = 1/1/0 2/1 µm) ig. S8. Distance maps or binarisation of images dependent on pre-processing. Top-to-bottom) Images of dual-labelled (A,B) HeLa cells targeting the actin cytoskeleton nd MeT5A cells targeting the (C,D) microtubule network, (E,F) caveolae and (G,H) focal dhesions (PXN), respectively. For N2N, a CNN was trained with two noise realisations of th espective label. For L2L, images of (A,B) AC-15/Phalloidin, (C,D) DM1A/YOL1/34, (E,F) D1P6W/4H312, (G,H) 5H11/Y113 are used as input/benchmark for the training. For the etwork trainings, a L1 (PXN)/L3S-SSIM (all else) loss function was used. (A-D) Distance map E-H) binarised images are shown for the respective pre-processed images (see also Fig. S7 orresponding raw images) L2L facilitates post-processing due to reduced non-structural sig Fig. S8. Distance maps or binarisation of images dependent on pre-processing. Image processing and analysis (Top-to-bottom) Images of dual-labelled (A,B) HeLa cells targeting the actin cytoskeleton, and MeT5A cells targeting the (C,D) microtubule network, (E,F) caveolae and (G,H) focal adhesions (PXN), respectively. For N2N, a CNN was trained with two noise realisations of the respective label. For L2L, images of (A,B) AC-15/Phalloidin, (C,D) DM1A/YOL1/34, (E,F) D1P6W/4H312, (G,H) 5H11/Y113 are used as input/benchmark for the training. For the network trainings, a L1 (PXN)/L3S-SSIM (all else) loss function was used. (A-D) Distance maps or (E-H) binarised images are shown for the respective pre-processed images (see also Fig. S7 for corresponding raw images). L2L facilitates post-processing due to reduced non-structural signal in the cell cytoplasm in the predictions (scale bar (top-to-bottom) = 1/1/0.2/1 µm). J. Cell Sci.: doi:10.1242/jcs.258994: Supplementary information Table S1. Evaluation of classical methods to enhance image contrast versus the deep learning-based image restoration methods noise2noise and label2label. Table S1. Evaluation of classical methods to enhance image contrast versus the deep learning-based image restoration methods noise2noise and label2label. Calculation of the average PSNR, NRMSE and MS-SSIM indices (M = 1, 3, 5) between (top panel) representative training input images, (middle panel) after applying a Gaussian filter, a Gaussian filter and rolling-ball background subtraction, a Gaussian filter and top-hat filter, or a Gaussian filter and contrast limited adaptive histogram equalization (CLAHE), or (bottom panel) predicted by a CNN after N2N/L2L training, and the images that are used as benchmark for L2L training with the (A) actin, (B) tubulin, (C) caveolae and (D) PXN (focal adhesion) dataset. For qualitative results see Fig. S3. On average, predicted images after L2L training exhibit, in comparison, the highest correlation to the respective benchmark images. The randomly selected image pairs (N = 1,000) to calculate the metrics were excluded from the network trainings. Actin Cytoskeleton Microtubule Network PSNR NRMSE PSNR NRMSE (dB) M=5 M=3 M=1 (dB) M=5 M=3 M=1 Input 16.73 0.149 0.140 0.547 0.365 0.139 Gaussian 18.25 0.127 0.124 Rolling BG 16.69 0.151 0.141 Tophat 16.55 0.153 0.144 CLAHE 18.01 0.130 0.127 0.627 0.435 0.180 0.544 0.388 0.153 0.535 0.370 0.144 0.631 0.441 0.182 N2N 17.99 0.131 0.124 L2L 20.05 0.103 0.437 0.333 0.150 17.23 0.526 0.439 0.254 18.31 0.406 0.355 0.186 17.13 0.403 0.342 0.173 17.00 0.525 0.435 0.247 18.05 0.528 0.444 0.258 18.31 0.667 0.562 0.338 18.27 0.124 0.647 0.459 0.196 0.661 0.477 0.206 Caveolae PAXILLIN Input 14.89 0.181 0.118 0.455 0.338 0.151 Gaussian 15.05 0.178 0.098 Rolling BG 14.91 0.181 0.100 Tophat 14.79 0.183 0.109 CLAHE 14.99 0.179 0.101 0.554 0.440 0.263 0.548 0.443 0.280 0.501 0.399 0.239 0.535 0.421 0.247 N2N 14.90 0.181 0.095 L2L 14.98 0.179 0.156 0.110 0.048 18.73 0.172 0.129 0.061 20.50 0.162 0.119 0.052 20.41 0.149 0.109 0.044 19.67 0.170 0.126 0.057 20.19 0.162 0.126 0.058 20.74 0.165 0.134 0.066 21.31 0.091 0.573 0.462 0.288 0.600 0.494 0.328 A B C D MS-SSIM MS-SSIM Journal of Cell Science • Supplementary information
https://openalex.org/W2138106523	https://atheneadigital.net/article/download/n18-flores-pons/720-pdf-ca	Catalan; Valencian	null	Review of IC-Investigación Cualitativa blog	Athenea digital	2,010	cc-by	677	gemmaflorespons@gmail.com La producció i publicació de materials que fan referència a la metodologia qualitativa ha anat incrementant de forma inseparable a la seva consolidació com a metodologia de recerca en ciències socials. Internet ha tingut un paper rellevant en aquest sentit, ja que ha multiplicat les formes de fer públic, de compartir i posar en relació tot allò que se sitúa en el marc de la metodologia qualitativa, de la mateixa manera que ha succeït amb molts altres àmbits. El blog IC-Investigación Cualitativa és una eina interessant que ofereix un recull de recursos, disponibles a la xarxa, per a la recerca qualitativa i els exposa organitzats temàticament. Així, les persones que treballem en recerca amb una perspectiva qualitativa podem trobar articles, capítols, llibres o pàgines d'autora així com vincles a revistes, grups de recerca, congressos, programes informàtics o altres portals web. Es tracta d'un blog que posa a la nostra disposició materials a través de links a les pàgines pròpies on es localitzen. No es tracta d'una centralització de materials, sinó de l'enxarxament de diferents recursos ja existents, ja que es mantenen els links a les pàgines on es localitzen. Aquest fet permet una doble tasca, difondre els propis materials i visibilitzar les fonts i recursos que els han fet disponibles, tot dibuixant així una xarxa d'alguns dels principals recursos per a la recerca qualitativa disponibles a internet. Alguns dels exemples dels apartats que hi podem trobar són, d'una banda, aquells que fan referència a mètodes d'anàlisi, com són l'anàlisi del contingut, l'anàlisi conversacional o l'anàlisi del discurs (sent aquest últim un dels apartats més àmplis del blog). D'altra banda, trobem els apartats que fan referència a mètodes i tècniques d'investigació, anant des de l'etnografia, la investigació acció participant o la història de vida fins a les entrevistes, els grups de discussió o grups focals. A més, hi ha un conjunt d'entrades que recullen materials útils pel disseny de recerques qualitatives com són les eines informàtiques o de reflexió ètica sobre la recerca. Finalment, hi ha un altre grup d'entrades que són aquelles que agrupen la informació segons temàtiques de recerca, de forma que trobem apartats d'investigació qualitativa en migració, salut, gènere, presons, educació o polítiques públiques. Aguayo, Francisco; Guajardo, Gabriel & Tomicic, Alemka (2010). IC-Investigación Cualitativa. Red de recursos en investigación cualitativ http://www.investigacioncualitativa.cl/ Gemma Flores-Pons Universitat Autònoma de Barcelona gemmaflorespons@gmail.com Gemma Flores-Pons Universitat Autònoma de Barcelona Athenea Digital - núm. 18: 121-122 (julio 2010) -RESEÑAS-ISSN: 1578-8946 Athenea Digital - núm. 18: 121-122 (julio 2010) -RESEÑAS-ISSN: 1578-8946 Aguayo, Francisco; Guajardo, Gabriel & Tomicic, Alemka (2010). IC-Investigación Cualitativa. Red de recursos en investigación cualitativa. http://www.investigacioncualitativa.cl/ gemmaflorespons@gmail.com Després d'aquesta esquematització, que no pot anar gaire més enllà del que ja el títol i subtítol del blog indiquen, no queda res més, si hi ha l'interès, que navegar una mica per la pàgina i explorar els recursos que ofereix per identificar allò que pugui resultar de més utilitat i interès a cada una. 121 Reseña del blog IC-Investigación Cualitativa Formato de citación Flores-Pons, Gemma (2010). Reseña del blog IC-Investigación Cualitativa. Athenea Digital, 18, 121-122. Disponible en http://psicologiasocial.uab.es/athenea/index.php/atheneaDigital/article/view/720. Este texto está protegido por una licencia Creative Commons. Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las siguientes condiciones: Reconocimiento: Debe reconocer y citar al autor original. No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar, o generar una obra derivada a partir de esta obra. Resumen de licencia - Texto completo de la licencia Este texto está protegido por una licencia Creative Commons. Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las siguientes condiciones: Reconocimiento: Debe reconocer y citar al autor original. No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar, o generar una obra derivada a partir de esta obra. Resumen de licencia - Texto completo de la licencia Este texto está protegido por una licencia Creative Commo Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las siguientes condiciones: 122
W4292566412.txt	https://www.frontiersin.org/articles/10.3389/fphy.2022.925523/pdf	en		Label-free observation of morphological alteration of irradiated-urothelial bladder carcinoma cells through digital holographic microscopy	Frontiers in physics	2,022	cc-by	5,281	Original Research 22 August 2022 10.3389/fphy.2022.925523 TYPE PUBLISHED DOI OPEN ACCESS EDITED BY Liangcai Cao, Tsinghua University, China REVIEWED BY Jun Ma, Nanjing University of Science and Technology, China Zhe Wang, Università degli Studi di Napoli Federico II, Italy CORRESPONDENCE Hao Wang, wanghaobysy@bjmu.edu.cn Feng Pan, panfeng@buaa.edu.cn † These authors have contributed equally to this work Label-free observation of morphological alteration of irradiated-urothelial bladder carcinoma cells through digital holographic microscopy Xi Xiao 1†, Leiping Che 2†, Yinjia Li 3,4, Ran Peng 1, Mingqing Wang 1, Wen Xiao 2, Feng Pan 2 and Hao Wang 1,5* 1 Department of Radiation Oncology, Peking University Third Hospital, Beijing, China, 2Key Laboratory of Precision Opto-Mechatronics Technology of Ministry of Education, School of Instrumentation Science and Optoelectronics Engineering, Beihang University, Beijing, China, 3Center of Basic Medical Research, Peking University Third Hospital Institute of Medical Innovation and Research, Beijing, China, 4 Medical Research Center, Peking University Third Hospital, Beijing, China, 5Cancer Center, Peking University Third Hospital, Beijing, China SPECIALTY SECTION This article was submitted to Optics and Photonics, a section of the journal Frontiers in Physics 21 April 2022 ACCEPTED 22 July 2022 PUBLISHED 22 August 2022 RECEIVED CITATION Xiao X, Che L, Li Y, Peng R, Wang M, Xiao W, Pan F and Wang H (2022), Labelfree observation of morphological alteration of irradiated-urothelial bladder carcinoma cells through digital holographic microscopy. Front. Phys. 10:925523. doi: 10.3389/fphy.2022.925523 COPYRIGHT © 2022 Xiao, Che, Li, Peng, Wang, Xiao, Pan and Wang. 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. Radiotherapy is an effective treatment for certain patients with muscle-invasive bladder cancer and radio-sensitivity detection plays a vital role during bladder cancer radio treatment because radiotherapy responses have profound inﬂuences on a patient’s prognosis. Although several potential biomarkers were investigated to assess the radio-response of bladder cancer, studies on detecting radio-sensitivity based on morphological characteristics of cancer cells at the single-cell level are rare. In fact, morphological parameters are vital characteristics of cells that could provide direct information to infer the physiological statuses of cells and evaluate the response of cells to the external stimulations. In this study, digital holographic microscopy was applied to quantify morphological parameters of bladder cancer cells (HT-1376) at the single-cell level and their alterations after exposure to four different radiation doses, i.e., 0 Gy (control), 4, 8, and 12 Gy. Based on the reconstructed phase images, four morphological parameters of cells, namely, cell phase volume (CPV), cell projected area (CPA), cell average phase height (CAPH), and cell maximum phase height (CMPH), were quantitatively calculated. The results show that the change rates of CPV, CAPH, and CMPH were increased with the radiation dose rising, while the change rate of CPA was decreased with the radiation dose increasing. Moreover, the change rates of CPV, CPA, CAPH, and CMPH were different between control group and 12 Gy treated group. The results demonstrate that morphological characteristics have the potential to be utilized to estimate the radio-sensitivity of bladder cancer cells, and it may provide new perspectives to establishing label-free methods to detect radio-sensitivity and guide radiotherapy in bladder cancer. KEYWORDS bladder cancer cell, cell morphology alteration by radiation, quantitative phase imaging, digital holography, radio-sensitivity Frontiers in Physics 01 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 1 Introduction In this study, we aimed to establish a method based on DHM to detect the radio-response of bladder cancer cells. DHM was applied to obtain the phase images of the cells and to retrieve the changes in cell morphological parameters, which were used to evaluate the radio-response of cells irradiated by different radiation dose. Cell morphological parameters including CPV, CPA, CAPH, and CMPH were quantitatively calculated from the reconstructed phase images. The change rates of morphological parameters manifest a closed relationship with radiation doses, where the change rates of CPV, CAPH, and CMPH were increased with the radiation doses rising, while the change rates of CPA were decreased with the radiation doses increasing. Moreover, the change rates of CPV, CPA, CAPH, and CMPH were different between control group and 12Gy treated group. Bladder cancer (BC) is the 10th most common cancer worldwide, with an estimated 200,000 deaths in 2018 GLOBOCAN statistics [1]. For muscle-invasive BC (MIBC), which occupies approximately 25% of all cases, radiotherapy is an alternative treatment for patients who demand bladder preservation or with unresectable lesions [2]. However, the heterogeneity of BC response to radiotherapy results in inconsistent treatment outcomes. Exploiting a predictive strategy to improve the accuracy of radiotherapy delivery is an urgent clinical problem to be solved. Several potential biomarkers were studied to access the radio-response of BC, but all failed to guide clinical decision-making due to unrevealed clinical validation and correlation [3]. At present, the choice of radiotherapy still depends on the preference of patients and the clinical experience of radiologists. Therefore, prospectively and precisely predicting the radio-response of BC to assist clinical decision-making is a practical issue the clinician faced. Ionizing radiation, as well as some other chemical drugs, could largely inﬂuence the cellular status and biological function. Meanwhile, comprehensive studies veriﬁed that the changes in biological alternation, including cell growth [4], intercellular communication [5], transcriptome, and proteome [6], were associated with cellular morphological phenotypes. Some speciﬁc morphological manifestations were even coordinated with the cell fate decisions [7]. These indicate that the response of cells to radiation may be evaluated via quantitative morphological observation. Digital holographic microscopy (DHM) is a novel optical microscopic imaging technique that combined qualities found in optical microscopy and digital holography with the capability of quantitative phase microscopic imaging [8]. As a quantitative phase imaging method, DHM images the sample by measuring the optical path length delays introduced by the sample. Thus, no labeling or staining is necessary for living cell observation. This also means that the three-dimensional morphology of a living cell could be objectively quantiﬁed using optical path length, and thus multiple morphological parameters could be deduced based on the phase image of the living cell to describe its morphology quantitatively. As the optical path length delays introduced by the living cell is the product of the geometric length of the living cell and the refractive index difference between the sample and the medium, the phase image encodes the refractive index information of the living cell. Also, the refractive index of a living cell is linearly proportional to dry mass density [9]. Therefore, the biochemical parameters of a living cell could be inferred from the phase image, such as cellular dry mass and cellular dry mass density. Furthermore, DHM arranged in an offaxis conﬁguration is capable of recording the phase image of the living cell in a single exposure with nanoscale sensitivity. Due to great advantages in living cell measurement, DHM has been widely applied in biomedical research, especially for measuring on morphological parameter changes in living cells [10]. Frontiers in Physics 2 Methods 2.1 Cell culture and treatments The human urothelial bladder cancer cell line HT-1376 (CRL1472) was purchased and validated from Beijing Biotides Biotechnology Co., Ltd. Cells were grown in Eagle’s Minimum Essential (EME) Medium (Gibco, #11095080) supplemented with 10% fetal bovine serum (FBS) (Gibco, #10099141) and 1% penicillin streptomycin (Gibco, #15140122) at 37°C in 5% CO2 humidiﬁed incubator (Thermo HERAcell150i). Monthly, mycoplasma tests ensured Mycoplasma-negative cultures (TransDetect, FM311-01). The Automated Cell Counter named Countess II FL (Invitrogen, #AMQAF1000) was used for live-cell counting. Cells (1.5−3×106/ dish) were seeded in 30-mm glass-bottom cell culture dishes (NEST Scientiﬁc #801001) and incubated for 12 h, followed by radiation using a Varian Trilogy (SSD = 100 cm). For the treatment group, the cells were exposed to different dose of 6-MV X-rays at a dose rate of 300 cGy/min. The irradiated cells were then used for further DH microscope or biology analyses. ™ 2.2 Biological characteristics of HT-1376 bladder cancer cell lines irradiated by different doses 2.2.1 Determining the viability of HT-1376 bladder cancer cell lines treated with different radiation doses The HT-1376 cells were cultured and irradiated as mentioned above, followed by the cell viability detection using the cell counting kit (CCK-8 kit). Three doses of irradiation (4, 8, and 12 Gy) and a control group were included in this experiment. After irradiation, cells should be cultured for 48-h and reseeded in clear bottom 96-well plates at the optimum concentration, followed by culturing again for 12-h till they grow adhering. All the steps were performed according to the manufacturer’s 02 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 FIGURE 1 (A) Schematic of the DH microscope; (B) a single hologram with a dimension of 1024 × 1024 pixels captured by (A); and (C) a reconstructed pseudo-three-dimensional phase map from (B). solid-state laser (532 nm, 100 mW, single-mode ﬁber output) is adopted and emits a coherent light that propagates through a collimating lens (L) to produce a plane wave. The polarizing beam splitter (PBS) splits this wave into two beams, one beam acts as the reference beam R, and the other beam is used as an illumination beam. In the object arm, the object beam O is formed after the illumination beam transmits through the condenser lens (CL), the object, the MO (Olympus UPLFLN, 20X, NA = 0.50), and the TL sequentially. A half-wave plate (HPW1) is positioned in front of the PBS to adjust the intensity ratio between R and O. Another half-wave plate (HPW2) is placed in the reference arm to make the polarization direction of O and R consistent. In the reference arm, a variable optical delay line (DL) is adopted to match both arms of the optical path precisely. After passing through the DL and being reﬂected by the mirror (M6) and the beam splitter (BS), R interferes with O with a small tile angle set by the M6. Thus, an off-axis hologram is formed and recorded by a charge-coupled device (CCD, 1024 × 1024 pixels, 5.86 μm, PointGrey, Canada). Figure 1B presents a hologram of HT-1376 cells captured by the DH microscope of Figure 1A, where high contrast interference fringes were observed. Figure 1C shows the reconstructed pseudo-three-dimensional phase maps of Figure 1B, it can be seen that CAPH varies in a very large range of about a few to dozen rad, manifesting the high heterogeneity of morphology of HT-1376 cells. instructions (CCK8 Dojindo CK04-05) and ﬁnally quantiﬁed on the microplate reader. 2.2.2 Determining proliferation ability of HT1376 bladder cancer cell lines treated with different radiation doses The HT-1376 cells were seeded and incubated in 6-well plates until adherent. After 8 Gy of X-ray radiation treatment, as described before, the cells were trypsinized to single-cell suspensions and seeded into 6-well plates in triplicate. At 14 days after seeding, colonies were stained with 0.5% crystal violet (Solarbio, #G1062), and the colonies with >50 cells were counted. 2.2.3 Determining migration ability of HT-1376 bladder cancer cell lines treated with different radiation doses The HT-1376 cells were also seeded and incubated in 6-well plates. After 8 Gy of X-ray radiation treatment as described before, a 10 µl pipette tip was used to scrape a wound on the cell monolayers, images of the wound region were taken, and distance was measured by ImageJ software. 2.3 Digital holographic microscopy setup and data processing 2.3.1 Digital holographic microscopy setup 2.3.2 Hologram recording and phase reconstruction In this study, holograms of HT-1376 cells were recorded by the DH microscope setup sketched in Figure 1A. This setup is built in an afocal conﬁguration, where the back focal plane of the microscope objective (MO) coincides with the front focal plane of the tube lens (TL), with the object placed at the front focal plane of the MO. A To reveal the morphological alteration of HT-1376 cells introduced by different Ionizing radiation doses, i.e., 0 Gy (control), 4, 8, and 12 Gy, each group was observed once before and after radiation. Each group was ﬁrst observed using a DH microscope and then taken to the radiation Frontiers in Physics 03 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 CPV instrument to receive Ionizing radiation. After radiating, each group was brought back and placed under DH microscope to perform the second observation. The time interval of each group from the ﬁrst observation to receive Ionizing radiation was about 30 min, and it also took about 30 min to bring back each group to perform the second observation. All observations were performed in cell culture dishes (NEST Scientiﬁc #801001). In the hologram reconstruction process, the hologram’s zeroorder and twin term was eliminated by the spatial-frequency domain ﬁltering method [11], and the refocusing distance was automatically detected by the Tamura criterion [12]. The phase images were then reconstructed by the angular spectrum method, and the phase aberrations introduced by the off-axis geometry and optical elements were removed by the background phase ﬁtting method [13]. After phase unwrapping, a phase offset operation was ﬁnally performed on the obtained phase images to make the average background phase value near the zero lines. The single-cell was extracted from the reconstructed phase images by the Otsu threshold segmentation algorithm [14], and the marker-based watershed segmentation algorithm was further applied when cells adhered together. CMPH CPV CPA (4) 1 SelectSortΔφx, y, 50 50 (5) 2.4.2 Change rate calculation To quantitatively compare the morphological changes among different radiation doses, the change rates of CPV, CPA, CAPH, and CMPH of every cell are calculated. The change rate of the parameter is deﬁned as the ratio of the change of the parameter before and after radiation to the parameter before radiation, which are expressed as: CPVA − CPVB CPVB CPAA − CPAB CRCPA CPAB CAPHA − CAPHB CRCAPH CAPHB CMPHA − CMPHB CRCMPH CMPHB CRCPV To quantitatively describe the change in cell morphology, four parameters were taken into consideration and calculated from the reconstructed phase images, including CPV, CPA, CAPH, and CMPH. DHM is a powerful quantitative phase imaging method that measures the optical phase delays to describe the sample quantitatively. In DHM, the sample is expressed by phase shift and could be written as: (6) (7) (8) (9) In these four equations, CR denotes change rate, subscript A denotes the parameter calculated after radiation, and subscript B denotes the parameter measured before radiation. (1) 2.4.3 Statistical analysis where λ is the wavelength of the laser source, nc(x, y) is the average cellular refractive index in the spatial position (x, y), nm is the refractive index of the cellular culture medium, and h(x, y) is cell height along observation direction. Phase shift at (x, y) is referred to as cell phase height at (x, y). It can be seen that cell phase height at (x, y) is determined by both the average cellular refractive index at (x, y) and the cell height at (x, y). Usually, the cellular refractive index changes slightly so that the change of cell phase height could reliably reﬂect the change of cell height. Since the phase shift of several nanometers could be sensed by interference, DHM could provide a highly precise way to detect the change along observation direction, that is, not achievable for traditional optical microscopes. Based on the phase image of cells, the four parameters, i.e., CPV, CPA, CAPH, and CMPH could be deduced as follows: Frontiers in Physics (3) In these four equations, Sc denotes the occupied region of the cell in the phase image, Sp denotes the real area of one pixel of the CCD camera, N denotes the total number of pixels in Sc, Sort(·) denotes the sort operation and Select{·} denotes the operation of selecting the top 50 largest numbers of a sequence. CPV, CPA, and CAPH could reﬂect the 3D, transverse and vertical morphology of living cell, respectively, while CMPH could reﬂect the vertical morphology of the cellular nucleus. 2.4.1 Calculation of morphological parameters 2π nc x, y − nm hx, y λ (2) CPA Sp × N CAPH 2.4 Calculation and analysis of cellular morphological parameters Δφx, y Δφx, y (x,y)∈sc Data were analyzed by GraphPad Prism 8 (GraphPad Software, Califnia, LLC) and displayed as a mean ± 95% conﬁdence interval. The comparisons between the two groups were analyzed by Student’s t-test. The level of signiﬁcant difference was set as two-tailed p < 0.05 (*p < 0.01, p < 0.05). 3 Results 3.1 Changes of the viability of HT-1376 bladder cancer cell lines treated with different radiation doses To establish the optimal radiation dose-response for the urothelial bladder carcinoma cell line, HT-1376 was treated 04 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 FIGURE 2 Cell viability was tested by CCK8 assay in untreated HT-1376 cells or different doses of irradiation. FIGURE 3 The irradiated cells were reseeded at a low density (400 cells/ well) in a petri dish (D = 3 cm). The Coomassie-staining and colonies (≥50 cells) counting were performed after 2 weeks. Representative photographs of colonies and the colony fraction were shown. with different doses of radiation. The cell viability was determined 48 h later by CCK8 assay. Radiation signiﬁcantly inhibited the viability of HT-1376 in a dose-dependent manner, as the cell number strongly decreased (Figure 2). once under the same environmental condition before and after radiation. As shown in Figure 5, a set of holographic phase images and their pseudo-three-dimensional images before and after radiation for 0, 4, 8, and 12 Gy were displayed in the ﬁrst two columns and later two columns, respectively. Phase images before and after radiation of each group were displayed with a uniﬁed color map, and color bars of each group were located on the right. The white line in Figure 5B indicates the scale of the phase images. According to Figure 5, no obvious change in phase height and cellular shape was observed in 0 Gy group, while phase height increase could be seen in 4, 8, and 12 Gy groups. The distributions of CPV, CPA, CAPH, and CMPH of each group before and after radiation were ﬁrst qualitatively compared. Figure 6 presents the histograms of CPV, CPA, CAPH, and CMPH of each group before and after radiation. The number of cells in 0Gy group, 4Gy group, 8Gy group, and 12Gy group are 94, 93, 67, 79, respectively. The histograms indicated by the blue color show the distributions of parameters before radiation, while the histograms indicated by the green color show the distributions of parameters after radiation. As can be seen in Figures 6C,D, the histograms of CPV, CAPH, and CMPH after radiation seen slightly shift right compared with those before radiation, while the histograms of CPA slightly shift left compared with those before radiation, which indicates that the morphology of cells is changed after radiation. The changes in CPV, CPA, CAPH, and CMPH of each group were quantitatively examined. Since higher radiation dose carries larger energy and would cause more severe damage to cells, increased changes in CPV, CPA, CAPH, and CMPH were expected with the rise of radiation doses. The change rates of these four parameters of each group were presented in Figure 7. 3.2 Changes of proliferation ability of HT1376 bladder cancer cell lines treated with different radiation doses Moreover, as the cytotoxic effect of radiation was the DNAdouble strand breakage, we investigated the alteration of cancer stem cell features via colony formation. The irradiated cells had a dramatically low colony-forming capacity when compared to the untreated group (p < 0.01) (Figure 3). 3.3 Changes of migration ability of HT1376 bladder cancer cell lines treated with different radiation doses As we had captured the phenomenon of cell movement under the DH microscope, we thus examined the migration ability of irradiated cells by wound-healing assay. The mean width of the wound remained unchanged in the radiation group after 24 h, while signiﬁcantly decreased in the untreated group (p = 0.05) (Figure 4). 3.4 Changes in morphological parameters of HT-1376 bladder cancer cell lines treated with different radiation doses HT-1376 cells were individually exposed to 0, 4, 8, and 12 Gy, where 0 Gy group acted as the control. All groups were measured Frontiers in Physics 05 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 FIGURE 4 The irradiated cells were recultured to 90% conﬂuence with adherence status, followed by a scratching assay. Closure of the wounded region was evaluated by light-microscopy every 3 h. The percentage of the healing area was evaluated via ImageJ software and displayed as a diagram. FIGURE 5 Phase images and corresponding pseudo-three-dimensional images of HT-1376 cells before and after radiation for 0, 4, 8, and 12 Gy. 1.92% vs. 0.98%, p = 0.0378) compared to the 0 Gy group, while 4 Gy group and 8 Gy group did not show a signiﬁcant decrease (0.86% vs. 0.98%, p = 0.9222 and ‒0.21% vs. 0.98%, p = 0.3598, respectively). As shown in Figure 7C, the change rate of the CAPH of 12 Gy group and 8 Gy group increased signiﬁcantly (4.74% vs. 0.95%, p = 0.0007 and 3.17% vs. 0.95%, p = 0.0239, respectively) compared to the 0 Gy group, while 4 Gy group did not show a signiﬁcant increase (1.96% vs. 0.95%, p = 0.2767). Moreover, the change rate of the CAPH of 12 Gy group increased As can be seen, the change rates of CPV, CAPH, and CMPH show increased trends with the radiation dose rising, while the change rate of CPA was decreased with the radiation increase. As shown in Figure 7A, the change rate of the CPV of 12 Gy group increased signiﬁcantly (4.49% vs. 2.28%, p = 0.0079) compared to the 0 Gy group, while 4 Gy group and 8 Gy group did not show a signiﬁcant increase (3.08% vs. 2.28%, p = 0.3447 and 3.19% vs. 2.28%, p = 0.3178, respectively). As shown in Figure 7B, the change rate of the CPA of 12 Gy group decreased signiﬁcantly (‒ Frontiers in Physics 06 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 FIGURE 6 The distributions of CPV, CPA, CAPH, and CMPH of each group before and after radiation. radiation and cell morphology is rare, especially the dynamic and quantitative morphological changes after radiation. This study, to our knowledge, is the ﬁrst record that observes and characterizes the cancer cell’s morphological changes after radiation based on phase imaging strategy via DH microscope. In experiments, bladder cancer cells were treated with three doses of radiation and imaged by the DH microscope. We have demonstrated that radiation could impair the growth and migration of cancer cells and thus cause remarkable morphological changes. Meanwhile, irradiation could not only attack the cell itself but also break the intercellular junctions and destroy cell communication. (PMID: 30570385, 34688032, signiﬁcantly (4.74% vs. 1.96%, p = 0.0168) compared to the 4 Gy group. As shown in Figure 7D, the change rate of CMPH in 12 Gy group increased signiﬁcantly (6.60% vs. 3.22%, p = 0.0390) compared to the 0 Gy group, while 4 Gy group and 8 Gy group did not show signiﬁcant (0.86% vs. 2.89%, p = 0.8407 and 5.43% vs. 2.89%, p = 0.1080, respectively) decrease. 4 Discussion Radiotherapy is one of the routine methods for tumor treatment. However, research on the relationship between Frontiers in Physics 07 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 FIGURE 7 The change rates in CPV, CPA, CAPH and CMPH of each group. indicating that the surface of cells bends and folds after radiation. Unfortunately, we only observed the radiological dose-dependent manner of cell viability and cell morphological change but not a time-dependent manner. Therefore, contrary to some studies before [15–17], there was no unique parameter under the DH microscope that could stand for post-radiation morphological features. However, despite the nonspeciﬁc morphological parameter, the DH microscope has several advantages over the light microscopes, including the collection of informative 3Dmorphology data, observation with nondestructive addition, and most importantly, tracing cells with the dynamic real-time view. To our knowledge, morphology data describing treatment for cancer cells is limited. Kemper et al. reported the ﬁrst case of drug inﬂuence on the morphology changes of pancreatic cancer cells via DH microscope. They treated the pancreatic cancer cells with Latrunculin B, which led to a decrease in their phase values and cell thickness [15]. Yunxin et al. identiﬁed that the low concentration of methanol (12%–25%) reduced the optical stickiness of cervical cancer cells, while the high concentration (50%) of methanol ﬁxed the cells immediately with tiny shape change [18]. Furthermore, chemotherapy etoposide improved the cell volume of prostate cancer cells [16], while cisplatin was unable to alter the cell volume of endometrial cancer cells [17]. As a result, the effect of chemical or physical interventions on the cell morphological change might depend on the treatmentspeciﬁc properties and the cytotoxic sensitivity of cells. 11149936, 33416428) In our experiment, we observed those two types of morphological phenomena for irradiated cells, which are cellular regulation and intercellular regulation. Single-cell response to radio-stimulation by self-alteration was deﬁned as cellular regulation, including cell division, cell death, and the disappearance of cytoplasmic vacuolization. The cell-to-cell interaction response to radio-stimulation was deﬁned as intercellular regulation, including cell communication, cell migration, endocytosis, and exocytosis. Notably, intercellular junctions are highly sensitive to ionizing radiation, and previous studies observed the irradiated cells increasing in size and cellular roundness due to cell-to-cell adhesion loss. (PMID: 30570385) This phenomenon is consistent with what we observed, that some cells separated quickly from a tight touched status and changed in shape after irradiation, as shown in Figure 5C. Although those phenomena we observed might not all be radio-speciﬁc, we noticed tremendous shape changes of cells in a short period leading to a rapid increase in phase height and, ultimately, cell death. Moreover, we obtained several parameters of irradiated cells, including phase height, cluster shade, and phase variance based on refractive index measurements. The average phase height for the conﬂuent monolayer cells after gradient radiation presented a good steady rising trend, which can be attributed to the shortage of pseudopodium and damage to adhesion ability. Correspondingly, the change rate of cell cluster shade rose over gradient radiation, Frontiers in Physics 08 frontiersin.org Xiao et al. 10.3389/fphy.2022.925523 statement is not provided on submission, a standard one will be inserted during the production process. The Author Contributions statement must describe the contributions of individual authors referred to by their initials, and in doing so, all authors agree to be accountable for the content of the work. Please see here for full authorship criteria. Therefore, the DH microscope parameter alone might not be sufﬁcient enough to distinguish the type of treatment. Our observation has some limitations. Firstly, no radiationrelated morphological parameter of cells was obtained due to the nonspeciﬁc shape alteration was captured. Secondly, only one type of cancer cell line was tested. Despite the limitations, our research has a speciﬁc signiﬁcance for the single irradiated-cell continuous monitoring and their 3D shape data obtaining. Funding 5 Conclusion This work is supported by the Beijing Municipal Natural Science Foundation (Grant No. M22017), Beijing Municipal Natural Science Foundation (Grant No.7192104), National Natural Science Foundation for Young Scholars (Grant No. 81402519) and Innovation and Transformation Fund of Peking University Third Hospital (Grant No. BYSYZHKC2021113). In conclusion, our study is the ﬁrst observation that deeply investigated the irradiated cancer cells based on the quantitative phase imaging strategy using a quantitative tool, i.e., DH microscopy. We not only veriﬁed that the irradiated-related biological phenomena can be captured and identiﬁed via DH microscopy but also obtained the pattern of cell morphological change in a radiological dose-dependent manner. More importantly, our results open the way for further studies focused on exploring the treatment-speciﬁc cell morphology or distinguishing the cell response via the previously discovered or reported quantitative morphology biomarkers. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Data availability statement 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. Author contributions The Author Contributions section is mandatory for all articles, including articles by sole authors. If an appropriate References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer J Clinicians (2018) 68(6):394–424. doi:10. 3322/caac.21492 5. Hughes BR, Mirbagheri M, Waldman SD, Hwang DK. Direct cell-cell communication with three-dimensional cell morphology on wrinkled microposts. Acta Biomater (2018) 78:89–97. doi:10.1016/j.actbio.2018. 07.053 2. El-Achkar A, Souhami L, Kassouf W. Bladder preservation therapy: Review of literature and future directions of trimodal therapy. Curr Urol Rep (2018) 19(12): 108. doi:10.1007/s11934-018-0859-z 6. Nassiri I, McCall MN. Systematic exploration of cell morphological phenotypes associated with a transcriptomic query. Nucleic Acids Res (2018) 46(19):e116. doi:10.1093/nar/gky626 3. Song YP, McWilliam A, Hoskin PJ, Choudhury A. Organ preservation in bladder cancer: An opportunity for truly personalized treatment. Nat Rev Urol (2019) 16(9):511–22. doi:10.1038/s41585-019-0199-x 7. Tatapudy S, Aloisio F, Barber D, Nystul T. Cell fate decisions: Emerging roles for metabolic signals and cell morphology. EMBO Rep (2017) 18(12):2105–18. doi:10.15252/embr.201744816 4. Goranov AI, Gulati A, Dephoure N, Takahara T, Maeda T, Gygi SP, et al. Changes in cell morphology are coordinated with cell growth through the TORC1 pathway. Curr Biol (2013) 23(14):1269–79. doi:10.1016/j.cub.2013.05.035 8. 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J Struct Biol (2015) 189(3):207–12. doi:10. 1016/j.jsb.2015.01.010 11. Cuche E, Marquet P, Depeursinge C. Spatial ﬁltering for zero-order and twinimage elimination in digital off-axis holography. Appl Opt (2000) 39(23):4070–5. doi:10.1364/ao.39.004070 16. Kemper B, Carl D, Schnekenburger J, Bredebusch I, Scha€fer M, Domschke W, et al. Investigation of living pancreas tumor cells by digital holographic microscopy. J Biomed Opt (2006) 11(3):034005. doi:10.1117/1.2204609 12. Memmolo P, Distante C, Paturzo M, Finizio A, Ferraro P, Javidi B. Automatic focusing in digital holography and its application to stretched holograms. Opt Lett (2011) 36(10):1945–7. doi:10.1364/OL.36.001945 17. Yao T, Cao R, Xiao W, Pan F, Li X. An optical study of drug resistance detection in endometrial cancer cells by dynamic and quantitative phase imaging. J Biophotonics (2019) 12(7):e201800443. doi:10.1002/jbio. 201800443 13. Leiping C, Xiao W, Xiaoping L, Liu J, Pan F, Ferraro P. Automatic removal of phase aberration in holographic microscopy for drug sensitivity detection of ovarian cancer cells. OSA Continuum (2020) 3(7):1856–68. doi:10.1364/OSAC. 391773 Frontiers in Physics 18. Wang Y, Yang Y, Wang D, Ouyang L, Zhang Y, Zhao J, et al. Morphological measurement of living cells in methanol with digital holographic microscopy. Comput Math Methods Med (2013) 2013:1–7. doi:10.1155/2013/715843 10 frontiersin.org
https://openalex.org/W3127463742	https://www.granthaalayahpublication.org/journals/index.php/granthaalayah/article/download/IJRG15_S09_58/3023	English	null	NEED OF GREEN GROWTH IN INDIA	International journal of research - granthaalayah	2,015	cc-by	985	NEED OF GREEN GROWTH IN INDIA Nancy Chauhan Govt. Girls P.G. College, Ujjain, (M.P.) The Economy of India is the seventh largest in the world by nominal GDP and the third largest by purchasing power parity with approximately 7% average growth rate for the last two decades. Although the years 2012-13 and 2013-14 have been disappointing. India needs the path of high economic growth quickly, which is essential because it will generate huge revenue for the government which can be utilized for social welfare and infrastructure program. But rapid growth is not enough, it must be of the environmental friendly nature. Many countries across the world have not taken into account the consequences of some environmental issues such as air and water pollution, climate change, energy use and natural resource depletion. In a recent global assessment approximately 60% of the world’s ecosystem services were found to be degraded or used unsustainably. This is the same case with India. Though its economic performance has brought immense benefits to its citizens as employment opportunities have increased and millions have been allowed to emerge from poverty; India’s remarkable growth record, however has been clouded by a degrading environment and growing scarcity of natural resources. In a recent survey of 178 countries whose environments were surveyed India ranked 155th overall and almost last in air pollution exposure. The survey also concluded that India’s environmental quality is for below all BRIC countries. Also according to another recent WHO survey across the G-20 economies, 13 out of the 20 most polluted cities are in India. As the population grows and urbanizes and consumption patterns change, pressure on the country’s natural resources air, water, land and forests will steadily increase. In fact, in the coming years, pressure on India’s environment driven by both poverty and prosperity, is projected to become highest in the world. In such scenario, there is a need of such low cost policy options that could significantly curtail environmental damage without compromising growth. [Social Issues and Environmental Problems, Vol.3 (Iss.9:SE): Sep, 2015] ISSN- 2350-0530(O) ISSN- 2394-3629(P) Impact Factor: 2.035 (I2OR) DOI: https://doi.org/10.29121/granthaalayah.v3.i9SE.2015.3139 [Social Issues and Environmental Problems, Vol.3 (Iss.9:SE): Sep, 2015] ISSN- 2350-0530(O) ISSN- 2394-3629(P) Impact Factor: 2.035 (I2OR) DOI: https://doi.org/10.29121/granthaalayah.v3.i9SE.2015.3139 WHY INDIA NEEDS GREEN GROWTH? For developing countries, like India, where economies are more dependent on natural resources, environmental issues such as air and water pollution, climate change, energy use and natural resources depletion are of primary importance. Environmental issues are one of the primary causes of disease, health issues and long term livelihood impact for India. Besides, Environmental degradation costs India about Rs.3.75 Trillian annually equivalent to 5.7 percent of the G.D.P with air pollution being a major contributor, according to a World Bank Report. There are significant benefits in moving towards a green economy. Green growth is the pursuit of economic growth and development while preventing costly environmental degradations, climate change, biodiversity loss and unsustainable natural resource use. One important element of green development would certainly target a lower level of intensity in emissions of greenhouse gases. WHAT IS GREEN GROWTH? The concept of Green Growth has its origin in the Asia and Pacific regions, at the fifth ministerial conference on environment and development held in March 2005 in Seoul. Green Growth is a term to describe a path of economic growth that uses natural resources in sustainable manner. It is used globally to provide an alternative concept to typical industrial economic growth. Green growth is a strategy for achieving sustainable development. It is focused on overhauling the economy in a way that synthesizes economic growth and environmental protection, building a green economy in which investments in resource savings as well as sustainable management of natural capital are drivers of growth. It emphasis economic progress to foster low carbon, social inclusive development. [1-2] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Social Issues and Environmental Problems, Vol.3 (Iss.9:SE): Sep, 2015] ISSN- 2350-0530(O) ISSN- 2394-3629(P) Impact Factor: 2.035 (I2OR) [Social Issues and Environmental Problems, Vol.3 (Iss.9:SE): Sep, 2015] ISSN- 2350-0530(O) ISSN- 2394-3629(P) Impact Factor: 2.035 (I2OR) [Social Issues and Environmental Problems, Vol.3 (Iss.9:SE): Sep, 2015] ISSN- 2350-0530(O) ISSN- 2394-3629(P) Impact Factor: 2 035 (I2OR) WHAT INDIA NEEDS TO DO?  Policy action and effective implementation is required to prevent obstacles to growth as well as to reduce the adverse impact upon unsustainable practices.  Policy action and effective implementation is required to prevent obstacles as well as to reduce the adverse impact upon unsustainable practices. y as well as to reduce the adverse impact upon unsustainable practices.  To increase energy efficiency in industries, vehicles, appliances.  To accelerate the transformation of its power sector including the adoption of clean technology, building green infrastructure for urban habitats and transportation and establishing sustainable agriculture and forestry practices. India can make green growth a reality by putting in place strategies to reduce environmental level of intensity in emissions of greenhouse gases. India can make green growth a reality by putting in place strategies to reduce environmental level of intensity in emissions of greenhouse gases. India can make green growth a reality by putting in place strategies to reduce environmental level of intensity in emissions of greenhouse gases. India is a rising power and can create new avenues of growth by trading in goods that economizes on resources consumption. India can also trade in labor intensive and skill intensive goods where we are competitive. The government of India has setup 2015 as the target to release green G.D.P. data. Green G.D.P is calculated by factoring in the environmental consequences of growth. REFERENCES REFERENCES 1. Diagnostic assessment of select environmental changes in India – World bank report, July 2013 2. Economy of India – http://en.wikipedia.org/wiki/Green-growth 3. India: Green Growth - Overcoming environmental challenges to promote development – - www.worldbank.org/en/news/feature/2014/03/06/green-growth-overcoming-india- environment-chanllenges-promote-development 4. Expansion vs Sustainability : India takes on the green growth challenges – mcknseyonsociety.com/india-taking-on-the-green-growth-challenge. [1-2] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH
W4224300629.txt	https://www.degruyter.com/document/doi/10.1515/bfp-2021-0093/pdf	de		Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? Eine empirische Untersuchung der Jahre 2010–2019	Bibliothek	2,022	cc-by	4,691	BIBLIOTHEK – Forschung und Praxis 2022; 46(1): 216–226 Christiane Strauß* und Ulla Wimmer Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? Eine empirische Untersuchung der Jahre 2010–2019 https://doi.org/10.1515/bfp-2021-0093 Zusammenfassung: Dieser Beitrag zur Themenwahl studentischer Abschlussarbeiten der Jahre 2010 bis 2019 präsentiert die Untersuchungsergebnisse von über 2 000 Titeldaten von sechs Hochschulen mit bibliotheksund informationswissenschaftlichen Studiengängen aus Deutschland und der Schweiz. Schwerpunktmäßig wird dargestellt, wie präsent Öffentliche Bibliotheken, wissenschaftliche Bibliotheken und andere Einrichtungen in Abschlussarbeiten vertreten sind und welche Themen von Studierenden gewählt werden. Schlüsselwörter: Abschlussarbeiten; Hochschulen; Bibliotheks- und Informationswissenschaft; Öffentliche Bibliotheken; wissenschaftliche Bibliotheken Topics of Library and Information Sciences Bachelor and Master Theses in Universities in Germany and Switzerland 2010–2019 Abstract: This article on the topic selection of student theses from 2010 to 2019 presents the analysis of more than 2,000 title data from six universities with library and information science programmes in Germany and Switzerland. The main focus is on the representation of public libraries, academic libraries and other institutions in these theses and which topics are chosen by students. Keywords: Theses; universities; library and information science; public libraries; academic libraries Inhalt 1 Einleitung und Fragestellung, Forschungsstand . 2 Methodik und Grenzen . . . . . . . . . . . . . . . . . . 3 Ergebnisse. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Wie präsent sind Bibliotheken in den Abschlussarbeiten? . . . . . . . . . . . . . . . . . . . . . . . . . . . . *Kontaktperson: Christiane Strauß, christiane.strauss@ibi.hu-berlin.de Ulla Wimmer, ulla.wimmer@ibi.hu-berlin.de 216 218 219 219 3.2 Gibt es beim Institutionen- und Spartenbezug Unterschiede zwischen den Hochschulen? . . . . . 220 3.3 Über welche Themen schreiben LIS-Studierende? 221 3.4 Themen der Arbeiten mit Institutionenbezug . . . . 222 3.5 Gibt es bei der Themenwahl Unterschiede zwischen den Hochschulen? . . . . . . . . . . . . . . . . . 223 4 Fazit und Ausblick . . . . . . . . . . . . . . . . . . . . . . 225 1 Einleitung und Fragestellung, Forschungsstand Wo sind die Öffentlichen Bibliotheken in Forschung und Lehre? Darüber diskutiert die Bibliothekscommunity immer wieder, seit vor ca. fünf Jahren sich der Stellenmarkt im Bereich der Öffentlichen Bibliotheken nach langer Zeit der Stellenknappheit endlich wieder öffnete.1 Diese Debatte bildete den Ausgangspunkt für die vorliegende Untersuchung.2 Die angeregte Diskussion um zeitgemäße Kompetenzen von BibliotheksmitarbeiterInnen, um die Curricula der bibliotheks- und informationswissenschaftlichen Studiengänge und um die Präsenz einer Bibliothekssparte in Studium und Wissenschaft repräsentiert sowohl die Veränderung des Berufsfelds „Bibliothek“ als auch des Fachs Bibliotheks- und Informationswissenschaft seit der Jahrtausendwende. Die Berufscommunity fordert einerseits vielseitig einsetzbare LIS3-AbsolventInnen mit ausgeprägten Schlüsselkompetenzen und einem breiten, flexibel verwendbaren Kompetenzprofil4, andererseits aber auch eine Ausrichtung der BerufsanfängerInnen auf konkrete, aktu- 1 Vgl. Konferenz der informations- und bibliothekswissenschaftlichen Ausbildungs- und Studiengänge (2018), Wimmer (2019a). 2 Die Arbeiten begannen im Januar 2020, wurden von April bis Juni 2021 weitergeführt und im Rahmen des 109. Deutschen Bibliothekartages 2021 präsentiert, vgl. Wimmer und Strauß (2021). 3 Die Abkürzung LIS (Library and Information Science) wird aus Gründen der Praktikabilität hier synonym mit der Bezeichnung „Bibliotheks- und Informationswissenschaft“ verwendet. 4 Vgl. Barbian und Vonhof (2017). Open Access. © 2022 Christiane Strauß und Ulla Wimmer, publiziert von De Gruyter. Creative Commons Namensnennung 4.0 International Lizenz. Dieses Werk ist lizensiert unter einer Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? elle Handlungsfelder der verschiedenen Bibliothekspraxen5. In Verbindung mit den kontinuierlichen Veränderungen im Gesellschafts-, Technologie- und Mediensektor ergibt sich daraus eine Neuausrichtung des Berufsbilds und der erforderlichen Kompetenzen und Curricula.6 Die Bibliotheks- und Informationswissenschaft wiederum, als die akademische Disziplin, in deren Forschungsbereich die Bibliotheken fallen, hat in den letzten Jahren ihren Fokus verbreitert: Sie untersucht vermehrt auch institutionsunabhängige Informationsthemen und vermittelt Kompetenzen, die für eine Tätigkeit in verschiedenen Berufsfeldern (von der Online-Agentur bis zur Forschung) qualifizieren.7 Die Kernfrage unseres Vorhabens war, ob und wie sich diese Veränderungen über einen längeren Zeitraum in den Abschlussarbeiten der LIS-Studierenden niederschlagen, ob sich also im Output der Hochschulen die Entwicklung des Faches Bibliotheks- und Informationswissenschaft und des Berufsbildes zeigt. Daraus ergaben sich die konkreten Fragestellungen unserer Untersuchung für die Jahre 2010–2019: Welcher Anteil der Abschlussarbeiten beschäftigt sich in diesem Zeitraum konkret mit Bibliotheken? In welchem Ausmaß und mit welchen Themen tauchen Bibliotheken überhaupt in diesen Arbeiten auf und inwiefern die verschiedenen Bibliothekssparten? Welcher Anteil behandelt dagegen ein Thema, das nicht an eine konkrete Institution gebunden ist, z. B. das Informationsverhalten von jungen Eltern oder das Publikationsverhalten der ForscherInnen einer bestimmten Disziplin? Welche Themenbereiche priorisieren die Studierenden? Wie präsent sind z. B. die klassischen Handlungsfelder wie Erwerbung und Erschließung im Vergleich zur Bildungsfunktion der Bibliothek, zu Managementthemen, zur Social-Media-Analyse oder den Möglichkeiten automatischen Datenmappings? Und lassen sich unterschiedliche Profile der Hochschulen dabei erkennen? Diese Fragen sind deshalb interessant, weil sich die Studierenden in der Regel ihre Themen selbständig wählen, und zwar auf Basis der Inhalte und Themen, die sie in der Lehre, aber auch oft im Praktikum besonders angesprochen haben. Man kann also an den Antworten ablesen, welcher Aspekt des Faches/des Berufsfelds die Studierenden interessiert und womit sie sich intensiv am Ende Ihres 5 Der Begriff „Praxis“ wird hier im Plural verwendet, weil „die Praxis“ in unterschiedlichen Organisationstypen (von der Universitätsbibliothek über die Mediendokumentation zur Gemeindebücherei) so unterschiedlich ist, dass von „einer Praxis“ im Singular nicht gesprochen werden kann. 6 Vgl. Fachbeirat ekz GmbH (2017), Georgy (2017), Gantert et al. (2018), Vonhof (2017), Petras et al. (2017). 7 Vgl. z. B. Ockenfeld (2020), Wimmer und Greifeneder (2018). 217 Studiums auseinandergesetzt haben. Daraus ergibt sich, mit welchem „Tiefenwissen“ und ggf. auch mit welcher beruflichen Orientierung (z. B. auf eine Bibliothekssparte) sie in den Beruf gehen – und ihn damit auf längere Sicht auch transformieren. Gleichzeitig stellt im „Kleinen Fach“ Bibliotheks- und Informationswissenschaft die Forschungsarbeit, die in Abschlussarbeiten geleistet wird, einen substantiellen Anteil der insgesamt geleisteten Forschungsarbeit dar. Man kann also umgekehrt auch an den Themen ablesen, welche Fragen und Probleme der Fachcommunity wissenschaftlich bearbeitet werden, wo also neues Wissen generiert wird, das dann der Community wieder zugutekommen kann. Es zeigt sich daran auch, inwieweit spezifische Fragestellungen einer Institution/ Sparte bearbeitet (oder vernachlässigt) werden und inwiefern sich das Profil der LIS verändert. Das Potential einer solchen Themenanalyse wurde bereits vor zehn Jahren erkannt: Für den Zeitraum von 2000 bis 2010 führte Prof. Dr. Gerhard Hacker (HTWK Leipzig) bereits eine ähnliche Untersuchung durch.8 Hier gab es allerdings ein etwas anderes Erkenntnisinteresse und daher ein anderes methodisches Vorgehen. Die wichtigsten Unterschiede zwischen der Untersuchung von Prof. Hacker und unserem Vorgehen fasst Tab. 1 zusammen. Der herausragende Unterschied besteht sicher darin, dass damals nur publizierte Arbeiten mit einem hohen Aufwand intellektuell ausgewertet wurden, während wir es darauf anlegten, rudimentäre Informationen über eine möglichst vollständige Liste der Arbeiten zu nutzen und so eine möglichst breite Datenbasis auszuwerten. Tab. 1: Unterschiede im Forschungsdesign zwischen Hacker (2010) und Strauß und Wimmer (2021) Hacker (2010) Strauß und Wimmer (2021) Forschungsperspektive/Ausgangspunkt Zusammenarbeit und Wissenstransfer zwischen Hochschule und Praxis Wo sind die (Öffentlichen) Bibliotheken in Forschung und Lehre? Zeitraum 2000–2009 2010–2019 Hochschulen HU Berlin, HS GraubünHU Berlin, FH den, HAW Hamburg, TH Darmstadt, HAW Hamburg, FH Hannover, Köln, HTWK Leipzig, HdM FH Köln, HTWK Leipzig, Stuttgart nur LIS-Institute, die explizit auch zu FH Potsdam, HdM Öffentlichen BibliotheStuttgart ken lehren, daher ohne: HS Darmstadt, HS Hannover, FH Potsdam 8 Hacker (2010). 218 Christiane Strauß und Ulla Wimmer Tab. 1: (fortgesetzt) Hacker (2010) Strauß und Wimmer (2021) Untersuchte Arbeiten 2 222 Abschlussarbeiten 676 Abschluss- und Projektarbeiten, für die alle, deren Titeldaten erVolltext, Abstract oder mittelt werden konnten. Inhaltsverzeichnis publiziert waren. Quellen HS-Webseiten, RepositoHS-Webseiten, Zeitschriften-Inhalts verz., rien, HS-BibliotheksDABI, Infodata E-Depot kataloge, interne Listen Methode Autopsie der publizierten Texte oder Abstracts Auswertung der Spartenbezug, Bezug Arbeiten nach auf eine namentlich genannte Institution (=Praxisbezug), Abschluss, Hochschule, Jahr. 5 thematische Cluster Limitationen Kodierung nur der Titeldaten Institutionenbezug und Sparte, Abschluss, Hochschule, Jahr, Autorengender. 29 ThemenKategorien in 9 Oberkategorien unterschiedliche und unterschiedliche und inkonsistente Publikati- inkonsistente Verzeichnungspraxis an den onspraxis an den Hochschulen und im Hochschulen (nichtZeitverlauf, Datenbasis publizierte Arbeiten teilweise nicht ganz nicht einbezogen) konsistent 2 Methodik und Grenzen Um die Forschungsfrage zu beantworten, haben wir Abschlussarbeiten aller Art aus den Jahren 2010–2019 untersucht, also Bachelor, Master, Diplom- und (unspezifische) „Abschlussarbeiten“. Ausgehend vom Forschungsinteresse wurden nur Hochschulen einbezogen, die explizit auch zu Öffentlichen Bibliotheken lehren – d. h. enthalten sind die HAW Hamburg, die HTWK Leipzig, Die FH Graubünden (Chur), die HdM Stuttgart, die TH Köln und die HU Berlin. Nicht enthalten sind die FH Darmstadt, FH Hannover und FH Potsdam. Im Sinne einer Diskursstrukturanalyse9 sollte die Datenbasis so umfassend wie möglich sein, also alle Arbeiten einbeziehen, deren Titeldaten auf irgendeine Art und Weise zugänglich waren. Dies stellte sich als das größte Problem – und die größte Limitation der Arbeit – heraus, denn unter den Hochschulen, und bei jeder Hochschule auch noch über einen Zeitraum von 10 Jahren, gab und 9 Vgl. Jäger (2000), Wimmer (2019b). gibt es sehr unterschiedliche Praktiken und „Policies“, wie Abschlussarbeiten verzeichnet werden. Verzeichnet werden z. B. „alle AutorInnen, die der Veröffentlichung nicht widersprechen“ (HU Berlin, TH Köln), „alle Arbeiten, die gedruckt in der Bibliothek vorliegen“ (HTWK Leipzig), „alle Arbeiten, die mindestens mit 2 bewertet wurden“ (diverse Hochschulen) bis hin zu „alle Arbeiten, die auf der institutsinternen Liste geführt werden“ (HdM Stuttgart). Öffentlich verzeichnet wird im Katalog, auf der Webseite oder im Repositorium, mit jeweils sehr unterschiedlichen Retrievalmöglichkeiten, was Auswirkungen auf die „Precision“ des Datensatzes hatte. Dabei gibt es beträchtliche Abgrenzungs- und Vollständigkeitsprobleme; einerseits weil sich die Verzeichnungspraxis immer wieder geändert hat oder einfach zeitlich hinterherhinkt. Zum anderen, weil in den Verzeichnissen teilweise auch Abschlussarbeiten aus angrenzenden Studiengängen enthalten sind, die man nicht ganz trennscharf von LIS-Abschlussarbeiten separieren kann. Das war vor allem bei der TH Köln der Fall mit den Studiengängen „Online-Redaktion“ und „Markt- und Medienforschung“, und bei der HAW Hamburg mit den Studiengängen „Digitale Kommunikation“ und „Digitale Transformation der Informations- und Medienwirtschaft“. Letztlich ist die organisatorische und inhaltliche Nähe zu anderen Studiengängen auch ein Ausdruck des sich verbreiternden Themenspektrums in der LIS. Insgesamt bedeutet das aber, dass die Datenbasis weder vollständig noch ganz konsistent ist.10 Das Ziel war jedoch, die größtmögliche Anzahl an Arbeiten auszuwerten. Um dies zu erreichen wurden einige Inkonsistenzen in Kauf genommen. Sie werden bei der Auswertung soweit möglich berücksichtigt. Eine größere Einschränkung besteht darin, dass Aussagen über eine Entwicklung innerhalb des Betrachtungszeitraums nur sehr eingeschränkt und nur in den Jahren 2013 bis 2017 möglich sind. Weiterhin bestehen Differenzen in der Anzahl der erfassten Abschlussarbeiten je Hochschule, die sich von 636 Titeln (HdM Stuttgart) bis zu 53 Titeln (FHGR Chur) erstrecken. Dies gilt es insbesondere hinsichtlich der Aussagekraft der Themenprofile der Hoch10 Es liegen z. B. folgende Limitationen vor: 1) Datenerhebung war im Januar 2020, daher ist das Jahr 2019 bei allen HS unvollständig außer TH Köln und HdM Stuttgart, 2) Teilweise gibt es starke Schwankungen in der Titelzahl zwischen den Jahren, für die es keine andere Erklärung als unterschiedliche Verzeichnungspraxis gibt. 3) Für das IBI der HU Berlin konnten erst ab 2013, für die HS Graubünden ab 2016 die Daten ermittelt werden. 4) 80 Arbeiten der TH Köln stammen definitiv aus dem Untersuchungszeitraum, haben aber keine Jahresangabe. 5) die Liste der HdM Stuttgart enthielt keine Autorenangabe, daher war keine Genderauswertung möglich. Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? schulen zu berücksichtigen. Tab. 2 zeigt die Verteilung der ermittelten und untersuchten Arbeiten auf die sechs Hochschulen. Tab. 2: Verteilung der untersuchten Titel auf Hochschulen. n = 2 222 Hochschule Anzahl Titel HdM Stuttgart 636 TH Köln 588 IBI HU Berlin 484 HAW Hamburg 239 HTWK Leipzig 222 FHGR Chur 53 219 Abschlussarbeiten ermöglichen. Im Laufe der Codierung wurde das Schema vollständig ausgeschöpft, d. h., es gibt kein Thema (unseres Kategorienschemas), das nicht besprochen wurde. Praktische Unsicherheiten in der Zuordnung wurden gemeinschaftlich analysiert und die Entscheidungen leitfadenähnlich festgehalten, beispielsweise bei Fällen, in denen zwei oder mehr Kategorien in Frage kamen oder ein sehr spezielles Thema einer Kategorie zugeordnet werden musste. Das Schema sowie die Codierhinweise können für spätere Untersuchungen wiederverwendet werden, um eine Wiederholung der Studie mit Vergleich- und Übertragbarkeit der Ergebnisse zu ermöglichen. 3 Ergebnisse Hinsichtlich der ermittelten Genderverteilung des Datensatzes ist festzuhalten, dass 70 % der Abschlussarbeiten von Frauen und 30 % von Männern verfasst wurden.11 Dies unterscheidet sich immerhin um fünf Prozentpunkte von der aktuellen Genderverteilung im Beruf (Frauen: 75 %, Männer: 25 %).12 Ob dies eine längerfristige Entwicklung in der Zusammensetzung der Berufsgruppe indiziert, konnte im Rahmen der Untersuchung nicht geklärt werden. Insgesamt wurden nach Bereinigung, Entfernung von Dubletten etc. 2 222 Datensätze ausgewertet. Standardmäßig wurden die Felder Titel, AutorIn und Jahr erfasst. Der Spartenbezug ließ sich über die Sichtung des Titels ermitteln: Sofern eine Institution explizit genannt oder implizit eindeutig gemeint war (Beispiel: Bibliotheksangebote für Vorschulkinder), konnte eine Zuordnung in die Bereiche Öffentliche Bibliotheken (ÖB), wissenschaftliche Bibliotheken (WB), Bibliotheken allgemein (kein Spartenbezug, auch bibliothekarische Sonderformen) und sonstige Institutionen (beispielsweise Verlage, Archive, Museen etc.) erfolgen. Arbeiten ohne BID-Institutionenbezug wurden in die Bereiche Akademische Lebenswelt (Hochschulen, Forschungseinrichtungen, Studium), Nichtakademische Lebenswelt (Wirtschaft/Arbeitswelt, Privatleben/Freizeit etc.) und Sonstiges aufgeteilt. Die thematische Codierung der Datensätze erfolgte anhand des von einer der Autorinnen entwickelten Kategorienschemas, welches auf der Themengliederung des BIBOPUS-Repositoriums basiert.13 Es umfasst 29 Kategorien, die ein differenziertes Bild der thematischen Vielfalt der 11 Die Ermittlung erfolgte auf Basis des Vornamens und resultiert in einer binären Geschlechtskategorie. 12 Institut für Arbeitsmarkt -und Berufsforschung (2018). Die Zahlen stammen aus dem Erhebungszeitraum 2013–2017. 13 Vgl. Wimmer (2019b) 182 f. 3.1 Wie präsent sind Bibliotheken in den Abschlussarbeiten? Zunächst geht es ganz grundlegend darum, wie präsent Bibliotheken (egal welcher Sparte) in den Abschlussarbeiten von LIS-Studierenden waren. Die Auswertung zeigt: 70 %–75 % aller Abschlussarbeiten im Untersuchungszeitraum bezogen sich explizit auf Bibliotheken oder angrenzende Institutionen. 25–30 % aller Arbeiten hatten keinen Bezug zu Bibliotheken oder Informationseinrichtungen. In dieses Segment fallen jedoch auch die Arbeiten, von denen wir annehmen, dass sie aus angrenzenden Studiengängen stammen. Abb. 1: Institutionen- und Spartenbezug der Abschlussarbeiten 2010–2019 (n = 2 222) 220 Christiane Strauß und Ulla Wimmer Abb. 2: Institutionen- und Spartenbezug bei den untersuchten Hochschulen. Ohne Arbeiten aus vermutlich angrenzenden Studiengängen schäftigen sich mit Themen aus allen Lebensbereichen. Diese Arbeiten können auch für Öffentliche Bibliotheken relevant sein, wenn es z. B. um das Informationsverhalten von SeniorInnen, von Eltern oder von Jugendlichen geht. Legende zu Abb. 2 Die weitere Differenzierung in Abb. 1 zeigt: Jeweils ein gutes Viertel (25 %–27 %) aller LIS-Abschlussarbeiten im Zeitraum 2010–2019 bezog sich explizit auf Öffentliche bzw. wissenschaftliche Bibliotheken. Sie waren in diesem Zeitraum somit zu gleichen Teilen vertreten. Dazu kommen noch einmal 15 % aller Arbeiten, die sich mit Bibliotheken allgemein (ohne Spartenbezug) beschäftigten. Die Arbeiten mit Bibliotheksbezug sind folglich zu fast 80 % auf eine konkrete Bibliothekssparte ausgerichtet. Die Arbeiten ohne Institutionsbezug beschäftigten sich zu einem guten Fünftel mit akademischen Themen: Studium, Forschung, wissenschaftliches Publizieren, Open Access, Forschungsdaten usw. Diese Arbeiten sind häufig auch für wissenschaftliche Bibliotheken relevant. Vier Fünftel der Arbeiten ohne Institutionenbezug be- 3.2 Gibt es beim Institutionen- und Spartenbezug Unterschiede zwischen den Hochschulen? Bezüglich der Präsenz von Bibliotheken und anderen Institutionen in den Abschlussarbeiten gibt es deutliche Unterschiede zwischen den Hochschulen. (Aus dieser Auswertung wurden die Datensätze ausgeschlossen, die vermutlich aus angrenzenden Studiengängen stammen.) Abb. 2 zeigt: Es gibt zwei Hochschulen, die HdM Stuttgart und die HTWK Leipzig, die einen besonders hohen Anteil an bibliotheksbezogenen Arbeiten aufwiesen: 80– 90 %. Bei den anderen vier Hochschulen lag der Anteil zwischen 50 und 70 %. Bei vier Hochschulen (TH Köln, HAW Hamburg, HTWK Leipzig, FHGR Chur) waren die bibliotheksbezogenen Arbeiten ganz grob gleich verteilt zwischen ÖB- und WBBezug. Das war anders bei der HdM Stuttgart und beim IBI der HU Berlin: In Stuttgart überwog klar der ÖB-Bezug, in Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? 221 Abb. 3: Die 10 am häufigsten gewählten Themen aller Abschlussarbeiten Worüber wird nun in den Abschlussarbeiten konkret geschrieben? Beim Blick auf die Top-10-Themen aller codierten Abschlussarbeiten – mit und ohne Institutionsbezug – zeigt sich, dass der Themenkomplex „Management und betriebliche Steuerung“ mit 13 % auf Position 1 liegt (s. Abb. 3). Die Arbeiten bespielen hierbei eine große Bandbreite von Management- und Marketingfragestellungen: Projektund Changemanagement, Wissens-, Personal- und Qualitätsmanagement sowie klassische Methoden und Instrumente der Lenkung und Führung für Institutionen und Unternehmen. Der Einsatz von Social Media, Online- und Content-Marketing sind weitere typische Themen in diesem Datensatz. Auf Position 2 befindet sich mit 7 %15 der Themenkomplex „Nutzerforschung, Informationsverhalten, Wissenschaftliches Arbeiten, Wissensgeschichte, Mediengeschichte“, fast gleichauf mit „Bibliothekarische Dienstleistungen, Bibliotheksbenutzung, Leihverkehr, Auskunft“ sowie „Erschließung, Linked Open Data, RDA“ an den Positionen 3 und 4. Weitere Top-Themen sind mit je 6 % „Leseförderung, Bildungspartner Bibliothek, Schulbibliothek, Kinder- und Jugendbibliothek“ und „Interkulturelle Bibliotheksarbeit, Diversity, spezielle Nutzergruppen“ sowie mit je 5 % die Themengebiete „Vermittlung von Informationskompetenz, Teaching Library, E-Tutorials, OPAC, Discovery-Systeme, Portale, virtuelle Bibliotheken“ und „Elektronische Publikationen, elektronisches Publizieren, Forschungsdaten, Open Data, virtuelle Forschungsumgebungen“. Fragestellungen aus dem Bereich „Technik, Automatisierung, IT allgemein“ machen 4 % der Abschlussarbeiten aus. Es dominieren insgesamt ‚handfeste‘, (bibliotheks-) praktische und handlungsorientierte Themen. Diese Top-10-Themen decken zwei Drittel der Abschlussarbeiten inhaltlich ab. Das verbliebene Drittel besteht aus Abschlussarbeiten, die den übrigen 19 Themenkategorien zugehörig sind. Bereinigt man den Datensatz um diejenigen Arbeiten, die vermutlich aus angrenzenden Studiengängen stammen (dies betrifft ca. 35 % der Management-Arbeiten und ca. 19 % der Arbeiten zur Nutzerforschung), verändert sich die Top 10 inhaltlich nicht. Der Anteil der Arbeiten mit Fragestellungen aus dem Bereich „Management, betriebli- 14 Hacker (2010) 180 (Abb. 6). 15 Zur Erinnerung: Bei 29 Themenkategorien würden bei Gleichverteilung auf jede Kategorie 3,4 % der Arbeiten entfallen. Berlin der WB-Bezug. Das reflektiert in gewisser Weise die Geschichte der beiden Einrichtungen; es erklärt sich aber vor allem auch durch die Art der Abschlüsse: Während die Bachelor-Abschlüsse im Datensatz insgesamt ganz klar überwogen, waren beim IBI eine hohe Zahl an Masterarbeiten aus dem Weiterbildenden Masterstudiengang Bibliotheks- und Informationswissenschaft eingeflossen. Diese Arbeiten haben einen starken WB-Schwerpunkt. Das Ergebnis bezüglich des Spartenbezugs entspricht für die HdM Stuttgart, das IBI und die HAW Hamburg den Ergebnissen, die Prof. Hacker für den Zeitraum 2000–2009 gefunden hat.14 Bei der TH Köln fand er einen deutlich stärkeren WB-Bezug, der in den Daten von 2010–2019 nicht mehr sichtbar war. 3.3 Über welche Themen schreiben LIS-Studierende? 222 Christiane Strauß und Ulla Wimmer Abb. 4: Themen, die am wenigsten für Abschlussarbeiten gewählt wurden che Steuerung“ verringert sich zwar von 13 % auf 8 %, führt jedoch weiterhin die Liste gewählter Themen an. Arbeiten mit dem Schwerpunkt „Nutzerforschung, Informationsverhalten etc.“ befinden sich weiterhin unter den Top-Themen (an Position 6 statt Position 2). Betrachtet man die Bottom 10, also diejenigen Themen, über die in Abschlussarbeiten am wenigsten geschrieben wird, so sind dies z. B. systembezogene, rechtliche und (wissenschafts-)theoretische Untersuchungen (vgl. Abb. 4). Arbeiten zu baulichen16/bibliothekstechnischen Fragestellungen sind ebenso selten vertreten wie Arbeiten über das Selbstverständnis und die Rolle von Bibliotheken in der Gesellschaft. Das „Bibliothekswesen des Auslands (allgemein)“ und „Verlage/Informationswirtschaft“ werden ebenfalls kaum als Gegenstand studentischer Abschlussarbeiten gewählt. Gemeinsam ist diesen Themen, dass sie einen starken Theoriebezug haben und ein komplexes methodisches Vorgehen erfordern, das mit Blick auf die Rahmenbedingungen studentischer Abschlussarbeiten (Bearbeitungszeit, Umfang, Zugang zu Daten etc.) wenig praxistauglich ist. Vorstellbar ist auch, dass die Themen als nicht förderlich für den Berufseinstieg bewertet werden. Das Thema Bibliotheksbau mag BachelorStudierenden noch weit entfernt erscheinen. Ein Vergleich dieser Daten mit den Ergebnissen der Untersuchung von Prof. Dr. Hacker ist insofern mit äußerster Vorsicht vorzunehmen, als sich die thematischen Auswertungskategorien im Zuschnitt unterscheiden. Es sind 16 Gemeint ist hier nicht die „Bibliothek als (Dritter) Ort“. Arbeiten mit diesem Schwerpunkt finden sich in der Kategorie „Bibliothekarische Dienstleistungen, Bibliotheksbenutzung“ wieder. keine belastbaren Aussagen über Themenentwicklungen möglich. Mit aller damit verbundenen Unschärfe scheinen die Themen in Hackers Themencluster „Management“ (das sind Bibliotheksbenutzung, Marketing, Öffentlichkeitsarbeit, zielgruppenspezifische Produktgestaltung17) etwas zugenommen, die in seinem Themencluster „Vermittlung“ (Elektronisches Publizieren, Informationsvermittlung, Lese- und Informationskompetenzvermittlung, Information Retrieval18) tendenziell etwas abgenommen zu haben. 3.4 Themen der Arbeiten mit Institutionenbezug Welche Themen wurden für Arbeiten gewählt, die einen Institutionenbezug (zu Bibliotheken, Archiven, Dokumentationseinrichtungen etc.) unabhängig von der Sparte haben? Von 1 538 Arbeiten behandeln zwei Drittel die Themen in Abb. 5. Wie zu erwarten war, ist die Themenwahl nun eindeutig stärker auf die Herausforderungen und Besonderheiten der jeweiligen Einrichtungen und Einrichtungstypen zugeschnitten: Fragen des Managements, der Benutzung und Dienstleistungen sind nach wie vor dominant. Hinzu kommen bei den Top-Themen die Kategorie „Erwerbung, Lizenzen, Bestandsaufbau, Pflicht, Makulieren“ sowie Arbeiten zu „Sonstige Bibliothekstypen und Sammlungen“, hier z. B. zu Patientenbibliotheken, OPLs, Speicher- oder 17 Vgl. Hacker (2010) 179. 18 Ebd. Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? 223 Abb. 5: Die 10 am häufigsten gewählten Themen aller Arbeiten mit Institutionenbezug (in orange: Themen, die im Vergleich zur Gesamtverteilung neu hinzugekommen sind) Gedenkstättenbibliotheken. Auch der Bereich der „Historischen Sammlungen, Provenienzforschung, Bestandserhaltung, Buchgeschichte“ mit konkreten Beschreibungen und Erschließungsarbeiten von und in überwiegend wissenschaftlichen Bibliotheken und Archiven/Museen ist hier vertreten. Die zuvor häufig vertretenen Themen „Nutzer(und Informationsverhaltens-)forschung“ und „Elektronisches Publizieren“ fallen hier ab, da sie zu einem Großteil (60 %) in Arbeiten ohne Institutionenbezug behandelt werden. Hierbei werden Fragen der Nutzer- und Informationsverhaltensforschung v. a. in nichtakademischen Lebensbereichen, Fragestellungen des „Elektronisches Publizierens“ im akademischen Bereich untersucht. Außerdem sind wir der Frage nachgegangen, ob es Unterschiede in der Themenwahl bei Arbeiten mit ÖB- und WB-Bezügen gibt. Es zeigt sich, dass diese Unterschiede sowohl bei den Themen an sich als auch bei der Verteilung der Themen bestehen. Abschlussarbeiten zu Öffentlichen Bibliotheken konzentrieren sich thematisch stark auf die Bereiche „Leseförderung, Bildungspartnerschaften, Kinder- und Jugendbibliotheken“ (21 %) sowie „Interkulturelle Bibliotheksarbeit, Diversity, spezielle Nutzergruppen“ (18 %) und „Bibliothekarische Dienstleistungen“ (10 %). Mit diesen drei Themen wird bereits rund die Hälfte der Arbeiten inhaltlich beschrieben (s. Abb. 6). Bei den Arbeiten zu wissenschaftlichen Bibliotheken ist die Themenwahl weniger konzentriert und gleichmäßiger verteilt (s. Abb. 7). An den Positionen 1-2 stehen mit je 10 % die Kategorien „Erschließung, Linked Open Data, RDA“ (bei Arbeiten zu ÖBs erwartungsgemäß kaum gewählt) sowie Arbeiten mit dem Fokus „Historische Sammlungen, Provenienzforschung, Bestandserhaltung, Buchgeschichte“. Fragen der „Bibliothekarischen Dienst- leistungen“ werden bei beiden Spartenbezügen (ÖB/WB) mit jeweils 10 % gleich häufig als Thema gewählt. Auch der Themenbereich „Vermittlung von Informationskompetenz“ kommt in Arbeiten beider Sparten in den Top-10Themen vor, ebenso die Kategorie „Management, betriebliche Steuerung“. Während bei den ÖB-Arbeiten „Interkulturelle Bibliotheksarbeit“ ein sehr prominentes Thema ist (Position 2), spielt es bei den WB-Arbeiten kaum eine Rolle (lediglich 1 % der Arbeiten mit WB-Bezug) und das, obwohl im Wintersemester 2019/20 über 400 000 ausländische Studierende an deutschen Hochschulen immatrikuliert waren.19 Hier böte sich Potential für interessante Abschlussarbeiten. Umgekehrt ist „Erwerbung“ eines der Top-Themen in WB-Arbeiten (Position 5), nicht jedoch in Arbeiten zu ÖBs (Position 10). Zusammenfassend lässt sich sagen, dass es eine eindeutige spartenspezifische Themenwahl in den Abschlussarbeiten gibt. 3.5 Gibt es bei der Themenwahl Unterschiede zwischen den Hochschulen? Bei Betrachtung der gewählten Abschlussarbeitsthemen je Hochschule lassen sich individuelle Profile beobachten (s. Abb. 8). Die Datensätze der Abschlussarbeiten aus vermutlich angrenzenden Studiengängen sind von der Auswertung ausgenommen. 19 Daten von Statista entnommen: https://de.statista.com/statistik/ daten/studie/301225/umfrage/auslaendische-studierende-in-deutsch land-nach-herkunftslaendern/. 224 Christiane Strauß und Ulla Wimmer Abb. 6: Über 75 % der Arbeiten mit ÖB-Bezug behandeln die acht farbig dargestellten Themen Abb. 7: Über 75% der Arbeiten mit WB-Bezug behandeln die zehn farbig dargestellten Themen Worüber schreiben LIS-Studierende ihre Abschlussarbeiten? 225 Abb. 8: Top-5-Themen von Abschlussarbeiten nach Hochschule Bei drei der sechs Hochschulen finden sich Themen auf den vorderen Positionen wieder, die über alle untersuchten Abschlussarbeiten hinweg keine Top-Themen sind, also als Profilelemente angesehen werden können. Arbeiten der HTWK Leipzig zeigen im Bereich „Historische Sammlungen, Provenienzforschung, Bestandserhaltung, Buchgeschichte“ eine Profilspitze (Position 1). An der HAW Hamburg werden besonders häufig Arbeiten zu Fragestellungen der „Kultur-, Veranstaltungs-, Öffentlichkeits- und Programmarbeit“ untersucht (Position 3). Für die FH Graubünden lassen sich aufgrund der geringen Anzahl von Datensätzen nur sehr limitiert Aussagen treffen. Erkennbar ist, dass „Nutzerforschung und Technik, Automatisierung, IT allgemein“ häufig gewählte Themen sind. Der Bereich „Verlage, Informationswirtschaft“ ist außerdem stark vertreten (an Position 3). Die Themenbereiche sind insgesamt breiter und weniger institutions- und spartenspezifisch angelegt. Bei zwei Hochschulen sind ebenfalls Themenschwerpunkte nachweisbar: an der HdM Stuttgart im Bereich „Leseförderung, Bildungspartnerschaft, Kinder- und Jugendbibliotheken“ (an Position 1), was mit der Möglichkeit der Schwerpunktbildung im Studienverlauf des BA-Studiengangs („Bibliotheks-, Kultur- und Bildungsmanagement“) zusammenhängt. Am IBI Berlin findet sich das Thema „Elektronisches Publizieren, Open Access, Open Science“ an der Top-Position. Zusammen mit den Positionen 2 („Erschließung“) und 3 („Nutzerforschung“) spiegelt diese Reihung die langjährige Zusammensetzung der Lehrstühle des Instituts wieder. Das Themenprofil der TH Köln liegt sehr nah an der allgemeinen Themenverteilung und bildet somit ein breites und vielfältiges Spektrum ab. Zu „OPACs, DiscoverySysteme, Portale, virtuelle Bibliotheken“ gibt es häufiger Arbeiten als im Durchschnitt. Es wird deutlich, dass kein Hochschulprofil dem anderen gleicht. Schwerpunkte und Spezifika sind eindeutig feststellbar. 4 Fazit und Ausblick Auch wenn der Begriff Bibliothek aus vielen Studiengangsbezeichnungen verschwunden ist, prägen Bibliotheken nach wie vor die Disziplin: Die Studierenden in den untersuchten LIS-Studiengängen mit ÖB-Bezug schreiben ca. drei Viertel aller Abschlussarbeiten mit einem direkten Bezug auf BID-Einrichtungen, und zwar zu gleichen Teilen über Öffentliche und wissenschaftliche Bibliotheken. Die Studierenden wählen ganz überwiegend handlungs- und praxisbezogene Themen aus, das zeigt sich in den Hauptthemenfeldern Management, Benutzung, Erschließung, Kompetenzförderung oder Arbeit mit besonderen Nutzergruppen. Dabei gibt es deutliche Themenunterschiede zwischen den Sparten der Öffentlichen und der wissenschaftlichen Bibliotheken. Es gibt darüber hinaus auch klare Profilunterschiede zwischen den sechs Hochschulen: die verschiedenen Studiengänge und Lehrstühle zeichnen sich deutlich ab. Aus unserer Sicht ergeben sich interessante Erkenntnisse bezüglich der Entwicklung sowohl von Disziplin und Profession und der Verschränkung der beiden Bereiche. Es würde daher Sinn machen, die Untersuchung in größeren Abständen zu wiederholen. Damit konsistentere Daten zur Verfügung stehen und Zeitreihen gebildet werden können, wäre es hilfreich, wenn sich die Hochschulen zu einer beständigeren, kontinuierlichen Verzeichnungspraxis für ihre Arbeiten entschließen könnten. 226 Christiane Strauß und Ulla Wimmer Literaturverzeichnis Barbian, Jan-Pieter; Vonhof, Cornelia (2017): „The Times they are achangin‘“. Welche Qualifikationen und Kompetenzen benötigen Bibliothekare in der Zukunft? In: BuB – Forum Bibliothek und Information, 462–69. Fachbeirat ekz GmbH (2017): Positionspapier „Berufsbild und Entwicklung“. In: Bibliotheksdienst, 51 (10/11), 876–77. Gantert, Klaus; Neher, Günther; Schade, Frauke (2018): Die digitale Transformation meistern: Aktuelle Entwicklungen in der bibliotheks- und informationswissenschaftlichen Aus- und Weiterbildung. In: BIBLIOTHEK – Forschung und Praxis, 42 (3), 441–52. Georgy, Ursula (2017): Bibliotheks- und informationswissenschaftliche Aus- und Weiterbildung: Herausforderungen und Perspektiven. In: Bibliotheksdienst, 51, (10/11), 864–75. Hacker, Gerhard (2010): Neue Qualitäten beim Wissenstransfer zwischen Hochschule und Berufspraxis? Eine Analyse deutscher LIS-Projekt- und Graduierungsarbeiten des letzten Jahrzehnts. In: Information – Wissenschaft & Praxis, 61 (3), 175–80. Institut für Arbeitsmarkt -und Berufsforschung (2018): Berufe im Spiegel der Statistik. Verfügbar unter http://bisds.iab.de/Defau lt.aspx?beruf=BG733&region=1&qualifikation=0. Jäger, Siegfried (2000): Theoretische und methodische Aspekte einer Kritischen Diskurs- und Dispositivanalyse. In: Handbuch Sozialwissenschaftliche Diskursanalyse. Opladen: Leske + Budrich. Verfügbar unter http://www.diss-duisburg.de/Internetbiblio thek/Artikel/Aspekte_einer_Kritischen_Diskursanalyse.htm#_ ftn14. Konferenz der informations- und bibliothekswissenschaftlichen Ausbildungs- und Studiengänge (2018): Konferenz der informations- und bibliothekswissenschaftlichen Ausbildungs- und Studiengänge (KIBA)/Sektion 7 im Deutschen Bibliotheksverband und Humboldt-Universität zu Berlin, Institut für Bibliotheks- und Informationswissenschaft. Paneldiskussion: Öffentliche Bibliotheken in Forschung und Lehre — Institut für Bibliotheks- und Informationswissenschaft. Institut für Bibliotheksund Informationswissenschaft. 4. Dezember 2018. Verfügbar unter https://www.ibi.hu-berlin.de/de/online-archiv/veranstal tungen/paneldiskussionoeb. Ockenfeld, Marlies (2020): Informationswissenschaft – Bandbreite und Ausrichtungen in der DACH-Region. In: Information Wissenschaft & Praxis. Petras, Vivien; Gäde, Maria; Rügenhagen, Melanie; Wimmer, Ulla (2017): Die bibliotheks- und informationswissenschaftlichen Masterstudiengänge an der Humboldt-Universität zu Berlin. In: Bibliotheksdienst, 51 (10/11), 891–900. Vonhof, Cornelia (2017): Bachelor und Berufspraxis – das Studienmodell der Hochschule der Medien Stuttgart. In: Bibliotheksdienst, 51 (10/11), 923–34. Wimmer, Ulla (2019a): Wo sind die Öffentlichen Bibliotheken in Forschung und Lehre? In: Bibliothek 2030. Humboldt-Universität zu Berlin. Verfügbar unter https://edoc.hu-berlin.de/handle/18 452/20928. Wimmer, Ulla (2019b): Die Geschichte vom großen Ö. Die Position der Öffentlichen Bibliotheken im Bibliotheksfeld und im bibliothekarischen Fachdiskurs der Bundesrepublik Deutschland seit 1964. Berlin: Humboldt-Universität. Verfügbar unter https://edoc.hu-b erlin.de/handle/18452/20588. Wimmer, Ulla; Greifeneder, Elke (2018): Die 90-jährige, die aus dem Bibliotheksfenster steigt – und bleibt. Forschung und Lehre am Institut für Bibliotheks- und Informationswissenschaft der Humboldt Universität zu Berlin. Vortrag. Gehalten auf: 107. Deutscher Bibliothekartag, Berlin. Verfügbar unter https://opus4.ko bv.de/opus4-bib-info/frontdoor/index/index/docId/15743. Wimmer, Ulla; Strauß, Christiane (2021): Worüber schreiben LISStudierende ihre Abschlussarbeiten? Vortrag. Gehalten auf: 109. Deutscher Bibliothekartag, Bremen. Verfügbar unter https:// opus4.kobv.de/opus4-bib-info/frontdoor/index/index/docId/ 17712. Christiane Strauß Humboldt-Universität zu Berlin Institut für Bibliotheks- und Informationswissenschaft Unter den Linden 6 D-10099 Berlin christiane.strauss@ibi.hu-berlin.de Ulla Wimmer Humboldt-Universität zu Berlin Institut für Bibliotheks- und Informationswissenschaft Unter den Linden 6 D-10099 Berlin ulla.wimmer@ibi.hu-berlin.de
https://openalex.org/W2040228980	https://www.scielo.br/j/spmj/a/HXnysWcyZtczznfYqcBSnwx/?lang=en&format=pdf	English	null	Twenty-first century: the beginning of the replacement parts era?	São Paulo medical journal	2,012	cc-by	1,185	Twenty-first century: the beginning of the replacement parts era? Século XXI: o início da era das peças de reposição? Alessandro Wasum MarianiI, Paulo Manuel Pêgo-FernandesII For many years, science fiction explored the idea of treating diseases by means of using artificial devices. References to replacement of human organs by implantable mechanical devices can be found in many books and films. The advances within medicine and bioengineering that have been achieved between the end of the twentieth century and the present date seem to be turn ing this dream into reality. IMD. Thoracic Surgeon, Instituto Dante Pazzanese de Cardiologia, and Postgraduate Student in the Discipline of Thoracic and Cardiovascular Surgery, Faculdade de Medicina da Universidade de São Paulo (FMUSP). IIMD, PhD. Associate Professor, Discipline of Thoracic Surgery, Instituto do Coração (InCOR), Hospital das Clínicas (HC), Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil. There are now six indexed scientific journals specifically focusing on this topic (Artificial Organs; Journal of Artificial Organs; Artificial Organs Today; Biomaterials, Artificial Cells, and Artificial Organs; Biomaterials, Medical Devices and Artificial Organs; and Trends in Bioma terials and Artificial Organs), and there is absolutely no doubt that this exemplifies the impor tance of this subject. Another indication of the seriousness of such research is that a search in the PubMed database using the key word “Artificial Organs” produces 43,411 retrieved results. The interest in this field has grown as materials with greater biocompatibility have become available and as device miniaturization has progressively accelerated.1 Private initiative, sepa rately or in association with university departments and research institutes, has funded a good proportion of this research, which has already resulted in patents and commercially marketed devices. The need for specialized professionals, whether as physicians, engineers, biologists or bioengineers, is growing and thus opening up new avenues for these professions. Among the organs that might be replaced, research is advancing particularly in relation to development of an artificial heart, kidney replacement therapy using “portable” machines, replacement limb orthoses and devices performing the endocrine function of the pancreas. Dialysis is the most widely used form of replacement of internal organ function with an artificial device. However, the need for dialysis sessions in which the patient remains literally a prisoner of the machine has a major impact on the quality of life of patients with kidney failure. editorial editorial editorial IMD. Thoracic Surgeon, Instituto Dante Pazzanese de Cardiologia, and Postgraduate Student in the Discipline of Thoracic and Cardiovascular Surgery, Faculdade de Medicina da Universidade de São Paulo (FMUSP). IIMD, PhD. Associate Professor, Discipline of Thoracic Surgery, Instituto do Coração (InCOR), Hospital das Clínicas (HC), Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil. Twenty-first century: the beginning of the replacement parts era? Século XXI: o início da era das peças de reposição? Miniaturization of dialysis systems has now made it possible to construct and, on an experimental basis, to use porta ble dialysis devices that can be attached to the patient’s body, thus allowing some activities to be per formed during the dialysis period. In 2008, Gura et al. published a paper on a non-implantable dialy sis device that was adapted to be inserted in a vest, thereby providing the patient with full mobility.2 Development of an artificial heart has now been studied for approximately 50 years. Today, this research has given rise to several long-term devices known as VADs (ventricular assist devices), which can be implanted. These devices enable life support through increased cardiac function for prolonged periods. Among these, the ones that have been most studied are: DeBakey VAD (MicroMed, Houston, United States), HeartMate II (Thoratec, Pleasanton, United States), DuraHeart (Terumo, Ann Arbor, United States), Incor (Berlin Heart, Germany) and HeartWare HVAD (HeartWare, Framingham, United States). Although complete artificial hearts now exist, the problems inherent to such devices (need for anticoagulation and battery life, among others) have so far meant that they are not used as the definitive treatment for most patients. Their use is often as a bridge to transplantation.3 Nevertheless, since research is heading towards solutions for these problems, many investi gators believe that such devices will be increasingly used in a definitive manner, within a few years. Another important point is that studies have already demonstrated that these devices, even with their limitations, have the capacity to increase not only the survival but also the quality of life of patients with advanced heart diseases.4 Sao Paulo Med J. 2012; 130(2):75-6 75 EDITORIAL \| Mariani AW, Pêgo-Fernandes PM Refinement of the bioengineering will also make it possible for organs with endocrine functions to be replaced. The best examples are systems capable of replacing the endocrine function of the pancreas. Devices known as “insulin bombs”, which have been devel oped and are already in clinical use, consist of microprocessor systems that measure blood glucose levels and automatically respond with insulin infusions. Twenty-first century: the beginning of the replacement parts era? Século XXI: o início da era das peças de reposição? No implantable models are currently commercially available, but the progressive reduction in volume and increase in precision of these devices indicate that, in the near future, an “implantable artificial endocrine pancreas” may become avail able for treating diabetes mellitus.5 Although ventilatory support and oxygenation machines (extracorporeal circulation) are well-known replacements for lung func tion, no prototypes for lungs that could be implantable yet exist. Nonetheless, the emergence of smaller-sized devices such as the New Lung,6 along with advances in tissue bioengineering, can be expected to motivate researchers in this field. Medicine and engineering are rapidly and consistently advancing together within the field of development of artificial organs. Recent papers like those described here signal that we will soon have new therapeutic alternatives available. However, the real impact of these devices on longevity and quality of life among the population can be expected to remain unknown for many years. The high estimated cost of using these products and what this will represent for the already far too high cost of modern medicine is the most worrying factor. 76 Sao Paulo Med J. 2012; 130(2):75-6 REFERENCES 1. Journal of Artificial Organs Editorial Committee. Journal of Artificial Organs 2010: the year in review. J Artif Organs. 2011;14(1):1-8. Date of first submission: December 6, 2011 Last received: December 6, 2011 Accepted: December 19, 2011 2. Gura V, Ronco C, Nalesso F, et al. A wearable hemofilter for continuous ambulatory ultrafiltration. Kidney Int. 2008;73(4):497-502. 3. Potapov EV, Krabatsch T, Ventura HO, Hetzer R. Advances in mechanical circulatory support: year in review. J Heart Lung Transplant. 2011;30(5):487-93. Address for correspondence: Alessandro Wasum Mariani Rua João Moura 690 — apto 121 Pinheiros — São Paulo (SP) — Brasil CEP 05412-001 E-mail: alessandro_mariani@hotmail.com 4. Allen JG, Weiss ES, Schaffer JM, et al. Quality of life and functional status in patients surviving 12 months after left ventricular assist device implantation. J Heart Lung Transplant. 2010;29(3):278-85. 5. Nishida K, Shimoda S, Ichinose K, Araki E, Shichiri M. What is artificial endocrine pancreas? Mechanism and history. World J Gastroenterol. 2009;15(33):4105-10. 6. Ota K. Advances in artificial lungs. J Artif Organs. 2010;13(1):13-6. 76 Sao Paulo Med J. 2012; 130(2):75-6
https://openalex.org/W2098060429	https://translational-medicine.biomedcentral.com/counter/pdf/10.1186/s12967-015-0441-0	English	null	Induction of tumor inhibitory anti-angiogenic response through immunization with interferon Gamma primed placental endothelial cells: ValloVax™	Journal of translational medicine	2,015	cc-by	8,192	* Correspondence: thomas.ichim@gmail.com †Equal contributors 1Batu Biologics Inc, San Diego, 9255 Towne Centre Drive, Suite 450, San Diego, CA 92121, USA Full list of author information is available at the end of the article RESEARCH Open Access Open Access Ichim et al. Journal of Translational Medicine (2015) 13:90 DOI 10.1186/s12967-015-0441-0 Abstract Background: While the concept of angiogenesis blockade as a therapeutic intervention for cancer has been repeatedly demonstrated, the full promise of this approach has yet to be realized. Specifically, drugs such as VEGF-blocking antibodies or kinase inhibitors suffer from the drawbacks of resistance development, as well as off-target toxicities. Previous studies have demonstrated feasibility of specifically inducing immunity towards tumor endothelium without consequences of systemic autoimmunity in both animal models and clinical settings. Method: Placenta-derived endothelial cells were isolated and pretreated with interferon gamma to enhance immunogenicity. Syngeneic mice received subcutaneous administration of B16 melanoma, 4 T1 mammary carcinoma, and Lewis Lung Carcinoma (LLC), followed by administration of control saline, control placental endothelial cells, and interferon gamma primed endothelial cells (ValloVax™). Tumor volume was quantified. An LLC metastasis model was also established and treated under similar conditions. Furthermore, a safety analysis in non-tumor bearing mice bracketing the proposed clinical dose was conducted. Results: ValloVax™immunization led to significant reduction of tumor growth and metastasis as compared to administration of non-treated placental endothelial cells. Mitotic inactivation by formalin fixation or irradiation preserved tumor inhibitory activity. Twenty-eight day evaluation of healthy male and female mice immunized with ValloVax™resulted in no abnormalities or organ toxicities. Conclusion: Given the established rationale behind the potential therapeutic benefit of inhibiting tumor angiogenesis as a treatment for cancer, immunization against a variety of endothelial cell antigens may produce the best clinical response, enhancing efficacy and reducing the likelihood of the development of treatment resistance. These data support the clinical evaluation of irradiated ValloVax™as an anti-angiogenic cancer vaccine. Induction of tumor inhibitory anti-angiogenic response through immunization with interferon Gamma primed placental endothelial cells: ValloVax™ Thomas E Ichim1†, Shuang Li2†, Hong Ma1, Yuliya V Yurova3, Julia S Szymanski1, Amit N Patel4, Santosh Kesari5,6, Wei-Ping Min7 and Samuel C Wagner1 © 2015 Ichim et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background Antitumor activity was also caused by the adoptive transfer of isolated spleen lymphocytes, thus demonstrating induction of tumor spe- cific immunity [30]. Other approaches have been utilized to induce immunity to VEGFR2, which resulted in in- duction of tumor regression without systemic toxicities [31-36]. Tumor endothelial marker 1 or endosialin is an- other antigen found selectively on the tumor vasculature. Facciponte et al. demonstrated that a DNA vaccination targeting endosialin reduced tumor vascularity, increased CD3+ T cell infiltration, and was correlated with signifi- cant inhibition of tumor growth. Epitope spreading to tumor antigens following the initial immune response against the tumor vasculature gives evidence that targeting the tumor endothelium may activate a cascade of path- ways conducive to tumor regression. Additionally, the DNA vaccination against endosialin did not affect other angiogenesis dependent physiological processes, exhibiting no adverse effects on menstruation, embryonic develop- ment, pregnancy, and wound healing in mouse models [14]. Other markers associated with tumor blood vessels have been utilized therapeutically in animal models for vaccination purposes including survivin [37-39], xenogen- eic FGF2R [40], VEGF [41], VEGF-R2 [42], MMP-2 [43], and endoglin [44,45]. Although tumor endothelial cells are more genetically bl h h ll h d i h ibili f exceedingly difficult to develop effective immunotherapies targeting tumor-derived antigens. A novel approach to- wards inducing anti-tumor immunity would be to target not the tumor itself, but the blood supply feeding the tumor, an essential mechanism of tumor growth. g Immunological targeting of tumor endothelium is ap- pealing based on: a) For every tumor endothelial cell thera- peutically neutralized approximately 200–300 tumor cells perish, thus reducing ability of tumors to lose expression of antigens; b) The immune system is in direct contact with the tumor endothelium, while immune access inside tumors is difficult due to areas of necrosis and high inter- stitial pressure; and c) Demonstrated prior efficacy of other anti-angiogenesis inhibitory compounds such as bevacizu- mab [11,12]. Furthermore, the elevated expression of Fas Ligand on the tumor endothelium mediates the selective killing of CD8+ Tumor Infiltrating Lymphocytes (TIL) allowing for a predominance of FoxP3+ T regulatory cells (Treg) to infiltrate the tumor microenvironment, demonstrating that the tumor blood vessels act as an immunological barrier promoting tumor tolerance [13]. Immune-mediated destruction of the tumor endothelium has been shown to significantly increase TILs in mouse models, which was correlated with tumor regression [14]. Background tumors lack expression of co-stimulatory molecules critical for the activation of naïve T cells, and suppress the expres- sion of these molecules on antigen presenting cells [9]. Tolerogenic means elaborated by the tumor inhibit T cell activation while creating a microenvironment conducive to T cell exhaustion. Poor T cell function in the tumor micro- environment allows tumors to escape immune-mediated destruction promoting the developent of treatment resist- ance through immunoediting [10]. The ability of tumors to escape immune pressure and sculpt their immuno- genic phenotype to evade immune destruction makes it Tumors utilize a variety of molecular mechanisms to evade the immune response, including loss of tumor specific an- tigens [1-3], suppression of antigen presenting machinery such as transporter associated protein and MHC expres- sion [4-7], and the production of immunosuppressive fac- tors, both soluble and surface bound [8]. Additionally, Correspondence: thomas.ichim@gmail.com †Equal contributors 1Batu Biologics Inc, San Diego, 9255 Towne Centre Drive, Suite 450, San Diego, CA 92121, USA Full list of author information is available at the end of the article Page 2 of 9 Ichim et al. Journal of Translational Medicine (2015) 13:90 response to Robo4, with no objectively detectable adverse effects on health, including normal menstruation and wound healing. Robo4 vaccinated mice showed im- paired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti- tumor effect of Robo4 vaccination was present in CD8 de- ficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody-dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism [23]. An- other antigen that is more ubiquitously found throughout the body, but with higher expression on tumor endothelial cells is the VEGF receptor 2 (VEGFR2) which is typ- ically found on hematopoietic stem cells and endothelial progenitor cells [24-29]. Despite expression on non- malignant tissue, successful induction of antitumor im- munity has been demonstrated using various immunization means against this antigen. Yan et al. utilized irradiated AdVEGFR2-infected cell vaccine-based immunotherapy in the weakly immunogenic and highly metastatic 4 T1 murine mammary cancer model. Lethally irradiated, virus-infected 4 T1 cells were used as vaccines. Vaccin- ation with lethally irradiated AdVEGFR2-infected 4 T1 cells inhibited subsequent tumor growth and pulmonary metastasis compared with challenge inoculations. Angio- genesis was inhibited, and the number of CD8+ T lympho- cytes was increased within the tumors. Preparation of vaccine Full term human placentas were collected from delivery room under informed consent. Fetal membranes were manually peeled back and the villous tissue is isolated from the placental structure. Villous tissue was subse- quently washed with cold saline to remove blood and scissors used to mechanically digest the tissue. Lots of 25 grams of minced tissue were incubated with approxi- mately 50 ml of HBSS with 25 mM of HEPES and 0.28% collagenase, 0.25% dispase, and 0.01% DNAse at 37 Celsius. The mixture of minced placental villus tissue and digest- ing solution was incubated under stirring conditions for three incubation periods of 20 minutes each. Ten minutes after the first incubation period and immediately after the second and third incubation periods, the DNAse was added to make up a total concentration of DNase, by vol- ume, of 0.01%. In the first and second incubations, the in- cubation flask is set at an angle, and the tissue fragments allowed to settle for approximately 1 minute, with 35 ml of the supernatant cell suspension being collected and re- placed by 38 ml (after the first digestion) or 28 ml (after the second digestion) of fresh digestion solution. After the third digestion the whole supernatant was collected. The supernatant collected from all three incubations was then pooled and is poured through approximately four layers of sterile gauze and through one layer of 70 micrometer poly- ester mesh. The filtered solution was then centrifuged for 1000 g for 10 minutes through diluted new born calf serum, said new born calf serum diluted at a ratio of 1 vol- ume saline to 7 volumes of new born calf serum. The pooled pellet was then resuspended in 35 ml of warm DMEM with 25 mM HEPES containing 5 mg DNase I. The suspension was subsequently mixed with 10 ml of 90% Percoll to give a final density of 1.027 g/ml and centri- fuged at 550 g for 10 minutes with the centrifuge brake off. The pellet was then washed in HBSS and cells incubated for 48 hours in complete DMEM media. After 3–4 pas- sages cells were incubating in media containing 100 IU of IFN-gamma per ml. Preparation of vaccine Subsequent to incubation cells were either used: a) unmanipulated; b) used as a lysate, with 10 freeze thaw cycles in liquid nitrogen, subsequent to which lysate was filtered through a 0.2 micron filter; c) mitotically inactivated by irradiation at 10 Gy; or d) inactivated by fix- ation in 0.5% formalin and subsequently washed. In this current study it was demonstrated that placen- tal endothelial cells that are interferon gamma primed potently inhibit tumor growth in 3 histologically distinct animal models, as well as suppress pulmonary metastasis subsequent to intravenous tumor administration. Fur- thermore, the therapeutic effect was retained when pla- cental endothelial cells have been mitotically inactivated by either formalin or irradiation. The success of this new approach may provide a new way to develop clinical ef- fective placental cells vaccination against a wide variety of tumors by targeting tumor angiogenesis. Background Additionally, the authors found that, immunity targeted to tumor vasculature was induced and was responsible for the anti-tumor activity, which was not associated with any noticeable toxicity to- ward non-malignant tissues [48,49]. From a clinical per- spective, a 17 patient trial demonstrated that HUVEC vaccine therapy significantly prolonged tumor doubling time and inhibited tumor growth in patients with recur- rent glioblastoma, inducing both cellular and humoral responses against the tumor vasculature without any adverse events or noticeable toxicities [50]. The clinical efficacy of using HUVEC vaccination to break tolerance to tumor angiogenesis has also been demonstrated in pa- tients with colorectal cancer and malignant brain tumors without any observable adverse effects on healthy angio- genesis [20]. CO2. The murine mammary carcinoma 4 T1 cells (ATCC) were grown DMEM medium (Sigma-Aldrich) with 10% FBS, l-glutamine, penicillin, and streptomycin at 37°C in 5% CO2. Lewis Lung Carcinoma (LLC) is a murine lung carcinoma originating from C57/BL6 mice. The cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine (Gibco-BRL, Life Technologies, Inc.). The cell line was cultured at 37°C in a 5% incubator. has been reported in tumor associated vascular cells [46,47]. Accordingly, a polyvalent vaccine approach tar- geting the immune system toward a plethora of endothe- lial cell antigens specific to the tumor endothelium may be more effective. With this approach comes a heightened theoretical risk of autoimmunity. Despite these theoretical concerns successful immunization against tumor endothe- lium has been performed utilizing Human Umbilical Vein Endothelial Cells (HUVEC). Wei et al. demonstrated that vaccination of mice with fixed xenogeneic whole endothe- lial cells (in the form of HUVEC) as a vaccine was effective in affording protection from tumor growth, inducing regression of established tumors, and prolonging the sur- vival of tumor-bearing mice. Additionally, the authors found that, immunity targeted to tumor vasculature was induced and was responsible for the anti-tumor activity, which was not associated with any noticeable toxicity to- ward non-malignant tissues [48,49]. From a clinical per- spective, a 17 patient trial demonstrated that HUVEC vaccine therapy significantly prolonged tumor doubling time and inhibited tumor growth in patients with recur- rent glioblastoma, inducing both cellular and humoral responses against the tumor vasculature without any adverse events or noticeable toxicities [50]. Background The clinical efficacy of using HUVEC vaccination to break tolerance to tumor angiogenesis has also been demonstrated in pa- tients with colorectal cancer and malignant brain tumors without any observable adverse effects on healthy angio- genesis [20]. Background Another further potential benefit of targeting the tumor associated vasculature is the potential of sensitizing tu- mors to radiotherapy [15], in part due to the selective thrombotic and apoptotic effects irradiation has on the tumor vasculature [16-19]. Current tyrosine kinase inhibi- tors blocking angiogenesis systemically inhibit pro- angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) or Angiopoetin, slowing blood vessel for- mation without differentiating between tumor and healthy angiogenesis. However, therapeutics that stimulate direct damage to the tumor endothelium have been shown to ac- tivate the coagulation cascade, effectively cutting off blood supply to the tumor and creating a hypoxic microenvir- onment conducive to necrosis and tumor regression [20]. A more effective anti-angiogenesis approach may be to stimulate selective killing of the tumor endothelium through immunotherapeutic vaccines. A fundamental question determining feasibility of vaccine- induced killing of tumor vasculature is whether antigens exist on the tumor endothelium that are not expressed on physiologically normal blood vessels, and whether immun- ity could be raised against such antigens. A few tumor endothelium-specific antigens have been reported. The roundabout receptor (ROBO)-4 is a transmembrane pro- tein that was originally found to orchestrate the neuronal guidance mechanism of the nervous system [21]. ROBO4 was found to be selectively expressed on tumor endothe- lial cells but not healthy vasculature [22]. Zhuang et al. demonstrated that mice immunized with the extracel- lular domain of mouse Robo4, showed a strong antibody Although tumor endothelial cells are more genetically stable then the tumor cells, thus reducing the possibility of immune mediated antigen loss, some mutational activity Page 3 of 9 Page 3 of 9 Ichim et al. Journal of Translational Medicine (2015) 13:90 has been reported in tumor associated vascular cells [46,47]. Accordingly, a polyvalent vaccine approach tar- geting the immune system toward a plethora of endothe- lial cell antigens specific to the tumor endothelium may be more effective. With this approach comes a heightened theoretical risk of autoimmunity. Despite these theoretical concerns successful immunization against tumor endothe- lium has been performed utilizing Human Umbilical Vein Endothelial Cells (HUVEC). Wei et al. demonstrated that vaccination of mice with fixed xenogeneic whole endothe- lial cells (in the form of HUVEC) as a vaccine was effective in affording protection from tumor growth, inducing regression of established tumors, and prolonging the sur- vival of tumor-bearing mice. Materials and methods Animals and cells Female C57BL/6 and BALB/c mice aged 8–12 weeks were purchased from The Jackson Laboratory. Animals were housed under conventional conditions at the Animal Care Facility, University of Western Ontario, and were cared for in accordance with the guidelines established by the Canadian Council on Animal Care. A murine melan- oma cell line established from a C57BL/6 mouse and designated B16F10 was obtained from the American Type Culture Collection (ATCC) and was maintained in RPMI 1640 medium (Sigma-Aldrich) with 10% FBS, l- glutamine, penicillin, and streptomycin at 37°C in 5% Page 4 of 9 Page 4 of 9 Ichim et al. Journal of Translational Medicine (2015) 13:90 administration of tumor challenge. As seen in Figures 1, 2 and 3, a trend towards reduction of tumor growth was ob- served with non-IFN-gamma pretreated endothelial cells, while a potent reduction of tumor growth was seen in ani- mals treated with cells that were first stimulated with interferon gamma. Interferon gamma pretreatment was shown to upregulate HLA I and HLA II (data not shown). Immunization schedules and tumor assessment For induction of tumor growth, 5 × 105 B16, LLC, or 4 T1 cells, American Type Culture Collection (Manassas, VA) cells were injected subcutaneously into the hind limb flank. Four weekly vaccinations of 5 × 105 test cells were administered subcutaneously on the contralateral side to which tumors were administered. Vaccination was per- formed on the day of tumor inoculation and on days 7, 14, and 21. Tumor growth was assessed every 3 days by two measurements of perpendicular diameters by a caliper, and animals were sacrificed when tumors reached a size of 1 cm in any direction. Tumor volume was calculated by the following formula: (the shortest diameter2 × the lon- gest diameter)/2. Mitotically inactivated endothelial cell vaccine retains antitumor activity across histologically different tumors For clinical development of a cancer angiogenesis vac- cine, it is imperative to generate cells that are mitotically inactivated. Part of the reason for this is that administra- tion of viable endothelial cells could potentially result in acceleration of tumor growth through enhancement of angiogenesis [59]. Furthermore, in previous clinical tri- als, endothelial cells were pretreated with a fixative to avoid this potential issue [20]. As seen in Figures 4, 5 and 6, for all tumor models tested, mitotic inactivation utilizing irradiation was mildly superior to formalin fix- ation. Materials and methods Animals and cells Additionally, no therapeutic effect was observed by administration of endothelial cell lysate, with activity being retained in activated endothelial cells. Successful utilization of placental endothelial cells in induction of anticancer immunity regardless of tumor type While it has previously been demonstrated that vaccination with autologous and allogeneic endothelial cells results in tumor regression [51-55], and safety of this approach has been reported in clinical studies [20], current means of extracting endothelial cells are limited to the need for tissue culture expansion. Generally endothelial cells proliferate poorly in vitro and require the addition of recombinant growth factors that add expense, as well as possibility of contamination during production of clinical grade produc- tion. Accordingly, a more practical source of endothelium would be the placental body, which contains up to 2–10 billion primary endothelial cells per placenta [56-58]. We immunized mice bearing LLC, B16 and 4 T1 cells. The immunization schedule was a therapeutic one in that time of immunization occurred concurrently with the Endothelial cell vaccine inhibit tumor metastasis Endothelial cell vaccine inhibit tumor metastasis In addition to reduction of tumor size growth, an im- portant aspect of cancer immunization is reduction in lung metastasis. Importantly, in the utilization of vac- cines against cancer, the ability of the immune system to seek and destroy metastatic cells is one of the key en- ticing factors of this approach. When intravenous ad- ministration of LLC was performed in C57BL/6 mice, immunization with ValloVax™resulted in inhibition of tumor lung metastasis colonies (Figure 7). The inhibition Figure 1 ValloVax™treatment inhibits B16 melanoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) B16 cells and quantified every third day. Figure 1 ValloVax™treatment inhibits B16 melanoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) B16 cells and quantified every third day. Ichim et al. Journal of Translational Medicine (2015) 13:90 Page 5 of 9 Figure 2 ValloVax™treatment inhibits 4 T1 mammary carcinoma growth. Female BALB/c mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) 4 T1 cells and quantified every third day. Figure 2 ValloVax™treatment inhibits 4 T1 mammary carcinoma growth. Female BALB/c mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) 4 T1 cells and quantified every third day. Figure 2 ValloVax™treatment inhibits 4 T1 mammary carcinoma growth. Female BALB/c mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Discussion was significantly more profound when placental endo- thelial cells were treated with interferon gamma. The concept of targeting tumor associated endothelium has been a holy grail of cancer therapists since the original work of Judah Folkman demonstrated that tumors cannot grow more than 1–2 millimeters without the stimulation of new blood vessel formation (angiogenesis) [60]. Specif- ically blocking angiogenesis is intellectually enticing be- cause the tumor-associated endothelium is derived from non-mutated tissue; therefore the possibility of devel- opment of a drug resistant phenotype is very low. Unfor- tunately several angiogenesis-targeting drugs that have demonstrated promising results in animal trials have failed in pivotal clinical trials. Examples include angiostatin, endostatin, and shark cartilage extract (Neovastat) [61,62]. Endothelial cell vaccine inhibit tumor metastasis Tumor growth was initiated by subcutaneous administration of 5 × 10(5) 4 T1 cells and quantified every third day. Safety evaluation More recent studies using the VEGF pathway blocking antibody, bevacizumab (Avastin), have demonstrated posi- tive results in specific types of tumors, which led to regu- latory approvals [63]. Unfortunately the use of these antibodies requires co-administration of chemotherapy, and is associated with signification toxicity [64]. Further- more, for reasons unknown, bevacizumab is ineffective in several tumor types, and in the tumors that it is effective, resistance often ensues, limiting long-term therapeutic utility [65]. More recent studies using the VEGF pathway blocking antibody, bevacizumab (Avastin), have demonstrated posi- tive results in specific types of tumors, which led to regu- latory approvals [63]. Unfortunately the use of these antibodies requires co-administration of chemotherapy, and is associated with signification toxicity [64]. Further- more, for reasons unknown, bevacizumab is ineffective in several tumor types, and in the tumors that it is effective, resistance often ensues, limiting long-term therapeutic utility [65]. placental derived endothelial cells as a source of antigen found on proliferating endothelium such as the tumor. The utilization of placenta derived tissues for immunization to cancer was initially introduced in the 1970s by Dr. Valentin Govallo (reviewed in Harandi [66]) who demonstrated that immunity to placental trophoblast extract resulted in re- duction of immune suppression using the PHA stimu- lation assay, as well as radiological tumor reductions. Dr. Govallo noted the immunological similarities between pregnancy and cancer. Later along with the advancement in molecular biology development founding placental cells and tumor endothelial cells share the molecules of angio- genesis such as VEGF, placental growth factor, angio- poietin, FGF, EGF, and TGF-beta, and as well placental endothelium expresses many of the novel tumor endothe- lial markers (TEM) such as ROBO4 [67], CLEC14A [68], The possibility of inducing selective immunity to pro- liferating blood vessels has been previously reported in animal models as well as pilot clinical trials. Unfortu- nately, a major limiting factor to clinical implementation has been the utilization of HUVEC cells as an antigenic source, which is limited in availability. Here we utilized Figure 5 Mitotically inactivated ValloVax™retains activity against 4 T1 mammary carcinoma growth. Female BALB/c mice (10 mice per group) were immunized with saline, ValloVax™, ValloVax™lysate, formalin fixed ValloVax™, or irradiated Vallovax™at a concentration of 5 × 10(5) cells or cell equivalents on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) 4 T1 cells and quantified every third day. Safety evaluation Seven male and female mice per group were treated with control, or 500,000, 2 million or 4 million ValloVax™ir- radiated cells subcutaneously. Cells were administrated as in the therapeutic protocol, in that they were given on day 0, 7, 14, and 21. Body weights were calculated every three days and organ sizes, biochemical and hematological parameters were evaluated. No significant deviation was noted, nor were signs of autoimmunity present (online Additional file 1). These preclinical data support the safety of the irradiated ValloVax™approach. Figure 3 ValloVax™treatment inhibits LCC lung carcinoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline (diamond) or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0 Figure 3 ValloVax™treatment inhibits LCC lung carcinoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) LLC cells and quantified every third day. Figure 3 ValloVax™treatment inhibits LCC lung carcinoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline (diamond), or 5 × 10(5) placental endothelium cells (square) or placental endothelium cells pretreated with interferon gamma (triangle) on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) LLC cells and quantified every third day. Ichim et al. Journal of Translational Medicine (2015) 13:90 Page 6 of 9 Figure 4 Mitotically inactivated ValloVax™retains activity against B16 melanoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline, ValloVax™, ValloVax™lysate, formalin fixed ValloVax™, or irradiated Vallovax™at a concentration of 5 × 10(5) cells or cell equivalents on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) B16 cells and quantified every third day. Figure 4 Mitotically inactivated ValloVax™retains activity against B16 melanoma growth. Female C57BL/6 mice (10 mice per group) were immunized with saline, ValloVax™, ValloVax™lysate, formalin fixed ValloVax™, or irradiated Vallovax™at a concentration of 5 × 10(5) cells or cell equivalents on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) B16 cells and quantified every third day. Safety evaluation Figure 5 Mitotically inactivated ValloVax™retains activity against 4 T1 mammary carcinoma growth. Female BALB/c mice (10 mice per group) were immunized with saline, ValloVax™, ValloVax™lysate, formalin fixed ValloVax™, or irradiated Vallovax™at a concentration of 5 × 10(5) cells or cell equivalents on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) 4 T1 cells and quantified every third day. Ichim et al. Journal of Translational Medicine (2015) 13:90 Page 7 of 9 Page 7 of 9 Figure 6 Mitotically inactivated ValloVax™Retains™activity against LLC lung carcinoma. Female C57BL/6 mice (10 mice per group) were immunized with saline, ValloVax™, ValloVax™lysate, formalin fixed ValloVax™, or irradiated Vallovax™at a concentration of 5 × 10(5) cells or cell equivalents on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) LLC cells and quantified every third day. Figure 6 Mitotically inactivated ValloVax™Retains™activity against LLC lung carcinoma. Female C57BL/6 mice (10 mice per group) were immunized with saline, ValloVax™, ValloVax™lysate, formalin fixed ValloVax™, or irradiated Vallovax™at a concentration of 5 × 10(5) cells or cell equivalents on days 0, 7, 14, and 21. Tumor growth was initiated by subcutaneous administration of 5 × 10(5) LLC cells and quantified every third day. Here we demonstrate the therapeutic activity of ValloVax™ against a wide range of histologically distinct tumor types, suggesting that the effect is acting against new blood ves- sels and not against shared tumor antigens. Additionally, the main concern of utilization of an antiendothelial vac- cine would be the possibility of inducing autoimmunity against the endothelium. This has not been observed in the 28 day safety study. We have demonstrated that tumor inhibiting activity was preserved when cells were mitoti- cally inactivated, however was substantially reduced when cell lysate was utilized. It is important to note that adju- vants were not administered as part of the vaccination inoculum. Accordingly, manipulation of the vaccine ad- ministration either by modification of dosage or frequency may induce more potent therapeutic responses. One of the deficiencies of the current study is the lack of direct demonstration that inhibition of endothelial proliferation was responsible for reduction in tumors. Although it is un- likely that direct tumor immunity was induced to all three tumor types assessed by the administration of ValloVax™, this possibility cannot be excluded. Safety evaluation Supporting the possi- bility that anti-endothelial immunity was induced is data demonstrating sera of immunized mice was able to inhibit proliferation of endothelial cells in vitro. These data are currently the subject of an additional manuscript looking at more detailed mechanisms of immunity and tumor biol- ogy. As well further studies on combination of ValloVax™ with standard of care treatments will be studied. and endosialin [69] suggested parallels between the pla- cental and tumor microenvironment, namely immune suppression, active angiogenesis, and the secretion of matrix metalloproteinase associated with metastasis, not just a functional, but also a molecular homology between placenta, tumor cells and tumor-associated endothelium. Based on the safety and possible efficacy of the Govallo vaccine, we sought to utilize placental endothelial cells as a polyvalent antigenic source for stimulation of immunity against proliferating endothelial cells that have been primed with interferon gamma to stimulate immunogen- icity. We termed this product “ValloVax™”. Figure 7 Reduction in lung metastasis after ValloVax™ immunization. Female C57BL/6 mice (10 mice per group) were immunized with saline, placental endothelial cells, and placental endothelial cells pretreated with IFN-gamma. Immunization was performed subcutaneously at same time as 5 × 10(5) LLC cells were administered intravenously. Mice were sacrificed after 3 weeks and lung colonies were quantified by counting per visual field. References 25. Chen C, Zeng L, Ding S, Xu K. Adult endothelial progenitor cells retain hematopoiesis potential. Transplant Proc. 2010;42(9):3745–9. 1. Kmieciak M, Payne KK, Idowu MO, Grimes MM, Graham L, Ascierto ML, et al. Tumor escape and progression of HER-2/neu negative breast cancer under immune pressure. J Transl Med. 2011;9:35. 1. Kmieciak M, Payne KK, Idowu MO, Grimes MM, Graham L, Ascierto ML, et al. Tumor escape and progression of HER-2/neu negative breast cancer under immune pressure. J Transl Med. 2011;9:35. 1. Kmieciak M, Payne KK, Idowu MO, Grimes MM, Graham L, Ascierto ML, et al. 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Chang CC, Campoli M, Restifo NP, Wang X, Ferrone S. Conclusion Figure 7 Reduction in lung metastasis after ValloVax™ immunization. Female C57BL/6 mice (10 mice per group) w Figure 7 Reduction in lung metastasis after ValloVax™ Figure 7 Reduction in lung metastasis after ValloVax™ immunization. Female C57BL/6 mice (10 mice per group) were immunized with saline, placental endothelial cells, and placental endothelial cells pretreated with IFN-gamma. Immunization was performed subcutaneously at same time as 5 × 10(5) LLC cells were administered intravenously. Mice were sacrificed after 3 weeks and lung colonies were quantified by counting per visual field. Based on existing preclinical and clinical data demonstrat- ing safety of endothelial cell vaccination, combined with the recent data described herein, ValloVax™appears to be a promising antiangiogenic vaccine platform. 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Author details 1 1Batu Biologics Inc, San Diego, 9255 Towne Centre Drive, Suite 450, San Diego, CA 92121, USA. 2Department of Endocrinology, The Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China. 3Nova Southeastern University, Fort Lauderdale, Florida, USA. 4Department of Surgery, University of Utah, Salt Lake City, Utah. 5Department of Neurosciences, University of California San Diego, 9500 Gilman Dr., MSC 0752, La Jolla, San Diego, CA 92093-0752, USA. 6Translational 1Batu Biologics Inc, San Diego, 9255 Towne Centre Drive, Suite 450, San Diego, CA 92121, USA. 2Department of Endocrinology, The Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China. 3Nova Southeastern University, Fort Lauderdale, Florida, USA. 4Department of Surgery, University of Utah, Salt Lake City, Utah. 5Department of Neurosciences, University of California San Diego, 9500 Gilman Dr., MSC 0752, La Jolla, San Diego, CA 92093-0752, USA. 6Translational Neuro-Oncology Laboratories, Moores Cancer Center, University of California San Diego, 3855 Health Sciences Dr., MSC 0819, La Jolla, San Diego, CA 92093-0819, USA. 7Department of Immunology, University of Western Ontario, London, Ontario, Canada. 20. 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https://openalex.org/W2950810015	https://zenodo.org/records/3257794/files/32608__1_203181_LE_311678.pdf	English	null	A citation-based map of concepts in invasion biology	NeoBiota	2,019	cc-by	9,200	Martin Enders1,2,3, Frank Havemann4, Jonathan M. Jeschke1,2,3 1 Freie Universität Berlin, Department of Biology, Chemistry, Pharmacy, Institute of Biology, Königin-Luise- Str. 1–3, 14195 Berlin, Germany 2 Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 310, 12587 Berlin, Germany 3 Berlin-Brandenburg Institute of Advanced Biodiversity Re search (BBIB), Altensteinstr. 34, 14195 Berlin, Germany 4 Humboldt-Universität zu Berlin, Philosophische Fakultät, Institut für Bibliotheks- und Informationswissenschaft, Dorotheenstr. 26, 10099 Berlin, Germany Corresponding author: Martin Enders (enders.martin@gmx.net) Academic editor: Robert Colautti \| Received 21 December 2018 \| Accepted 23 May 2019 \| Published 19 Ju Citation: Enders M, Havemann F, Jeschke JM (2019) A citation-based map of concepts in invasion biology. NeoBiota 47: 23–42. https://doi.org/10.3897/neobiota.47.32608 Abstract Invasion biology has been quickly expanding in the last decades so that it is now metaphorically flooded with publications, concepts, and hypotheses. Among experts, there is no clear consensus about the rela tionships between invasion concepts, and almost no one seems to have a good overview of the literature anymore. Similar observations can be made for other research fields. Science needs new navigation tools so that researchers within and outside of a research field as well as science journalists, students, teachers, practitioners, policy-makers, and others interested in the field can more easily understand its key ideas. Such navigation tools could, for example, be maps of the major concepts and hypotheses of a research field. Applying a bibliometric method, we created such maps for invasion biology. We analysed research papers of the last two decades citing at least two of 35 common invasion hypotheses. Co-citation analysis yields four distinct clusters of hypotheses. These clusters can describe the main directions in invasion biol ogy and explain basic driving forces behind biological invasions. The method we outline here for invasion biology can be easily applied for other research fields. 23 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 23 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 23 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota NeoBiota 47: 23–42 (2019) doi: 10.3897/neobiota.47.32608 http://neobiota.pensoft.net NeoBiota 47: 23–42 (2019) doi: 10.3897/neobiota.47.32608 http://neobiota.pensoft.net n-based map of invasion RESEARCH ARTICLE A citation-based map of concepts in invasion biology Martin Enders1,2,3, Frank Havemann4, Jonathan M. Jeschke1,2,3 Copyright Martin Enders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction When you are visiting a city, you can usually find some important places by yourself, for example the central station, a supermarket, and maybe even a few touristic high lights. A better way, however, would be that a friend draws you a map with the places in the city you are interested in. Then you would also find the small French café, the little arthouse cinema, and the restaurant serving delicious oriental food. But this map will be limited by your friend’s knowledge of her district. What if you want to visit an other part of the city? You will find yourself in the same position as before. Therefore, an even better way is to ask several people who live in different areas of the city. In this way, you can get a detailed picture of the whole city and, if you are lucky, even find the best brewed coffee in the city.hi f The same is true when you start in a new research field. Enders et al. (2018) showed that the field of invasion biology can be seen as such a big city in which many of its in habitants, i.e. invasion biologists, have no clear picture of the whole city; their knowl edge seems to be limited to their immediate field of interest within invasion biology. What is the solution for a problem like this? Suppose you have no good tourist guide at hand, then you need to observe where other tourists go to and follow them. For a research field, this would be an analysis of citations made by specialists. i Authors of a scholarly paper cite publications and other sources they assume to be relevant for the topic of their paper. Thus, scholarly papers form a huge network, a view already propagated by one of the fathers of bibliometrics (de Solla Price 1965). The identification of topics in bibliographies is an old problem in bibliometrics. Start ing with co-citation analysis (Marshakova 1973; Small 1973; Small and Sweeny 1985), important recent developments include hybrid approaches that combine citation- based and term-based techniques (Glenisson et al. 2005; Glänzel and Thijs 2017), and term-based probabilistic methods (topic modelling, Yau et al. 2014). The 21st century brought the advance of many methods for clustering in networks (Fortunato 2010; Xie et al. 2013; Amelio and Pizzuti 2014). Some of these methods were also applied to citation networks (Gläser et al. Keywords bibliometric methods, biological invasions, concepts, invasion biology, invasion science, map, navigation tools, network of invasion hypotheses Copyright Martin Enders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 24 Martin Enders et al. / NeoBiota 47: 23–42 (2019) Martin Enders et al. / NeoBiota 47: 23–42 (2019) Introduction 2017; Velden et al. 2017), and topic identification is often accompanied by visualization of the topic landscape (Börner 2015). For this publication, we analysed co-citations of invasion hypotheses in research papers of the last two decades. Co-citation analysis was independently introduced by Irina Marshakova (1973) and Henry Small (1973) (see also Havemann 2016). Because there are no strict rules for citing, they had to solve the problem of noise in co-citation data. Irina Marshakova compared the observed absolute co-citation numbers with ex pected numbers in a null model of independent random citing and only accepted co- citation links between cited sources that are more frequently co-cited than in 95% of random trials in the null model. In other words, she assumed binomial distributions of co-citation numbers and chose a significance level of 95%. Henry Small, on the other hand, reduced noise by using thresholds of relative co-citation measures (Jaccard and Salton index). Also, other relative measures of co-citation strengths were used (Gmür 2003; Egghe and Leydesdorff 2009; Boyack and Klavans 2010). In a recent study, Tru Citation-based map of invasion biology 25 jillo and Long (2018) used absolute co-citation numbers as a similarity measure and created a sequence of nested co-citation networks by setting different thresholds for this measure. In invasion biology or related research fields, however, no citation-based map of major concepts and hypotheses does, to our knowledge, currently exist. Invasion biology is a discipline that grew very slowly at first. In the 19th century, early concepts on non-native species were mentioned (Cadotte 2006), for example in Darwin’s (1859) book “On the origin of species by means of natural selection”. Further concepts were introduced by the Swiss botanist Albert Thellung (Kowarik and Pyšek 2012), Elton (1958) and others until the 1950s; however, there was still too little work on the topic to recognize a distinct research field. Possibly due to a growing conscious ness for ecosystems in a changing world (Meadows et al. 1972) and in human respon sibilities (Jonas 1979), interest in invasion biology strongly increased since the late 20th century (Richardson and Pyšek 2008). It has also influenced other research fields; for example, concepts and hypotheses of invasion biology are used in restoration ecology, landscape ecology, urban ecology, or risk assessments of genetically modified organisms (Jeschke et al. 2013; Lowry et al. 2013).ii Our study aims were twofold. Introduction First, we wanted to find a suitable map of the field of invasion biology based on co-citation analysis. Second, we aimed to compare this map to those created with two other approaches: a map based on an assessment of the characteristics (“traits”) of hypotheses (Enders and Jeschke 2018), and one based on an online survey (Enders et al. 2018). Methods We defined 35 common concepts and hypotheses in invasion biology and their rep resenting key publications (Table 1). This list is based on Enders and Jeschke (2018) and Enders et al. (2018), which are in turn based on Catford et al. (2009). For clarity, we only give one key publication per hypothesis. One paper is the key publication for four hypotheses (EI, ERD, IS, NAS), and another paper for two hypotheses (SG, BID) (Table 1). Thus, Table 1 includes 31 key publications. h A first hint about relationships between our key publications can be obtained from their direct citation links, but this approach is limited by the small sample size of publications. As there is some randomness in the act of citation, a larger sample size is useful. Using bibliographic coupling relations between key papers, i.e., analysing to which degree their reference lists overlap, has the same drawback. An alternative approach, which we applied here, is co-citation analysis, where joint citations of key papers are analysed, using all publications of the field. This approach can thus draw from a much larger dataset. We downloaded all 10,430 records citing any of our key publications from the Web of Science (WoS, as licensed for Freie Universität Berlin, March 2017). Vari ants of referencing key papers were identified semi-automatically with the help of an R-script provided by Felix Mattes. For example, missing or wrong author initials or Martin Enders et al. / NeoBiota 47: 23–42 (2019) 26 wrong page numbers were corrected in this way. Then we determined the yearly cita tion and co-citation numbers of all key publications. We expect higher numbers of key papers cited in review papers which diminishes the weight of each co-citation. There fore, we excluded reviews from the analysis. Key invasion papers are also cited outside of invasion biology. We excluded such outside-of-the-field papers from co-citation analysis, as invasion hypotheses are pri marily applied in invasion biology and we expect that peculiarities of their relation ships are discussed within the field, whereas joint citations by publications outside of the field are less reliable for assessing such relationships. We defined papers belonging to the field as those that are returned by the term search proposed by Vaz et al. Methods (2017): “Ecological invasion” or “Biological invasion” or “Invasion biology” or “Invasion ecology” or “Invasive species” or “Alien species” or “Introduced species” or “Non-native spe cies” or “Nonnative species” or “Nonindigenous species” or “Non-indigenous species” or “Al lochthonous species” or “Exotic species”. Using this term search on 28.08.2017 in the WoS returned 30,731 records. After excluding 1,769 review papers, 28,962 papers remained in the sample. These are mainly primary research communications (28,295) and have mainly been published after 1990 (28,841; i.e. 99.6%). Figure 1 displays the time distribution of the sample of these 28,841 invasion biology papers in the WoS. In the 1990s, the number of papers in the field has remained small. We therefore restricted our analysis to the time period 1999–2017. Thus, we ended up with a sample of 1,518 invasion biology papers that cite at least two of our key publications listed in Table 1. The sample includes 1501 research articles, mainly in journals but also 39 in conference proceedings and five in books. In addition, we have eight letters and nine editorials. The time distribution of the sample is displayed in Figure 2. Salton’s cosine In the n-dimensional vector space with one dimension per citing paper, each cited source i can be represented by a vector vik (k = 1, ..., n) with vik = 1 if paper k cites source i and vik = 0 otherwise. The Salton index S(i, j) of two sources is a similarity measure defined as the cosine of the angle between the two source vectors (Hamers et al. 1989). Translated into the language of set theory, it can be calculated as: S i j c c c c i j i j ( , ) · , (1) (1) where ci is the set of papers citing source publication i. Salton’s cosine gives values in the interval [0, 1]. Co-citations are usually determined within reference lists of citing publications ci published during a given year. Due to heavily fluctuating citation num where ci is the set of papers citing source publication i. Salton’s cosine gives values in the interval [0, 1]. Co-citations are usually determined within reference lists of citing publications ci published during a given year. Due to heavily fluctuating citation num Citation-based map of invasion biology 27 Figure 1. Numbers of publications in invasion biology, using the same search term as Vaz et al. (2017) in the Web of Science. The number of publications in 2017 is relatively low because the search was per formed within this year, on 28 August 2017. 95 109 96 113 142 190 188 273 260 319 417 469 590 804 974 1158 1211 1479 1629 1968 2130 2175 2317 2417 2772 2791 1755 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Figure 1. Numbers of publications in invasion biology, using the same search term as Vaz et al. (2017) in the Web of Science. The number of publications in 2017 is relatively low because the search was per formed within this year, on 28 August 2017. 3 12 17 13 26 50 48 81 91 105 104 123 143 119 146 135 165 128 9 2000 2002 2004 2006 2008 2010 2012 2014 2016 Figure 2. Number of publications per year that cite at least two of the key papers given in Table 1. This sample of 1518 publications was analysed in detail here; it is a subset of the publications shown in Figure 1. Figure 2. Salton’s cosine Number of publications per year that cite at least two of the key papers given in Table 1. This sample of 1518 publications was analysed in detail here; it is a subset of the publications shown in Figure 1. bers, we combined several years to get broader citation windows. Due to this change, a challenge was that two key papers i and j published within the citation window in different years yi < yj have different chances to be cited: older papers have more oppor tunities to be cited than younger papers. We made their chances to be cited as equal as possible by reducing the set ci to citing papers published from year yj on. Martin Enders et al. / NeoBiota 47: 23–42 (2019) 28 . List of 35 common invasion hypotheses and how we defined them (cf. Catford et al. 2009; End Jeschke 2018; Enders et al. 2018). Table 1. List of 35 common invasion hypotheses and how we defined them (cf. Catford et al. 2009; End ers and Jeschke 2018; Enders et al. 2018). Hypothesis Description Key reference ADP Adaptation The invasion success of non-native species depends on the adaptation to the conditions in the exotic range before and/or after the introduction. Non-native species that are related to native species are more successful in this adaptation. Duncan and Williams (2002) BA Biotic acceptance aka “the rich get richer” Ecosystems tend to accommodate the establishment and coexistence of non-native species despite the presence and abundance of native species. Stohlgren et al. (2006) BID Biotic indirect effects Non-native species benefit from different indirect effects triggered by native species. Callaway et al. (2004) BR Biotic resistance aka diversity-invasibility hypothesis An ecosystem with high biodiversity is more resistant against non- native species than an ecosystem with lower biodiversity. Levine and D’Antonio (1999) DEM Dynamic equilibrium model The establishment of a non-native species depends on natural fluctuations of the ecosystem, which influences the competition of local species. Huston (1979) DN Darwin’s naturalization The invasion success of non-native species is higher in areas that are poor in closely related species than in areas that are rich in closely related species. Daehler (2001) DS Disturbance The invasion success of non-native species is higher in highly disturbed than in relatively undisturbed ecosystems. Salton’s cosine Hobbs and Huenneke (1992) EE Enemy of my enemy aka accumulation- of-local-pathogens hypothesis Introduced enemies of a non-native species are less harmful to the non-native as compared to the native species. Eppinga et al. (2006) EI Enemy inversion Introduced enemies of non-native species are less harmful for them in the exotic than the native range, due to altered biotic and abiotic conditions. Colautti et al. (2004) EICA Evolution of increased competitive ability After having been released from natural enemies, non-native species will allocate more energy in growth and/or reproduction (this re-allocation is due to genetic changes), which makes them more competitive. Blossey and Nötzold (1995) EN Empty niche The invasion success of non-native species increases with the availability of empty niches in the exotic range. MacArthur (1970) ER Enemy release The absence of enemies in the exotic range is a cause of invasion success. Keane and Crawley (2002) ERD Enemy reduction The partial release of enemies in the exotic range is a cause of invasion success. Colautti et al. (2004) EVH Environmental heterogeneity The invasion success of non-native species is high if the exotic range has a highly heterogeneous environment. Melbourne et al. (2007) GC Global competition A large number of different non-native species is more successful than a small number. Colautti et al. (2006) HC Human commensalism Species that are living in close proximity to humans are more successful in invading new areas than other species. Jeschke and Strayer (2006) HF Habitat filtering The invasion success of non-native species in the new area is high if they are pre-adapted to this area. Weiher and Keddy (1995) IM Invasional meltdown The presence of non-native species in an ecosystem facilitates invasion by additional species, increasing their likelihood of survival or ecological impact. Simberloff and Von Holle (1999) IRA Increased resource availability The invasion success of non-native species increases with the availability of resources. Sher and Hyatt (1999) IS Increased susceptibility If a non-native species has a lower genetic diversity than the native species, there will be a low probability that the non-native species establishes itself. Colautti et al. (2004) ISH Island susceptibility hypothesis Non-native species are more likely to become established and have major ecological impacts on islands than on continents. Jeschke (2008) IW Ideal weed The invasion success of a non-native species depends on its specific traits (e.g. life-history traits). Salton’s cosine Rejmánek and Richardson (1996) Citation-based map of invasion biology 29 Hypothesis Description Key reference LS Limiting similarity The invasion success of non-native species is high if they strongly differ from native species, and it is low if they are similar to native species. MacArthur and Levins (1967) MM Missed mutualisms In their exotic range, non-native species suffer from missing mutualists. Mitchell et al. (2006) NAS New associations New relationships between non-native and native species can positively or negatively influence the establishment of the non- native species. Colautti et al. (2006) NW Novel weapons In the exotic range, non-native species can have a competitive advantage against native species because they possess a novel weapon, i.e. a trait that is new to the resident community of native species and therefore affects them negatively. Callaway and Ridenour (2004) OW Opportunity windows The invasion success of non-native species increases with the availability of empty niches in the exotic range, and the availability of these niches fluctuates spatio-temporally. Johnstone (1986) PH Plasticity hypothesis Invasive species are more phenotypically plastic than non-invasive or native ones. Richards et al. (2006) PP Propagule pressure A high propagule pressure (a composite measure consisting of the number of individuals introduced per introduction event and the frequency of introduction events) is a cause of invasion success. Lockwood et al. (2005) RER Resource-enemy release The non-native species is released from its natural enemies and can spend more energy in its reproduction, and invasion success increases with the availability of resources. Blumenthal (2006) RI Reckless invader aka “boom-bust” A non-native species that is highly successful shortly after its introduction can get reduced in its population or even extinct over time due to different reasons (such as competition with other introduced species or adaptation by native species). Simberloff and Gibbons (2004) SDH Shifting defence hypothesis After having been released from natural specialist enemies, non-native species will allocate more energy in cheap (energy- inexpensive) defenses against generalist enemies and less energy in expensive defenses against specialist enemies (this re-allocation is due to genetic changes); the energy gained in this way will be invested in growth and/or reproduction, which makes the non- native species more competitive. Doorduin andVrieling (2011) SG Specialist-generalist Non-native species are more successful in a new region if the local predators are specialists and local mutualists are generalists. Callaway et al. Salton’s cosine (2004) SP Sampling A large number of different non-native species is more likely to become invasive than a small number due to interspecific competition. Also, the species identity of the locals is more important than the richness in terms of the invasion of an area. Crawley et al. (1999) TEN Tens rule Approximately 10% of species successfully take consecutive steps of the invasion process. Williamson and Brown (1986) Communities in networks Clusters of highly cited sources containing often co-cited sources are assumed to rep resent knowledge bases of current research fronts (Small and Sweeny 1985). Such clusters are particularly useful for constructing conceptual maps that should serve as navigation tools for research fields, as they group similar concepts and hypotheses in one cluster. Especially in the last two decades, several clustering methods have been developed in network science (see Fortunato (2010) for a review). Clusters (also called modules or communities) of nodes in networks should have many internal links and comparatively few external links. In the case of weighted networks, not the number of external and internal links is compared but the sum of their weights. Identifying clusters in a network is a way of investigating its inner structure. 30 Martin Enders et al. / NeoBiota 47: 23–42 (2019) For the case of disjoint communities, Newman and Girvan (2004) introduced “modularity” as an evaluation function of a graph partition. It compares the actual number of internal edges of each community with the number expected in a null model without community structure. In the usual null model, each vertex is expected to have the same degree as in the original graph.f We compared the results of different algorithms for community detection from the packages SNA (Handcock et al. 2003) and igraph (Csardi and Nepusz 2006) in R (R Development Core Team 2008), which can be categorized into several types. (1) The Girvan and Newman (2002) algorithm is an example of divisive clustering (igraph func tion cluster_edge_betweenness). It recursively detects links with high edge betweenness and removes them from the network. The clustering dendrogram is cut at the partition with maximum modularity. (2) Clauset et al. (2004) proposed to set each node as a cluster and then merge those two subgraphs that give the highest gain in modularity; this is repeated until there is no gain in modularity anymore (igraph function cluster_ fast_greedy). Again, the clustering dendrogram is cut at the partition with maximum modularity. (3) Quite similar is the approach introduced by Brandes et al. (2008) (igraph function cluster_optimal). It maximizes modularity applying an optimization algorithm from integer linear programming. (4) We also applied the Louvain algorithm designed by Blondel et al. Communities in networks (2008) that very quickly maximizes partition modularity (igraph function cluster_louvain), (5) the “walk trap” algorithm suggested by Pons and Latapy (2005) that assumes a random walker gets trapped in communities and calculates these “traps” (ig raph function cluster_walktrap), and (6) a divisive spectral algorithm suggested by New mann (2006) which also maximizes modularity (igraph function cluster_leading_eigen). Beside global evaluation functions like modularity, there are also functions that evaluate cohesion and separation of each community. A community C is well separated from the rest of the network if the escape probability of a random walker is small (For tunato 2010). It is given by the ratio of the sum of external degrees of a community’s nodes to the sum of their total degrees: P C k C k C esc out ( ) ( ) ( ) = . (2) (2) The weak definition of a community after Radicchi et al. (2004) is fulfilled when the total internal degree is greater than the total external degree. The requirements are fulfilled if Pesc < 0.5. The strong community definition requires that every node has a stronger internal than external connection. Results We analysed different time periods (time steps of 1–5 years), but the results varied too much to get a clear picture. This means that the edges between the nodes varied from pe riod to period. Obviously, in different years relationships between different concepts and Citation-based map of invasion biology 31 Citation-based map of invasion biology 31 Figure 3. Partition of co-citation network M with maximum modularity q = 0.520. Links are weighted with significant co-citation numbers of hypothesis papers (significance level 95%, cf. text). For acronyms of hypotheses see Table 1. LS 1967 DEM 1979 OW 1986 TEN 1986 DS 1992 EICA 1995 HF 1995 IW 1996 BR 1999 SP 1999 DN 2001 ER 2002 ADP 2002 NW 2004 EI ERD IS NAS 2004 SG BID 2004 RI 2004 PP 2005 RER 2006 MM 2006 PH 2006 BA 2006 GC 2006 EE 2006 HC 2006 EVH 2007 ISH 2008 SDH 2011 EN 1970 IRA 1999 IM 1999 Darwin's cluster Propagule cluster Resistance cluster Enemy cluster Propagule cluster Propagule cluster Enemy cluster Figure 3. Partition of co-citation network M with maximum modularity q = 0.520. Links are weighted with significant co-citation numbers of hypothesis papers (significance level 95%, cf. text). For acronyms of hypotheses see Table 1. hypotheses were discussed in the literature. We decided to accumulate the data from 1999 to 2017 to get a clearer, cumulative picture of relationships between invasion hypotheses. We constructed two co-citation networks of our 31 key papers. Network M is based on Marshakova (1973) where accepted links are weighted by co-citation num bers (Fig. 3). Following Small and Sweeny (1985) in network S, we weighted all links with Salton’s cosine and omitted links with a cosine below a threshold of 0.1 (Fig. 4). This threshold was chosen to receive a clearer picture of the graph and to have no un connected nodes. We compared the results obtained with different clustering algorithms (Table 2). In both networks, maximum modularity was achieved by a partition with four clusters. The partitions in M and S differ only in the membership of the plasticity hypothesis (PH), which switches between two clusters. We named the four clusters obtained in both networks by the most prominent principle of their hypotheses (Figs 3, 4; Ta ble 3): Darwin’s cluster, resistance cluster, propagule cluster and enemy cluster. Martin Enders et al. / NeoBiota 47: 23–42 (2019) 32 Martin Enders et al. Results / NeoBiota 47: 23 42 (2019) 32 Table 2. Partitions of co-citation networks M and S obtained by different algorithms maximizing modularity. Algorithm Number of clusters Modularity M-network S-network M-network S-network Cluster_optimal 4 4 0.520 0.463 Fast_greedy 4 4 0.520 0.463 Louvain 4 4 0.520 0.463 Leading_eigen 6 4 0.502 0.441 Edge_betweenness 3 5 0.464 0.428 Walktrap 4 5 0.520 0.430 LS 1967 DEM 1979 OW 1986 TEN 1986 DS 1992 EICA 1995 HF 1995 IW 1996 BR 1999 SP 1999 DN 2001 ER 2002 ADP 2002 NW 2004 EI ERD IS NAS 2004 SG BID 2004 RI 2004 PP 2005 RER 2006 MM 2006 PH 2006 BA 2006 GC 2006 EE 2006 HC 2006 EVH 2007 ISH 2008 SDH 2011 EN 1970 IRA 1999 IM 1999 Darwin's cluster Propagule cluster Resistance cluster Enemy cluster Figure 4. Partition of co-citation network S with maximum modularity q = 0.463. Links are weighted with Salton’s cosine of co-citation numbers of hypothesis papers above a minimum threshold of 0.1 (cf. text). For acronyms of hypotheses see Table 1. . Partitions of co-citation networks M and S obtained by different algorithms maximizing modularity. Darwin's cluster Figure 4. Partition of co-citation network S with maximum modularity q = 0.463. Links are weighted with Salton’s cosine of co-citation numbers of hypothesis papers above a minimum threshold of 0.1 (cf. text). For acronyms of hypotheses see Table 1. Figure 4. Partition of co-citation network S with maximum modularity q = 0.463. Links are weighted with Salton’s cosine of co-citation numbers of hypothesis papers above a minimum threshold of 0.1 (cf. text). For acronyms of hypotheses see Table 1. The plasticity hypothesis (PH) switches between the enemy and the propagule cluster. In the M-network, PH has no links to the propagule cluster because the num bers of co-citations with papers of the cluster are not significant on the 95%-level.h The plasticity hypothesis (PH) switches between the enemy and the propagule cluster. In the M-network, PH has no links to the propagule cluster because the num bers of co-citations with papers of the cluster are not significant on the 95%-level.h The best partition of network M has modularity 0.520 (see Fig. 3) and its clusters are communities in the weak and strong sense. The best partition of network S (Fig. Results 4) has modularity 0.463 and its clusters are communities in the weak sense, two of them also in the strong sense (Darwin’s and enemy cluster). Since the key papers for IW (ideal weed) Citation-based map of invasion biology 33 Table 3. Partitions of co-citation networks M and S with maximum modularity. The key papers (see Ta ble 1 for details) are ranked in their clusters by internal strength. Also, escape probability Pesc is displayed for each cluster (cf. Figs 3, 4). Table 3. Partitions of co-citation networks M and S with maximum modularity. The key papers (see Ta ble 1 for details) are ranked in their clusters by internal strength. Also, escape probability Pesc is displayed for each cluster (cf. Figs 3, 4). for each cluster (cf. Figs 3, 4). Results Network M Network S Hypothesis-paper Internal strength Hypothesis-paper Internal strength Darwin´s cluster Pesc(C) = 0.27 Pesc(C) = 0.24 Adaptation (ADP 2002) 54 Darwin‘s naturalization (DN 2001) 0.77 Darwin‘s naturalization (DN 2001) 53 Adaptation (ADP 2002) 0.76 Limiting similarity (LS 1967) 19 Limiting similarity (LS 1967) 0.57 Habitat filtering (HF 1995) 6 Habitat filtering (HF 1995) 0.32 Niche cluster Pesc(C) = 0.02 Pesc(C) = 0.25 Biotic resistance (BR 1999) 243 Biotic resistance (BR 1999) 1.75 Disturbance (DS 1992) 163 Disturbance (DS 1992) 1.28 Sampling (SP 1999) 63 Sampling (SP 1999) 0.57 Invasional meltdown (IM 1999) 44 Biotic acceptance (BA 2006) 0.45 Increased resource availability (IRA 1999) 38 Dynamic equilibrium model (DEM 1979) 0.41 Opportunity windows (OW 1986) 37 Empty niche (EN 1970) 0.35 Biotic acceptance (BA 2006) 36 Invasional meltdown (IM 1999) 0.34 Empty niche (EN 1970) 33 Opportunity windows (OW 1986) 0.34 Dynamic equilibrium model (DEM 1979) 31 Increased resource availability (IRA 1999) 0.29 Environmental heterogeneity (EVH 2007) 24 Environmental heterogeneity (EVH 2007) 0.20 Propagule cluster Pesc(C) = 0.01 Pesc(C) = 0.38 Propagule pressure (PP 2005) 186 Propagule pressure (PP 2005) 1.28 Global competition (GC 2006) 141 Global competition (GC 2006) 0.78 Human commensalism (HC 2006) 38 Ideal weed (IW 1996) 0.66 Tens rule (TEN 1986) 28 Tens rule (TEN 1986) 0.54 Island susceptibility hypothesis (ISH 2008) 11 Island susceptibility hypothesis (ISH 2008) 0.50 Ideal weed (IW1996) 10 Human commensalism (HC 2006) 0.46 Plasticity hypothesis (PH 2006) 0.40 Enemy cluster Pesc(C) = 0.02 Pesc(C) = 0.14 Enemy release (ER 2002) 652 Enemy release (ER 2002) 2.41 Evolution of increased competitive ability (EICA 1995) 465 Evolution of increased competitive ability (EICA 1995) 1.98 Enemy inversion, Enemy reduction, Increased susceptibility, New associations 357 Enemy inversion, Enemy reduction, Increased susceptibility, New associations (EI; ERD; IS; NAS 2004) 1.58 Missed mutualism 196 Missed mutualism (MM2006) 1.37 Novel weapons 192 Novel weapons (NW 2004) 1.30 Resource-enemy release (RER 2006) 81 Specialist-generalist, Biotic indirect effects (SG; BID 2004) 1.04 Specialist-generalist, Biotic indirect effects (SG; BID 2004) 67 Enemy of my enemy aka accumulation- of-local-pathogens hypothesis (EE 2006) 0.69 Enemy of my enemy aka accumulation-of- local-pathogens hypothesis (EE 2006) 60 Resource-enemy release (RER 2006) 0.62 Plasticity hypothesis (PH 2006) 41 Shifting defence hypothesis (SDH 2011) 0.58 Shifting defence hypothesis (SDH 2011) 35 Reckless invader aka “boom-bust” (RI 2004) 0.24 Reckless invader aka “boom-bust” (RI 2004) 20 Martin Enders et al. Discussion The clusters of networks M and S are remarkably similar. Two of the four clusters in each network are even identical, namely the concept clusters focused on eco-evolution ary and phylogenetic relationships between non-native and resident species (Darwin’s cluster) and the concept cluster focused on biotic resistance of ecosystems against non- native species (resistance cluster). Comparing these two networks further, one can see that the other two concept clusters differ just in the membership of PH, the plasticity hypothesis. In one case (M-network), PH is in the concept cluster focused on species relationships (enemy cluster). In the other case (S-network), PH is a member of the concept cluster focused on introduction and species traits (propagule cluster). Results / NeoBiota 47: 23–42 (2019) 34 and IM (invasional meltdown) have stronger external than internal connections, the niche and propagule cluster do not meet the strong definition here. In general, the centrality of a node in an unweighted graph can be measured by its degree. The analogy in weighted networks is called the strength of the node and is defined as the sum of weights of its links. The centrality within a subgraph is then the sum of weights of the node’s internal links and can be called its internal strength which we use for ranking papers in Table 3. What are the implications from the networks? The networks visualize how invasion biologists have seen their research field during the last two decades. Essentially, the networks suggest four broad themes that are represented by the four clusters. One core idea comes from evolutionary biology; it highlights the importance of eco-evolutionary relationships between non-native and resident species, and the capability of species to adapt to new environments (evolutionary perspective, Darwin’s cluster). A second core idea is the possibility that ecosystems can be resistant, or not, against non-native species based on their char acteristics (ecosystem perspective, resistance cluster). A third core idea is that species interactions such as host-parasite or predator-prey interactions (including the loss of such interactions in the exotic environment, i.e., enemy release) are very important for understanding biological invasions (species-interactions perspective, enemy clus ter). Finally, the most recent core idea is that human action is principally influencing biological invasions, which can thus only be understood by studies bridging different research fields (Richardson and Pyšek 2008; Kueffer 2017) (interdisciplinary per spective; propagule cluster). Following this line of thought, the discipline of invasion biology is now sometimes called invasion science, reflecting that it is not simply a biological subdiscipline but stretches towards other disciplines including social sci ences and economics (Richardson and Ricciardi 2013). Citation-based map of invasion biology 35 Strongly connected hypothesis pairs Some of the hypotheses in our networks are particularly strongly connected. In this section, we highlight one strongly connected hypothesis pair for each of the four clus ters, and outline whether these connections are reasonable. In Darwin’s cluster, the two hypotheses adaptation (ADP) and Darwin’s naturali zation hypothesis (DN) are very strongly connected. The two key publications for these hypotheses included in Table 1 were published at roughly the same time (2001 and 2002). However, DN has its origin in the mid-19th century in what is probably biology’s most famous publication of all times (Darwin 1859). As Darwin’s book is mainly cited for other reasons than DN, we used another publication as the key paper for DN. The main reason for the strong connection between the hypotheses DN and ADP based on their co-citation in so many papers might be that both hypotheses are contradicting each other (Table 1) and are jointly called Darwin’s naturalization conun drum (Diez et al. 2008). In the resistance cluster, there is a particularly strong connection between biotic resistance (BR) and the disturbance hypothesis (DS). These two hypotheses are in fact logically linked. According to DS, the invasion success of non-native species is higher in highly disturbed than in relatively undisturbed ecosystems (Table 1). In other terms, highly disturbed ecosystems show lower resistance against non-native species than relatively undisturbed ecosystems. Thus, both hypotheses focus on the resistance of ecosystems against non-native species; BR does so with a focus on biodiversity, and DS with a focus on disturbance (Jeschke and Heger 2018). Another link between the two hypotheses is that disturbance can reduce biodiversity. In the propagule cluster, the propagule pressure hypothesis (PP) is very strongly connected to global competition (GC). The latter hypothesis is actually based on PP (Catford et al. 2009), which explains that these concepts are often jointly cited. Finally in the enemy cluster, the enemy release hypothesis (ER) and EICA hypoth esis are particularly strongly connected. This can also be easily explained, as EICA uses enemy release as an underlying assumption (Table 1). Comparing the network to previous works In comparison to the other two networks published by Enders and Jeschke (2018) and Enders et al. (2018), the networks of this publication are way clearer. Com pared with the similarity-dissimilarity network in Enders et al. (2018), which was created based on an online survey, the M- and S-networks have fewer connections and clearer, distinct clusters. Apparently, the survey participants had different views on the relationships between invasion hypotheses, possibly because invasion biology has so many hypotheses now that it is hard for researchers to know them all; the similarity-dissimilarity network in Enders et al. (2018) used direct responses given by the survey participants when being asked for hypothesis pairs how similar they are. If participants often simply guessed the similarity of hypothesis pairs, one would expect a random network to emerge from the answers, and this is what Enders et al. (2018) found. This problem was circumvented for two other networks in Enders et al. (2018), joint-mentions networks A and B, which are only based on hypotheses that the survey participants indicated to know best. These networks are clearer than the similarity-dissimilarity network; however, they do not seem to be as useful maps as the networks M and S presented here. They are not as clear, there clusters have a lower modularity (ca 0.25 for both networks; Enders et al. 2018), and their clusters are not communities in the strong sense. The network in Enders and Jeschke (2018), which was created by traits of the con cepts and hypotheses, has three clusters consisting of concepts with a focus on (i) human interference, (ii) mutualisms, and (iii) enemies (predators or parasites). The modularity is relatively high (ca 0.4) but still lower than for the two networks shown here. Also, the clus ters are not communities in the strong sense. This network also seems to be less suitable to serve as a map of the field than the networks shown here, particularly the M-network. Which network is the better map? Although the clusters of the two networks are very similar, the better map is in our opinion the M-network. This is due to the following two reasons. First, the M-network has 25% less edges compared to the S-network which results in a clearer picture. Sec ond, the clusters in the M-network are better separated from each other than in the S-network; all clusters in the M-network are communities in the strong sense, but this is only true for two clusters in the S-network. Martin Enders et al. / NeoBiota 47: 23–42 (2019) 36 Data accessibility The R script underlying this study is available via Dryad (https://doi.org/10.5061/ dryad.d2q07t6). The R script underlying this study is available via Dryad (https://doi.org/10.5061/ dryad.d2q07t6). Conclusions and outlook The co-citation approach has proven useful to construct conceptual maps of the field of invasion biology. These maps, particularly the M-network, are clearer than previ ous maps created with other approaches. Efforts to create such conceptual maps that highlight relationships between major concepts within a research field are currently limited. In fact, we are unaware of other attempts to create such maps. This lack of conceptual maps means that researchers lack navigation tools which would help them identify where their work is located within a given research field such as invasion biol ogy. The results of a recent online survey among >350 invasion biologists suggest that the participants lack a “joint vision how invasion hypotheses are related to each other” (Enders et al. 2018). This resembles the situation that invasion biologists lack a com mon map of the field, which also implies that they do not know where their own work is located in comparison to other studies in the field. The utility of conceptual maps and other navigation tools for research fields thus seems obvious, and it is of course not Citation-based map of invasion biology 37 restricted to invasion biology. Such maps can be provided as interactive visualization tools (https://www.hi-knowledge.org, Jeschke et al. 2018). But the conceptual maps constructed for this study are early steps on the way towards advanced navigation tools. An important next step would be to allow for con cepts and hypotheses to be included in more than one cluster, so that they can take the role of cluster-connecting concepts. To take this next step, a cluster-finding-algorithm that allows overlapping communities should be considered. Furthermore, we have thus far applied three different approaches to create conceptual maps. Other approaches can be imagined as well, for example based on a Delphi-approach in which a group of experts follows multiple iterative steps to create a consensus map. Further work should also involve the expansion of the network to include maps of related fields. 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https://openalex.org/W3082251670	https://durham-repository.worktribe.com/preview/1258580/31654.pdf	English	null	Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes	Inorganic chemistry	2,020	cc-by	24,940	doi.org/10.26434/chemrxiv.12195084.v1 Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes File list (2) download file view on ChemRxiv PorSil paper final.pdf (2.97 MiB) download file view on ChemRxiv PorSil SI final.pdf (18.18 MiB) Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes Burhan A. Hussein, Zainab Shakeel, Andrew T. Turley, Aisha N. Bismillah, Kody Wolfstadt, Julia Pia, Melanie Pilkington, Paul R. McGonigal, Marc Adler Submitted date: 24/04/2020 • Posted date: 27/04/2020 Licence: CC BY-NC-ND 4.0 Citation information: Hussein, Burhan A.; Shakeel, Zainab; Turley, Andrew T.; Bismillah, Aisha N.; Wolfstadt, Kody; Pia, Julia; et al. (2020): Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12195084.v1 Burhan A. Hussein, Zainab Shakeel, Andrew T. Turley, Aisha N. Bismillah, Kody Wolfstadt, Julia Pia, Melanie Pilkington, Paul R. McGonigal, Marc Adler Submitted date: 24/04/2020 • Posted date: 27/04/2020 Licence: CC BY-NC-ND 4.0 Citation information: Hussein, Burhan A.; Shakeel, Zainab; Turley, Andrew T.; Bismillah, Aisha N.; Wolfstadt, Kody; Pia, Julia; et al. (2020): Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12195084.v1 Submitted date: 24/04/2020 • Posted date: 27/04/2020 Licence: CC BY-NC-ND 4.0 Citation information: Hussein, Burhan A.; Shakeel, Zainab; Turley, Andrew T.; Bismillah, Aisha N.; Wolfstadt, Kody; Pia, Julia; et al. (2020): Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12195084.v1 Citation information: Hussein, Burhan A.; Shakeel, Zainab; Turley, Andrew T.; Bismillah, Aisha N.; Wolfstadt, Kody; Pia, Julia; et al. (2020): Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12195084.v1 Porphyrins are cornerstone functional materials that are useful in a wide variety of settings ranging from molecular electronics to biology and medicine. Their applications are often hindered, however, by poor solubilities that result from their ex- tended, solvophobic aromatic surfaces. Attempts to counteract this problem by functionalizing their peripheries have been met with only limited success. Here, we demonstrate a versatile strategy to tune the physical and electronic properties of porphyrins using an axial functionalization approach. Porphyrin silanes (PorSils) and bissilyloxy PorSils (SOPS) are prepared from porphyrins by opera- tionally simple κ4N-silylation protocols, introducing bulky silyloxy “caps” that are central and perpendicular to the planar porphyrin. While porphyrins typically form either J- or H-aggregates, SOPS do not self-associate in the same manner: the silyloxy axial substit- uents dramatically improve solubility by inhibiting aggregation. Moreover, axial porphyrin functionalization offers convenient han- dles through which optical, electronic, and structural properties of the porphyrin core can be modulated. We observe that the identity of the silyloxy substituent impacts the degree of planarity of the porphyrin in the solid state as well as the redox potentials. Supporting Information Placeholder Supporting Information Placeholder ABSTRACT: Porphyrins are cornerstone functional materials that are useful in a wide variety of settings ranging from molecular electronics to biology and medicine. Their applications are often hindered, however, by poor solubilities that result from their ex- tended, solvophobic aromatic surfaces. Attempts to counteract this problem by functionalizing their peripheries have been met with only limited success. Here, we demonstrate a versatile strategy to tune the physical and electronic properties of porphyrins using an axial functionalization approach. Porphyrin silanes (PorSils) and bissilyloxy PorSils (SOPS) are prepared from porphyrins by opera- tionally simple κ4N-silylation protocols, introducing bulky silyloxy “caps” that are central and perpendicular to the planar porphyrin. While porphyrins typically form either J- or H-aggregates, SOPS do not self-associate in the same manner: the silyloxy axial substit- uents dramatically improve solubility by inhibiting aggregation. Moreover, axial porphyrin functionalization offers convenient han- dles through which optical, electronic, and structural properties of the porphyrin core can be modulated. We observe that the identity of the silyloxy substituent impacts the degree of planarity of the porphyrin in the solid state as well as the redox potentials. accomplished through peripheral functionalization of the por- phyrin with i) hydrophilic or non-conjugated lipophilic groups to interact favorably with the solvent medium and/or ii) bulky groups that sterically disfavor intermolecular association. In na- ture, porphyrins are buried in biomolecular hydrophobic clefts which circumvents the solubility obstacle; synthetic systems mimicking this approach have recently been explored to im- prove photophysical and electrochemical performance.14 File list (2) download file view on ChemRxiv download file view on ChemRxiv Control of Porphyrin Planarity and Aggregation by Covalent Capping: Bissilyloxy Porphyrin Silanes Burhan A. Hussein,†,‡ Zainab Shakeel,† Andrew T. Turley,‡ Aisha N. Bismillah,‡,§ Kody M. Wolfstadt,† Julia E. Pia,† Melanie Pilkington,⊥ Paul R. McGonigal,‡ and Marc J. Adler†,* † Department of Chemistry & Biology, Ryerson University, 350 Victoria St., Toronto, ON, M5B 2K3, Canada ‡ Department of Chemistry, Durham University, Lower Mountjoy, Stockton Road, Durham, DH1 3LE, United Kingdom § Department of Chemistry, Dartmouth College, Hanover, New Hampshire, 03755, USA ⊥ Department of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON, L2S 3A1, Canada Supporting Information Placeholder ABSTRACT: Porphyrins are cornerstone functional materials that are useful in a wide variety of settings ranging from molecular electronics to biology and medicine. Their applications are often hindered, however, by poor solubilities that result from their ex- tended, solvophobic aromatic surfaces. Attempts to counteract this problem by functionalizing their peripheries have been met with only limited success. Here, we demonstrate a versatile strategy to tune the physical and electronic properties of porphyrins using an axial functionalization approach. Porphyrin silanes (PorSils) and bissilyloxy PorSils (SOPS) are prepared from porphyrins by opera- tionally simple κ4N-silylation protocols, introducing bulky silyloxy “caps” that are central and perpendicular to the planar porphyrin. While porphyrins typically form either J- or H-aggregates, SOPS do not self-associate in the same manner: the silyloxy axial substit- uents dramatically improve solubility by inhibiting aggregation. Moreover, axial porphyrin functionalization offers convenient han- dles through which optical, electronic, and structural properties of the porphyrin core can be modulated. We observe that the identity of the silyloxy substituent impacts the degree of planarity of the porphyrin in the solid state as well as the redox potentials. Supporting Information Placeholder Supporting Information Placeholder INTRODUCTION Porphyrins are a long-known and well-studied class of com- pounds.1 Naturally occurring variants are vital for the biochem- istry of both plants (e.g. chlorophyll, a photoredox catalyst for photosynthesis) and animals (e.g. heme, an oxygen carrier).2 Accordingly, significant efforts have been dedicated to estab- lishing laboratory syntheses of this class of compounds.3 Both porphyrins and κ4N,N′,N′′,N′′′-porphyrin–metal complexes (i.e. metalloporphyrins)4 possess optical, electronic, and chemical properties that make them useful in a range of applied fields, including photodynamic therapy,5,6 biological imaging,7 or- ganic photovoltaics,8 photoredox catalysis,9 and analytical chemistry.10 However, their applications can be hindered by their suboptimal physical properties. A constraining drawback of working with porphyrins is that they are difficult to handle and process owing to their poor solubilities, which can be as- cribed to the favorable J- or H-aggregation of their exposed ar- omatic surfaces.11 Efforts have been made to prepare deriva- tives that exhibit improved solubilities in aqueous12 and or- ganic13 solutions. Inhibition of aggregation has generally been Phthalocyanines (Pcs)15 have been used for many of the same applications as porphyrin compounds.16 Like porphyrins, Pcs are planar and suffer from poor solubilities. However, an ele- gant approach to improve their physical properties has been to silylate the center of the Pc, introducing axially oriented groups that shield the aromatic surface. Beyond its effect on solubility, silylation modifies the optical and electronic profiles of Pcs, which has led to over 1000 papers discussing the synthesis, properties, and applications of silylated Pcs (Si-Pcs).17 Curi- ously, however, there is a relative dearth of literature describing such functionalization of the parent porphyrin compounds, which is presumably due to the relatively harsh conditions and operationally complex protocols used to incorporate the central silicon atom. While Si-Pcs are formed in the presence of SiCl4 hands as the modified porphyrin covalently reacted with the solid support. We accomplished the silylation of 2 using HSiCl3 with Et3N, a common laboratory reagent (Scheme 1). After aqueous workup, residual Et3N can be readily removed by evap- oration. Using this method we were able to avoid the necessity for chromatography, and instead were able to isolate our desired product by simple liquid-liquid extraction, resulting in up to an 85% yield of 2-SiCl2. The generalization of this reaction in combination with the simplification of the purification proce- dure are steps forward for the prospects of developing this class of molecules. INTRODUCTION To synthesize the SOPS, 2-SiCl2 was converted quantitively to 3 by refluxing in a THF/water mixture, and sub- sequently silylated using Et3N and the respective silyl chloride (Scheme 1). These final compounds were air-stable and purified by column chromatography using silica gel and alkaline eluents (see Supporting Information); large scale purification could be accomplished by recrystallization. to i) template the macrocyclization and ii) silylate in situ, por- phyrin silanes (PorSils) have been synthesized by first making the porphyrin and then inserting the silicon by forming the [por- phyrin]2- species with a strong and difficult-to-handle amide base under strictly anhydrous conditions.18,19 Sun and co-work- ers were able to silylate two porphyrins using HSiCl3 and a ter- tiary amine base. However, purification of the resulting PorSil dichloride requires chromatography on neutral alumina,20 which makes the protocol too inefficient and expensive to be applied as a general method on a large scale. Furthermore, this protocol can be difficult to reproduce as the PorSil dichloride can react with the alumina sorbent via nucleophilic substitution of a chloride. This lack of development of PorSils is unfortunate given that peripheral ring functionalization – allowing for useful tuning of the electronic properties – is much easier to achieve for PorSils than for Si-Pcs. There have been relatively few reports of pe- ripherally functionalized Si-Pcs.21 The substituted pyrroles and phthalic acids required as starting materials are synthetically challenging and scarcely commercially available. Porphyrins, on the other hand, possess bridging methines connecting their pyrrole rings that serve as convenient handles for meso substi- tution. This meso substitution is readily achieved by simply forming the porphyrin from commercially available aldehydes, of which there are many. Therefore, a robust procedure for κ4N- silylation of porphyrin derivatives is an appealing target that would provide access to PorSil materials that combine the elec- tronic tuneability of porphyrins with the improved physical properties of Si-Pcs. It is worth commenting on the more practical aspects of this synthesis, particularly in reference to key intermediate 2-SiCl2. While stable in open air and during neutral aqueous workup, the Si-Cl bonds in the PorSil are labile enough that reaction quickly and quantitatively occurs between 2-Si(Cl)2 and silica gel (in both column and thin-layer chromatography (TLC)). Neither the precursor (2) nor subsequent product (3) display such reac- tivity. INTRODUCTION This allows for facile tracking of reaction progress in ei- ther the generation of 2-SiCl2 from 2 or consumption of 2-SiCl2 to make 3, as 2-SiCl2 is completely immobile on a silica TLC plate.22 NMR Analysis. The NMR spectra of the synthesized SOPS provide insight into the structural and electronic characteristics of these molecules (Figure 1). The quadrupole of the aromatic porphyrin shields the alkyl/aryl protons of the silyloxy axial substituents resulting in marked upfield shifts of these signals. This was not unexpected: N-H protons in 2 – which also lie in the shielded porphyrin core – are observed at -2.75 ppm and similar upfield shifts are observed for axial substituents in re- lated PorSils and SiPcs.23 This shielding falls off with distance from the silane (and thus the center of the quadrupole); for ex- ample, in TPP-Si(OSiPh3)2 (1i), the signal for the ortho protons (HE) is visible at 4.69 ppm, significantly upfield from the usual aromatic region, and the signal for the meta protons (HF) is ob- served at 6.57 ppm (Figure 1). An additional consequence is that while phenylsilanes typically have overlapping signals for the meta (HF) and para (HG) protons, such protons in these com- pounds (i.e. 1f-i) are completely resolved, with Δδ of 0.26-0.32 ppm. A similar effect is observed in the 13C NMR spectra, with significant upfield shifts observed for the axial silane substitu- ents (see Supporting Information, Figures S1-18). Here, we describe a straightforward and general synthetic ap- proach to PorSils (Scheme 1), which has allowed us to elabo- rate them into a new class of porphyrins, bissilyloxy PorSils (SOPS). Investigation of this series of SOPS (1a-i) based on 5,10,15,20-tetraphenylporphyrin (TPP, 2) revealed that the ax- ial substituents not only serve to solubilize the porphyrin but also to tune their optical and electronic properties. RESULTS AND DISCUSSION Synthesis and Reactivity of PorSils and SOPS. Tetra- phenylporphyrin (TPP, 2) was synthesized by the Adler–Longo method.3b Previous methodology for accessing PorSils relied on the use of a highly reactive lithium amide to deprotonate the porphyrin prior to reaction with HSiCl3, which was found to be a better porphyrin silylating reagent than the perhaps more in- tuitive building block SiCl4.18 Sun and co-workers found that porphyrin silylation could be accomplished by combining the porphyrin and HSiCl3 in the presence of Pr3N.20 They relied on chromatography to purify the resulting TPP-Si(Cl)2 (2-SiCl2), an inefficient approach that also notably did not work in our pp y Scheme 1. Synthesis of the target library of bissilyloxy porphyrin silanes (SOPS). Reagents and conditions: i. HSiCl3, Et3N, rt, 24 h, 85%; ii. tetrahydrofuran, water, reflux, 2 h, quantitative; iii. chlorosilane (RCl), Et3N, 1,2-dichloroethane, reflux, 16 h, 82–97%. Reagents and conditions: i. HSiCl3, Et3N, rt, 24 h, 85%; ii. tetrahydrofuran, water, reflux, 2 h, quantitative; iii. chlorosilane (RCl), Et3N, 1,2-dichloroethane, reflux, 16 h, 82–97%. Figure 1. Partial 1H NMR spectra (400 MHz, CDCl3, 298 K) of 2, 3, and 1i showing i) that aromatic resonances of the axial substitu- ent are shifted upfield by their proximity to the porphyrin ring and ii) that HB is shifted significantly upfield in aromatic SOPS (1i). Signal from residual CHCl3 in CDCl3. Impact of Axial Substitution on Porphyrin Planarity. Por- Sils have previously been observed to have distortion in the pla- narity of the ring system in the solid state, which is due to the small size of silicon relative to the porphyrin cavity size.24 In these instances, the porphyrin core slightly contracts to allow for sufficient orbital overlap between the pyrrole nitrogen atoms and the electron-deficient silicon atom. These non-planar por- phyrins25 can adopt a variety of conformations, including ruf- fled and waved, 26 which each have characteristic patterns of displacement. Of the 10 previously reported porphyrin silane crystal structures, four were close to planar (∆r < 0.15 Å: TPP- Si(OH)227 TPP-Si(CH2TMS)2, TPP-Si(CHCH2)2, and TPP- Si(CCPh)228) and six were clearly non-planar (∆r>>0.15 Å: [TPP-Si(THF)2]2+•2Cl-,29 TPP-SiOTf2,18 TPP-SiMePh,30 TPP- SiPh2,28 tetrakis(p-tolyl)porphyrin-SiF2, and tetrakis(p-trifluo- romethylphenyl)porphyrin-SiF231). RESULTS AND DISCUSSION From this set it seems as though planarity can be achieved if both i) the axial substituents are sufficiently small so as to not abut the porphyrin and ii) the axial ligand is capable of π-back- bonding to provide the silicon with extra electron density and allow Si-N bonds to lengthen to planarity. For example, while a fluorine atom is certainly small, it does not effectively provide electron density to the silicon via unshared electrons. On the other hand, the phenyl substituents can more readily π-back- bond to the silicon, however the ortho hydrogens have a delete- rious steric interaction with the macrocyclic ring, preventing porphyrin planarization. This theory is also supported by the observed Si-N and Si-X (X = first atom of axial substituent) bond lengths: planar PorSils feature longer Si-N bonds com- pared to non-planar structures and the Si-X bond lengths are similar to those in tetracoordinate silanes as opposed to the elongated bonds expected in a hexacoordinate silane.32 Figure 1. Partial 1H NMR spectra (400 MHz, CDCl3, 298 K) of 2, 3, and 1i showing i) that aromatic resonances of the axial substitu- ent are shifted upfield by their proximity to the porphyrin ring and ii) that HB is shifted significantly upfield in aromatic SOPS (1i). Signal from residual CHCl3 in CDCl3. While the proton resonance for HB appears at approximately 8.1-8.2 ppm (Figure 1) in the spectra of 2 and alkyl capped de- rivatives 1a-e, it is shifted upfield to 7.4-8.0 ppm for 1f-i. We attribute this effect to through-space interactions between HB and the aromatic ring(s) of the capping groups in 1f-i; such in- teractions are also observed in the solid state (see Supporting Information, Figures S49-53). All NMR spectra acquired at am- bient temperature were well-resolved; this evidence of rapid conformational sampling suggests that these intramolecular contacts do not restrict bond rotation. There is, however, a dis- tinct broadening of the aromatic peaks of the silyloxy cap in 1f at low temperatures, indicating anisotropy due to slow confor- mational sampling. We were unable to reach a temperature low enough to sufficiently slow the rate of interconversion such that two distinct species could be detected (see Supporting Infor- mation, Figure S19). To systematically probe the impact of axial substitution on solid-state conformation of the PorSils, single crystals of the four species that possessed aromatic silyl caps 1f-i were ana- lyzed by X-ray diffraction. RESULTS AND DISCUSSION In general, we were gratified to see that modification of the easily incorporated axial silyloxy substitu- ents can control the degree to which the porphyrin deviates from planarity in the crystal structure. This demonstrates that axial substitution is a powerful tool for control of solid-state porphy- rin conformation. unique amongst PorSils that have been examined by X-ray crys- tallography: it has an Si-X bond distance similar to that of a tetracoordinate silane, yet the porphyrin in the crystal structure is still highly ruffled. The other derivatives seem to fit into the construct derived from previous studies of porphyrin silanes (as discussed above). One last interesting note is that while the silyloxy substituents in 1g, 1h, and 1i are arranged in an anti configuration with re- spect to the Si-O(-Si1-)O-Si bond, the highly ruffled 1f has a Si-O(-Si1-)O-Si dihedral angle of 86.72°. This may be a conse- quence of the unique conformation of this PorSil, or it could be that the packing in the crystal lattice impacts this arrangement. Influence of Axial Substitution on Solubility. Owing to their large, three-dimensional hydrophobic surfaces, SOPS (1a-i) have much greater solubility in organic solvents than TPP (2), as seen in Table 2. There were also noticeable differences in solubility between the series of aliphatic SOPS and the series Table 2. Solubility of TPP (2), aliphatic SOPS (1e), and ar- omatic SOPS (1h). Influence of Axial Substitution on Solubility. Owing to their large, three-dimensional hydrophobic surfaces, SOPS (1a-i) have much greater solubility in organic solvents than TPP (2), as seen in Table 2. There were also noticeable differences in solubility between the series of aliphatic SOPS and the series In terms of electronics, the iterative replacement of methyl for phenyl groups on the silyloxy cap (1f to 1g to 1i) should increase the electron density on the silane allowing for length- ening of the O1-Si2/O2-Si3 bond (Figure 2); the excess electron density on the oxygen could translate to a shorter Si1-O dis- tances and allow the Si-N bonds to lengthen to accommodate the preferred return to planarity of the ring. No such effect is observed. RESULTS AND DISCUSSION This data confirmed the presumed structures in all cases: in each of the four structures the hyper- coordinate silicon core adopts an octahedral structure with the two silyloxy ligands observed in a trans (diaxial) arrangement (Figure 2). Figure 2. a) X-ray crystal structures of SOPS 1f-1i confirm the presence and structure of capping groups on either face of the porphyri units. Both independent molecules in the unit cell of 1i are shown. b) Near-linear Si1–O–Si linkages (bond angles 150-168°) connect th bulky silyloxy caps to the PorSil. The porphyrin system of 1f is ruffled while 1i is slightly bent. Nitrogen atoms are shown in blue, silico atoms in yellow, oxygen atoms in red, and carbon atoms in gray and purple. Hydrogen atoms are omitted for clarity. Figure 2. a) X-ray crystal structures of SOPS 1f-1i confirm the presence and structure of capping groups on either face of the porphyrin units. Both independent molecules in the unit cell of 1i are shown. b) Near-linear Si1–O–Si linkages (bond angles 150-168°) connect the bulky silyloxy caps to the PorSil. The porphyrin system of 1f is ruffled while 1i is slightly bent. Nitrogen atoms are shown in blue, silicon atoms in yellow, oxygen atoms in red, and carbon atoms in gray and purple. Hydrogen atoms are omitted for clarity. Figure 2. a) X-ray crystal structures of SOPS 1f-1i confirm the presence and structure of capping groups on either face of the porphyrin units. Both independent molecules in the unit cell of 1i are shown. b) Near-linear Si1–O–Si linkages (bond angles 150-168°) connect the bulky silyloxy caps to the PorSil. The porphyrin system of 1f is ruffled while 1i is slightly bent. Nitrogen atoms are shown in blue, silicon atoms in yellow, oxygen atoms in red, and carbon atoms in gray and purple. Hydrogen atoms are omitted for clarity. Table 1. Selected data from crystal structures of SOPS 1f-i. RESULTS AND DISCUSSION Compound Si1-O bond lengthsa (Å) Si1-O-Si bond anglesa (º) Δrb (Å) Displacement of individual meso carbons from N1-Si1-N2 planea,b (Å) TPP-Si(OSiMe2Ph)2 (1f) 1.688(2) (Si1-O1) 1.681(2) (Si1-O2) 150.8(2) (Si1-O1-Si2) 155.2(2) (Si1-O2-Si3) 0.469 -0.431 (C5), +0.507 (C16), -0.549 (C27), +0.389 (C38) TPP-Si(OSiMePh2)2 (1g) 1.6758(14) 157.75(10) 0.006 0 (C5), +0.011 (C16), 0 (C5), -0.011 (C16) TPP-Si(OSiPh2tBu)2 (1h) 1.6801(12) 167.57(9) 0.111 -0.152 (C5), -0.069 (C16), +0.152, (C5), +0.069 (C16) TPP-Si(OSiPh3)2 (1i) Pose 1c (1i′) 1.684(3) 163.9(2) 0.058 -0.006 (C5), -0.109 (C16), +0.006 (C5), +0.109 (C16) Pose 2c (1i′′) 1.690(3) 159.8(2) 0.085 -0.025 (C45), -0.145 (C56), +0.025 (C45), +0.145 (C56) a Atoms labeled according to designation in the crystal structure: Si1 is the central silicon atom; Si2 and Si3 are the silicon atoms in the silyloxy caps; O1 and O2 are the linking oxygens of the bissilyl ethers. N1 and N2 refer to two cis-coordinated nitrogens. For full numbering scheme see Supporting Information, Figures S41, 43, 45, and 47. b See reference 25; (+) and (–) designations represent relative orientation. c The asymmetric unit in 1i contains two molecules, each in a different conformational pose (1i′ and 1i′′). a Atoms labeled according to designation in the crystal structure: Si1 is the central silicon atom; Si2 and Si3 are the silicon atoms in the silyloxy caps; O1 and O2 are the linking oxygens of the bissilyl ethers. N1 and N2 refer to two cis-coordinated nitrogens. For full numbering scheme see Supporting Information, Figures S41, 43, 45, and 47. b See reference 25; (+) and (–) designations represent relative orientation. c The asymmetric unit in 1i contains two molecules, each in a different conformational pose (1i′ and 1i′′). Of the four compounds we examined (seen in Table 1), all deviated from planarity. Compound 1g is the most planar mol- ecule with two meso carbon atoms located trans to each other displaced above and below the plane by ±0.011 Å. This is the most planar porphyrin silane crystal structure reported to date. The meso carbons in 1h and in both poses of 1i were each out of plane, though only slightly (≤0.152 Å), and porphyrins main- tained overall symmetry around the central silicon. Unique amongst the studied set, compound 1f displayed the least planar structure and was surprisingly devoid of symmetry around the central silicon atom. a Limiting concentration determined by titrating 0.1 mL aliquots of solvent into samples of the porphyrins (5-15 mg) followed by sonication until full dissolution. b No dissolution observed up to 10 mg in 15 mL. RESULTS AND DISCUSSION From a steric perspective, the Si1-O-Si bond angle of the least planar porphyrin (1f) is the most acute so one might think the steric interaction is influencing ring structure, how- ever i) this is also the smallest of the studied substituents and arranged in the sterically least impacting way in the solid state (with the phenyl pointing away from the porphyrin) and ii) there is no general correlation between that bond angle and planarity in the studied compounds. Table 2. Solubility of TPP (2), aliphatic SOPS (1e), and ar- omatic SOPS (1h). Limiting concentrationa (mM) Solvent 2 1e 1h Hexanes 0.0b 8.0 1.7 Toluene 2.5 7.3 4.4 Dichloromethane 4.7 13 6.5 Ethyl acetate 0.41 6.7 1.2 Acetonitrile 0.20 0.05 0.05 Ethanol 0.11 0.70 0.28 There does appear to be a correlation between axial silyloxy substituent size and Si1-O-Si angle, tending towards linear as the cap size increases in this set. The SOPS bearing the largest substituent (OSitBuPh2, 1h) displays a remarkably wide bond angle of 168°. The Si1-O bond length also varies based on the silyl cap, though no clear trend is observed. While shorter Si1- O bond distances would be expected in the more planar SOPS, there is a complex interplay amongst these derivatives with re- spect to the steric and electronic demands of the silyloxy cap substituents. From this perspective the Me2Ph derivative (1f) is a Limiting concentration determined by titrating 0.1 mL aliquots of solvent into samples of the porphyrins (5-15 mg) followed by sonication until full dissolution. b No dissolution observed up to 10 mg in 15 mL. Figure 3. The solid-state superstructure of 1h, which shows that the silyloxy cap shields the porphyrin core from intermolecular ar- omatic interactions. A Hirshfeld surface plot reveals that the inter- atomic distances between neighboring molecules are almost en- tirely at (white) or above (blue) the sum of the van der Waals radii. (Close contacts shown in red.) none come into close contact with the surface of the porphyrin ring system itself; they contact the cap instead. A Hirshfeld sur- face plot33 of 1h (Figure 3) and interaction energy calculations (see Supporting Information, Tables S3-12 and Figures S56-70) confirm that these interactions between the neighboring mole- cules are dominated by van der Waals forces. RESULTS AND DISCUSSION The SOPS contact one another at distances that correspond closely to the sum of the respective van der Waals radii, as can be seen by the large areas of white in the Hirshfeld surface. Impact of Axial Substitution on Optoelectronic Proper- ties. As expected, all the investigated porphyrins are UV-active compounds (Table 3, Figures 4 and 5a). Silylation of TPP (2) to make TPP-Si(OH)2 (3) does not significantly impact observed λmax – though there is a small general red-shift – and a J-aggre- gation-type Soret band is observed around 450 nm. Q1 is the strongest Q-band of both 2 and 3, indicating that the dissym- metry that exists in 2 giving rise to this optical phenomenon is also present in 3. This spectral feature lies in stark contrast to what is typically seen in two-dimensional metallated (i.e. Zn(II), Ni(II), Cu(II), and Fe(II)) porphyrins, which have only one or two Q-bands (with Q2 and/or Q3 persisting) due to higher levels of symmetry.34 Interestingly, when the silyloxy caps are installed we observe Q-band structures more like the metallated porphyrins, with Q2 being the most intense (Figure 4). There is some differentiation amongst the relative molar extinction co- efficients of the Q bands for SOPS; for example, 1a has almost no Q4 band, while 1b has a Q4 band that is greater in intensity than Q1. Notably, in all SOPS some degree of dissymmetry is evidenced by the presence of all four Q bands, and the respec- tive λmax (Soret and Q bands) of the SOPS are generally red- shifted compared to that of 2, with the notable exception of Q4 (Table 3). Both of these observations can be attributed to the likely ruffling of the porphyrin in solution.35 This hypothesis supported by a density functional theory structural study (M06- 2X/6-31G with a polarizable continuum model for dichloro- methane solvent) that shows non-planarity in the calculated ground state minimized structure of SOPS in solution (see Sup- porting Information, Figures S54 and 55). Variance amongst the SOPS λmax (Soret and Q bands) is modest (Table 3); the largest range can be seen in Q4, where the lowest (TPP- Si(OSitBuMe2)2, 1c) and highest (TPP-Si(OSiPh3)2, 1i) λmax are Figure 3. The solid-state superstructure of 1h, which shows that the silyloxy cap shields the porphyrin core from intermolecular ar- omatic interactions. a Low energy visible transitions from UV-Vis in dichloromethane. b Data collected using 0.1 M NBu4PF6 dichloromethane solutions at 100 mV s-1 and referenced to a ferrocene ([Fc]/[Fc]+) internal standard followed by conversion to NHE; [Fc]/[Fc]+ = +765 mV vs. NHE in dichloromethane. c A second oxidation peak was observed for 2 at 1.63 V vs. NHE. RESULTS AND DISCUSSION b) Normalized emission profiles of porphy- rins in i) dichloromethane (10 µM, solid lines) and ii) dispersed in optically clear amorphous polymer films (0.1 wt.% ZEONEX film, dotted lines). Figure 4. Comparison of normalized absorption for 1a-i, 2, and 3. separated by 15 nm (618 and 633 nm, respectively). This ab- ti d t d t t th t th l ti f ti l i Figure 4. Comparison of normalized absorption for 1a-i, 2, and 3. Figure 4. Comparison of normalized absorption for 1a-i, 2, and 3. separated by 15 nm (618 and 633 nm, respectively). This ab- sorption data demonstrates that the selection of a particular si- lyloxy cap can be used to control the structure of the porphyrin ring. separated by 15 nm (618 and 633 nm, respectively). This ab- sorption data demonstrates that the selection of a particular si- lyloxy cap can be used to control the structure of the porphyrin ring. The examined porphyrins were fluorescent in a dichloro- methane solution (Table 3 and Figure 5b). For 2 we observed a very strong emission band at 650 nm and a much smaller emis- sion at 711 nm. This two-emission profile is typical of porphy- rins, and is attributed to two tautomeric states.36 In toluene, 2 has the same λmax as in dichloromethane but emits relatively more intensely at 711 nm.37 This demonstrates that environmen- tal effects (i.e. solvent) impact the population distribution of the two tautomers but not the energy of the individual HOMO- LUMO gaps. When the SOPS (1a-i) were excited at their re- spective Soret bands in solution, two emission signals were noted: one around 600 nm and another closer to 650 nm (Table 3 and Figure 6b). The ~50 nm blue shift of the SOPS with re- spect to 2 corresponds to what is seen in fluorescent metallopor- phyrins such as TPP-Zn.38 Interestingly, 3 fluoresces at three distinct λmax: 600, 650, and 711 nm. This finding suggests that compound 3 either i) possesses optical character similar to both free base and metalloporphyrin and/or ii) has an additional red- shifted emission due to aggregation. The population distribution between the emission bands at ~600 and ~650 nm differs from that seen in the SOPS. It is clear that the silyloxy cap is impact- ing ring electronics, perhaps as a consequence of inhibition of aggregation and/or influence on porphyrin planarity. Figure 5. RESULTS AND DISCUSSION Absorption and emission spectra of a representative set of porphyrins in dichloromethane: 1d (aliphatic SOPS), 1i (aro- matic SOPS), 2, and 3. a) Absorption spectra of porphyrins, with inset zoom on Q bands. b) Normalized emission profiles of porphy- rins in i) dichloromethane (10 µM, solid lines) and ii) dispersed in optically clear amorphous polymer films (0.1 wt.% ZEONEX film, dotted lines). of the identity of the silyloxy cap. However, the SOPS have slightly higher oxidation potentials (1.31-1.35 V) that those ob- served for 2 (1.31 V), and significantly lower than that observed for 3 (1.52 V). This increase in the oxidation potential indicates a stabilization of the HOMO of the PorSils in solution. Samples of 2, aliphatic SOPS 1d, and aromatic SOPS 1i were also embedded into a ZEONEX polymer matrix and examined by fluorescence (Figure 5b). The emission spectrum of 2 was significantly red-shifted in the ZEONEX film. Given that gen- eral environmental effects do not cause such a phenomenon in 2, this appears to be evidence of aggregation of the porphyrin in the polymer.39 While the SOPS did show an altered popula- tion distribution of the two tautomeric states, as evidenced by a change in relative intensities of the two emission bands, the en- ergies of these transitions, however, were not affected. These findings support our hypothesis that silyloxy caps discourage aggregation. There was more variance in the reduction potential of the SOPS, with values ranging from -1.35 V (1e) to -1.06 V (1a). In all cases the SOPS were reduced at potential greater than ei- ther 2 or 3, which we observed as -0.92 and -0.93 V, respec- tively. A small trend was observed for the C3-symmetrical lin- ear trialkylsilyloxy caps (1a, 1d, and 1e), with longer chain lengths being associated with reduction potentials of greater magnitude; this could be due to electron repulsion by the si- lyloxy substituents. No other obvious trends were observed, however it is clear that the LUMO energies of the SOPS are sensitive to the identity of the silyloxy cap. The electrochemical behavior of these compounds was stud- ied using cyclic voltammetry (CV) for the full range of synthe- sized SOPS (Table 3; for CV spectra see Supporting Infor- mation, Figures S35-40). All SOPS underwent a reversible one- electron oxidation with similar oxidizing potentials regardless RESULTS AND DISCUSSION A Hirshfeld surface plot reveals that the inter- atomic distances between neighboring molecules are almost en- tirely at (white) or above (blue) the sum of the van der Waals radii. (Close contacts shown in red.) of aromatic SOPS; these differences were used to our advantage in the purification process (see Supporting Information). The improved solubilities of the SOPS compared to 2 can be ration- alized by considering the non-covalent interactions between the molecules in the solid state. Porphyrins that lack axial capping groups, such as 2, are known to experience favorable face-to- face aromatic interactions between their large, exposed π-sur- faces. These interactions cause aggregation, which in turn can red-shift absorption and quench fluorescence. On the other hand, analysis of the packing structures of 1f-i (see Supporting Information, Figures S42, 44, 46, and 48) shows that the bulky axial caps prevent this interaction. As an illustrative example, there are three molecules near the axial cap of 1h (Figure 3), but Table 3. Physicochemical data of studied porphyrins. Compound Absorptiona λmax [nm] Emission λmax [nm] (lex [nm]) E1/2b [V vs. NHE] Soret Q1 Q2 Q3 Q4 Oxidation Reduction 1a 421 513 552 589 625 605, 643 (430) 1.33 - 1.06 1b 422 512 550 591 623 592, 627 (430) 1.36 - 1.21 1c 418 512 549 586 618 605, 646 (427) 1.34 - 1.08 1d 424 514 555 596 627 598, 645 (424) 1.32 - 1.18 1e 423 514 555 595 627 602, 649 (423) 1.32 - 1.35 1f 423 514 552 591 628 605, 649 (427) 1.35 - 1.10 1g 422 514 553 592 628 600, 648 (423) 1.36 - 1.08 1h 423 512 550 587 628 607, 648 (431) 1.35 - 1.11 1i 425 515 553 592 633 602, 645 (427) 1.33 - 1.13 2 417 514 549 590 646 650, 711 (418) 1.34c - 0.92 3 418, 446 514 550 590 649 600, 650, 711 (418) 1.52 - 0.93 nd 3. s ab- ar si- hyrin loro- ved a mis- phy- ne, 2 vely men- f the MO- r re- were Table h re- opor- three that both red- ution Figure 5. Absorption and emission spectra of a representative set of porphyrins in dichloromethane: 1d (aliphatic SOPS), 1i (aro- matic SOPS), 2, and 3. a) Absorption spectra of porphyrins, with inset zoom on Q bands. Author Contributions The manuscript was written through contributions of all authors. (8) a) Martinez-Diaz, M.V.; de la Torre, G.; Torres, T. Lighting Porphyrins and Phthalocyanines for Molecular Photovol- taics. Chem. Commun. 2010, 46(38), 7090-7108. b) Walter, M.G.; Rudine, A.B.; Wamser, C.C. Porphyrins and Phthal- ocyanines in Solar Photovoltaic Cells. J. Porphyrins Phthal- ocyanines 2010, 14(9), 759-792. c) Lu-Lin Li, L.-L.; Diau, E. W.-G. Porphyrin-Sensitized Solar Cells. Chem. Soc. Rev. 2013, 42(1), 291-304. CONCLUSIONS AND OUTLOOK Edge-functionalized porphyrin derivatives are reliable and widely used building blocks for functional organic materials. We have demonstrated that the straightforward, high-yielding modification of porphyrins at their core is a viable alternative 2932. b) Shanmugam, S.; Xu, J.; Boyer, C. Exploiting Met- alloporphyrins for Selective Living Radical Polymeriza- tion Tunable over Visible Wavelengths. J. Am. Chem. Soc. 2015, 137(28), 9174-9185. c) Huang, X.; Groves, J.T. Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins. Chem. Rev. 2018, 118(5), 2491-2553. way to tune their properties. By silylating the porphyrin and capping with axial silyloxy substituents, we have increased por- phyrin solubility and prevented aggregation-induced physico- chemical phenomena. The identity of the silyloxy cap can be used to tune porphyrin planarity and molecular orbital energies. This approach offers opportunities for the customization of por- phyrins through axial tuning and an improvement in the pro- cessability of porphyrins via increased solubility. (5) Kou, J.; Dou, D.; Yang, L. Porphyrin Photosensitizers in Photodynamic Therapy and its Applications. Oncotarget. 2017, 8(46), 81591-81603. (6) a) Dougherty, T. J.; Kaufman, J. E.; Goldfarb, A.; Weishaupt, K. R.; Boyle, D. G.; Mittelman, M. Photoradiation Therapy for the Treatment of Malignant Tumors. Cancer Res. 1978, 38(8), 2628-2635. b) Santoro, A.M.; Lo Giudice, M.C.; D’Urso, A.; Lauceri, R.; Purrello, R.; Milardi, D. Cationic Porphyrins are Reversible Proteasome Inhibitors. J. Am. Chem. Soc. 2012, 134(25), 10451-10457. ACKNOWLEDGMENT The authors would like to thank Ryerson University for funding. B.A.H. acknowledges the Engineering and Physical Sciences Re- search Council (EPSRC) Centre for Doctoral Training in Soft Mat- ter and Functional Interfaces (SOFI) for a PhD studentship. A.T.T. and A.N.B. acknowledge EPSRC Doctoral Training Grants. MP acknowledges NSERC RTI for support. The authors also thank Profs. Bryan Koivisto and Stefania Impellizzeri (Ryerson Univer- sity) for advice and support. (9) a) Eriksson, K.; Göthelid, E.; Puglia, C.; Bäckvall. J.; Oscars- son, S. Performance of a Biomimetic Oxidation Catalyst Immobilized on Silica Particles. J. Catal. 2013, 303, 16-21. b) Sheng, W.; Jiang, Q.; Luo, W.; Guo, C. Oxidative Rear- rangement of Internal Alkynes to Give One-Carbon- Shorter Ketones via Manganese Porphyrins Catalysis. J. Org. Chem. 2013, 78(11), 5691-5693. c) Rybicka-Jasinśka, K.; Shan, W.; Zawada, K.; Kadish, K.M.; Gryko, D. Porphy- rins as Photoredox Catalysts: Experimental and Theoreti- cal Studies J. Am. Chem. Soc. 2016, 138(47), 15451-15458. d) Rybicka-Jasińska, K.; König, B.; Gryko, D. Porphyrin- Catalyzed Photochemical C–H Arylation of Heteroarenes. Eur. J. Org. Chem. 2017, 2104-2107. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. (7) ( ) a) Shi, J.; Liu, T.W.B.; Chen, J.; Green, D.; Jaffray, D.; Wil- son, B.C; Wang, F.; Zheng, G. Transforming a Targeted Porphyrin Theranostic Agent into a PET Imaging Probe for Cancer. Theranostics 2011, 1, 363-370. b) Huynh, E.; Leung, B.Y; Helfield, B.L.; Shakiba, M.; Gandier, J.A.; Jin, C.S.; Master, E.R.; Wilson, B.C.; Goertz, D.E.; Zheng, G. In Situ Conversion of Porphyrin Microbubbles to Nanoparti- cles for Multimodality Imaging. Nature Nanotech. 2015, 10(4), 325-332. c) Varchi, G.; Foglietta, F.; Canaparo, R.; Ballestri, M.; Arena, F.; Sotgiu, G.; Guerrini, A.; Nanni, C.; Cicoria, G.; Cravotto, G.; Fanti, S.; Serpe, L. Engineered Porphyrin Loaded Core-Shell Nanoparticles for Selective Sonodynamic Anticancer Treatment. Nanomedicine 2015, 10(23), 3483-3494. Experimental and synthetic details; 1H, 13C, and VT-NMR spectra; UV-Vis, fluorescence, and CV spectra; X-ray crystallographic data and Hirshfeld analysis; DFT structural studies; Hirshfeld surfaces brief description; mass spectra (PDF) Corresponding Author Corresponding Author * marcjadler@ryerson.ca (11) REFERENCES Swallowtail Porphyrins: Synthe- sis, Characterization and Incorporation into Porphyrin Dyads. J. Org. Chem. 2004, 69(11), 3700-3710. c) Oda, K.; Akita, M.; Hiroto, S.; Shinokubo, H. Silylethynyl Substitu- ents as Porphyrin Protecting Groups for Solubilization and Selectivity Control. Org. Lett. 2014, 16(6), 1818-1821. (14) a) Liu, C.; Liu, K.; Wang, C.; Liu, H.; Wang, H.; Su, H.; Li, X.; Chen, B.; and Jiang, J. Elucidating Heterogeneous Pho- tocatalytic Superiority of Microporous Porphyrin Organic Cage. Nat. Commun. 2020, 11(1), 1047. b) Liu, W.; Lin, C.; Weber, J.A.; Stern, C.L.; Young, R.M.; Wasielewski, M.R.; Stoddart, J.F. Cyclophane-Sustained Ultrastable Porphy- rins. 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REFERENCES (22) Interestingly, while both i) the exposed hydroxy functionality of the silica gel surface and ii) methanol form stable bonds to the PorSil to make their respective adducts, attempts to form and isolate the TPP-Si(OEt)2 derivative resulted instead in hy- drolysis and the formation of 3. This suggests that the Si-O-C in this porphyrin system is more reactive than Si-Cl in 2- SiCl2. Attempts to synthesize and observe bulkier alkoxy var- iants, namely TPP-Si(OiPr)2 and TPP-Si(OtBu)2, were also unsuccessful. Also noteworthy to mention is that the bis(tri- methylsilyloxy) derivative proved hydrolytically unstable when exposed to the atmosphere and thus was not evaluated in this study. These findings highlight the vital nature of the steric bulk of the axial substituent with respect to the proper- ties of this family of compounds. (15) Sakamoto, K.; Ohno-Okumura, E. Syntheses and Func- tional Properties of Phthalocyanines. Materials 2009, 2(3), 1127-1179. 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Actuators B: Chem. 2014, 202, 1137-1147. f) Colomban, C.; Kudrik, E.V.; Afanasiev, P.; Sorokin, A.B. Degradation of Chlorinated Phenols in Water in the Presence of H2O2 and Water-Soluble μ-Ni- trido Diiron Phthalocyanine. Catal. Today 2014, 235, 14- 19. g) Matsuzaki, H.; Murakami, T.N.; Masaki, N.; Furube, A.; Kimura, M.; Mori, S. Dye Aggregation Effect on Inter- facial Electron-Transfer Dynamics in Zinc Phthalocya- nine-Sensitized Solar Cells. J. Phys. Chem. C. 2014, 118, 17205-17212. h) Arıcan, D.; Erdogmuş, A.; Koca, A. Elec- trochromism of the Langmuir–Blodgett Films Based on Monophthalocyanines Carrying Redox Active Metal Cen- ters. Thin Solid Films. 2014, 550, 669-676. i) Melville, O.A.; Lessard, B.H.; Bender, T.P. Phthalocyanine-Based Organic Thin-Film Transistors: A Review of Recent Advances. ACS Appl. Mater. REFERENCES (1) a) Sessler, J.L.; Seidel, D. Synthetic Expanded Porphyrin Chemistry. Angew. Chem. Int. Ed. 2003, 42(42), 5134-75. b) da GH Vicente, M.; Smith, K.M. 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REFERENCES (25) Planarity can be quantified by evaluating the position of the meso carbons of the porphyrin compared to the plane defined by the pyrrole nitrogen atoms; to ac-quire our data, we estab- lished a plane defined by N1, Si1, and N2 (i.e. the central sil- icon and two cis-coordinated porphyrin nitrogen atoms) and measured the shortest distance between this plane and each me-so carbon. These distances are then averaged to give the common descriptor Δr. (26) (17) (26) Shelnutt, J.A.; Song, X.-Z.; Ma, J.-G.; Jia, S.-L.; Jentzen, W.; Medforth, C.J. Nonplanar Porphyrins and their Signifi- cance in Proteins. Chem. Soc. Rev. 1998, 27(1), 31-41. pp f ( ) a) Lessard, B.H.; Dang, J.D.; Grant, T.M. Gao, D.; Seferos, a) Lessard, B.H.; Dang, J.D.; Grant, T.M. Gao, D.; Seferos, D.S.; Bender, T.P. Bis(tri-n-hexylsilyl oxide) Silicon Phthalocyanine: A Unique Additive in Ternary Bulk Het- erojunction Organic Photovoltaic Devices. ACS Appl. Ma- ter. Interfaces 2014, 6(17), 15040-15051. b) Lim, B.; Bloking, J. T.; Ponec, A.; McGehee, M. D.; Sellinger, A. Ter- nary Bulk Heterojunction Solar Cells: Addition of Soluble NIR Dyes for Photocurrent Generation beyond 800 nm. ACS Appl. Mater Interfaces 2014, 6(9), 6905-6913. c) Les- sard, B.H.; White, R.T.; AL-Amar, M.; Plint, T.; Castrucci, J.S.; Josey, D.S.; Lu, Z.; Bender, T.P. Assessing the Poten- tial Roles of Silicon and Germanium Phthalocyanines in Planar Heterojunction Organic Photovoltaic Devices and How Pentafluoro Phenoxylation Can Enhance π–π Inter- actions and Device Performance. ACS Appl. Mater. Inter- faces 2015, 7(9), 5076-5088. (27) (27) Jian-Yu, Z; Katsuaki, K.; Aida, T. Dioxygen Insertion into the Axial Si-C Bonds of Organosilicon Porphyrins. Chem. Lett. 1998, 5, 453-454. (28) (28) Zheng, J-Y.; Konishi, K.; Aida, T. Crystallographic Studies of Organosilicon Porphyrins: Stereoelectronic Effects of Axial Groups on the Nonplanarity of the Porphyrin Ring. Inorg. Chem. 1998, 37(10), 2591-2594. g (29) See paper in ref. 23. (30) Ishida, S.; Yoshimura, K.; Matsumoto, H.; Kyushin, S. Selec- tive Si–C Bond Cleavage on a Diorganosilicon Porphyrin Complex Bearing Different Axial Ligands. Chem. Lett. 2009, 38(4), 362-363. (31) (31) Kane, K.M.; Lemke, F.R.; Petersen, J.L. trans-Difluorosili- con(IV) Complexes of Tetra-p-tolylporphyrin and Tetrakis(p-(trifluoromethyl)phenyl)porphyrin: Crystal (18) f ( ) Kane, K.M.; Lemke, F.R.; Petersen J.L. Bis(trifluoro- methanesulfonato)(tetra-p-tolylporphyrinato)sili- Structures and Unprecedented Reactivity in Hexacoordi- nate Difluorosilanes1. Inorg. Chem. 1997, 36(7), 1354-1359. (38) REFERENCES Observed bond lengths for Si-X in crystal structure of por- phyrin silane (PorSil) and a tetracoordinated (4c) silane (with reference): X = OH, PorSil = 1.67 Å, 4c = 1.66 Å (ref. 23); CHCH2, PorSil = 1.83 Å (ref. 24), 4c = 1.86 Å (Bartkowska, B.; Krüger, C. Tetravinylsilane. Acta Cryst. 1997, C53, 1066-1068.); CCPh, PorSil = 1.82 Å (ref. 24), 4c = 1.83 Å (Wrackmeyer, B.; Khana, E.; Bayer, S.; Toka, O.L.; Klim- kina, E.V.; Miliusc, W.; Kempe, R. Alkynylsilanes and Al- kynyl(vinyl)silanes. Synthesis, Molecular Structures and Multinuclear Magnetic Resonance Study. Z. Naturforsch. 2010, 65b, 725-744.); CH2TMS, PorSil = 1.93 Å (ref. 24), 4c = 1.92 Å (Buttrus, N.H.; Eaborn, C.; Hitchcock, P.B.; Lickiss, P.D.; Najim, S.T. 1,3 Silicon to Silicon Migration of the Methoxy Group in Solvolysis of (bromodiphenylsi- lyl)(methoxydimethylsilyl)bis(trimethylsilyl)methane. Crystal Structures of (ethoxydimethylsilyl)(methoxydi- phenylsilyl)bis(trimethylsilyl)-methane and (methoxydi- methylsilyl)(methoxydiphenylsilyl)bis(trimethylsilylme- thane). J. Chem. Soc. Perkin Trans. II 1987, 1753-1757.). As a point of reference, here are two examples for non-planar porphyrins: Ph, PorSil = 1.94 Å (ref. 24), 4c = 1.86 Å (Párká- nyi, L.; Sasvári, K. Crystal structure of tetraphenylsilane. Period. Polytechnic. Chem. Eng. 1973, 17(3), 271-276.); F, PorSil = 1.64 Å (ref. 26), 4c = 1.60 Å (Dell, S.; Ho, D.M.; Pascal, R.A. in- and out-Cyclophanes Bearing Non-Hydro- gen Bridgehead Substituents. J. Org. Chem. 1999, 64(15), 5626-5633.) Pascal, R.A. in- and out-Cyclophanes Bearing Non-Hydro- gen Bridgehead Substituents. J. Org. Chem. 1999, 64(15), 5626-5633.) Structures and Unprecedented Reactivity in Hexacoordi- nate Difluorosilanes1. Inorg. Chem. 1997, 36(7), 1354-1359. (32) (33) ) Spackman, M. A.; Jayatilaka, D. Hirshfeld Surface Analy- sis. CrystEngComm 2009, 11(1), 19-32. (34) Ralphs, K.; Zhang, C.; James, S. L. Solventless Mechano- chemical Metalation of Porphyrins. Green Chem. 2017, 19(1), 102-105. (35) ) Valicsek, Z.; Horváth, O. Application of the Electronic Spectra of Porphyrins for Analytical Purposes: The Effects of Metal Ions and Structural Distortions. Microchem. J. 2013, 107, 47-62. (36) Uttamlal, M.; Holmes-Smith, A.S. The Excitation Wave- length Dependent Fluorescence of Porphyrins. Chem. Phys. Lett. 2008, 454(4-6), 223-228. (37) y ( ) (37) Ghosh, M.; Nath, S.; Hajra, A.; Sinha, S. Fluorescence Self- Quenching of Tetraphenylporphyrin in Liquid Medium. J. Lumin. 2013, 141, 87-92. Crystal Structures of (ethoxydimethylsilyl)(methoxydi- phenylsilyl)bis(trimethylsilyl)-methane and (methoxydi- methylsilyl)(methoxydiphenylsilyl)bis(trimethylsilylme- thane). J. Chem. Soc. Perkin Trans. II 1987, 1753-1757.). As a point of reference, here are two examples for non-planar porphyrins: Ph, PorSil = 1.94 Å (ref. (37) REFERENCES 24), 4c = 1.86 Å (Párká- nyi, L.; Sasvári, K. Crystal structure of tetraphenylsilane. Period. Polytechnic. Chem. Eng. 1973, 17(3), 271-276.); F, PorSil = 1.64 Å (ref. 26), 4c = 1.60 Å (Dell, S.; Ho, D.M.; (38) Zheng, W.; Shan, N.; Yu, L.; Wang, X. UV–visible, Fluores- cence and EPR Properties of Porphyrins and Metallopor- phyrins. Dyes Pigm. 2008, 77, 153-157. (39) (39) Chen, Z.; Lohr, A.; Saha-Möller, C.R.; Würthner, F. Self-As- sembled π-Stacks of Functional Dyes in Solution: Struc- tural and Thermodynamic Features. Chem. Soc. Rev. 2009, 38(2), 564-584. PorSil paper final.pdf (2.97 MiB) download file view on ChemRxiv Supporting Information Supporting Information Burhan A. Hussein,†,‡ Zainab Shakeel,† Andrew T. Turley,‡ Aisha N. Bismillah,‡,§ Kody M. Wolfstadt,† Julia E. Pia,† Melanie Pilkington,⊥ Paul R. McGonigal,‡ and Marc J. Adler†,* † Department of Chemistry & Biology, Ryerson University, 350 Victoria St., Toronto, ON, M5B 2K3, Canada ‡ Department of Chemistry, Durham University, Lower Mountjoy, Stockton Road, Durham, DH1 3LE, United Kingdom § Department of Chemistry, Dartmouth College, Hanover, New Hampshire, 03755, USA ⊥ Department of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON, L2S 3A1, Canada Burhan A. Hussein,†,‡ Zainab Shakeel,† Andrew T. Turley,‡ Aisha N. Bismillah,‡,§ Kody M. Wolfstadt,† Julia E. Pia,† Melanie Pilkington,⊥ Paul R. McGonigal,‡ and Marc J. Adler†,* † Department of Chemistry & Biology, Ryerson University, 350 Victoria St., Toronto, ON, M5B 2K3, Canada ‡ Department of Chemistry, Durham University, Lower Mountjoy, Stockton Road, Durham, DH1 3LE, United Kingdom Department of Chemistry, Dartmouth College, Hanover, New Hampshire, 03755, USA ⊥ Department of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON, L2S 3A1, Canada 1 Table of Contents 1. GENERAL METHODS.............................................................................................................................. 3 2. SYNTHETIC DETAILS............................................................................................................................... 5 3. 1H, 13C, AND VARIABLE-TEMPERATURE NMR SPECTROSCOPIC CHARACTERIZATION OF SYNTHESIZED COMPOUNDS . 15 4. UV-VIS SPECTROSCOPY ....................................................................................................................... 34 5. PHOTOLUMINESCENCE SPECTROSCOPY .................................................................................................... 44 6. CYCLIC VOLTAMMETRY ........................................................................................................................ 49 7. X-RAY CRYSTALLOGRAPHIC ANALYSIS...................................................................................................... 57 7.1 INTRAMOLECULAR CONTACTS IN SOLID STATE......................................................................................... 63 8. IN SILICO MODELLING ......................................................................................................................... 69 9. HIRSHFELD SURFACE ANALYSIS OF SOLID-STATE STRUCTURES ...................................................................... 71 10. CALCULATION OF INTERMOLECULAR INTERACTION ENERGIES IN THE SOLID STATE .......................................... 81 11. COMPOUND IDENTITY VALIDATION BY LOW RESOLUTION MASS SPECTRA ..................................................... 87 12. REFERENCES .................................................................................................................................... 92 2 2 1. General Methods Materials: All reagents were purchased from commercial suppliers (Sigma-Aldrich, Alfa Aesar, or Oakwood Chemicals) and used without further purification. Instrumentation and Analytical Techniques: Purification by column chromatography was carried out using silica (Silicycle: ultrapure flash silica). Analytical thin-layer chromatography (TLC) was performed on aluminum-backed sheets pre-coated with silica 60 F254 adsorbent (0.25 mm thick; Silicycle) and visualized under UV light (254 nm), if needed. Routine 1H and 13C{1H} nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz and 101 MHz respectively, on a Bruker AV 400 instrument at ambient temperatures. Variable-temperature (VT) NMR was recorded on a Varian Inova 500 (1H 500.130 MHz) at range of temperatures. For VT measurements, operating temperatures were calibrated using an internal calibration solution of MeOH and glycerol. Chemical shifts (δ) are reported in parts per million (ppm) from low to high field and referenced to a residual non-deuterated solvent (CHCl3) peak for 1H and 13C nuclei. Coupling constants (J) are reported in Hertz (Hz). Standard abbreviations indicating multiplicity are used as follows: s = singlet; d = doublet; m = multiplet; br = broad). 13C NMR experiments were proton decoupled. NMR spectra were processed using MestReNova version 11.0. Data are reported as follows: chemical shift; multiplicity; coupling constants; integral and assignment. Low resolution mass spectrometry (MS) results were obtained on a Waters GCT Premier instrument using electron impact (EI) for ionization. High-resolution electrospray (HR- ESI) mass spectra were measured using a Waters LCT Premier XE high resolution, accurate mass UPLC ES MS with ASAP ion source. Cyclic voltammetry (CV) data were collected using a Metrohm µ-Autolab Type III potentiostat/galvanostat and are reported relative to the potential of the normal hydrogen electrode (NHE). UV-Vis spectroscopy data was obtained using a Cary 3 3 Series UV-Vis Spectrophotometer from Agilent Technologies. Steady-state photoluminescence (PL) solution data were obtained using a Perkin-Elmer LS 50 B Luminescence Spectrometer. Steady-state PL of films and solutions were measured using Jobin Yvon Fluoromax and Fluorolog with machine-specific calibration curves. The low temperature and temperature- dependent PL spectra were measured using a Janis Research Co. Inc. nitrogen filled cryostat. Thus, at low temperature, films were in a nitrogen atmosphere and solutions in a sealed long- neck cuvette. Suitable single crystals of all four compounds 1f-i were mounted on a cryoloop with paratone oil and examined on a Bruker APEX-II CCD diffractometer. 1. General Methods All four compounds have been deposited in the Cambridge Structural Database with numbers CCDC 1977452- 1977455. 4 4 2. Synthetic Details 2. Synthetic Details N N N N Ph Ph Ph Si OSi SiO Et Et 1 2 3 4 5 6 Et Et Et N N N N Ph Ph Ph Si OSi SiO Et Et 1 2 3 4 5 6 Et Et Et 1a: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of triethylchlorosilane (40 mg, 0.37 mmol). The reaction was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.70. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1a as a purple solid (127 mg, 0.14 mmol, 95%). 1H NMR (400 MHz, CDCl3) d 8.83 (s, 8H, H1), 8.18 – 8.13 (m, 8H, H2), 7.79 – 7.70 (m, 12H, H3, H4), -1.36 (t, J = 7.9 Hz, 18H, H6), -2.68 (q, J = 8.0 Hz, 12H, H5). 13C NMR (101 MHz, CDCl3) d 143.9, 141.7, 134.1 130.5, 127.7, 126.8, 117.7, 4.49, 2.80. IR (cm-1) 3057, 2942, 2867, 2797, 2378, 2265, 2101, 2081, 1944, 1881, 1758, 1597, 1534, 1491, 1439, 1409, 1354, 1226, 1205, 1173, 1060, 1006, 882, 845, 801, 748. UV-Vis (CH2Cl2): λmax (nm) 421 (Soret band), 513, 552, 589, 625 (Q bands); Fluorescence (CH2Cl2, λex = 430): λem (nm) 605, 643. HRMS-ASAP m/z [M]+ calculated for C56H58N4O2Si3 902.3868, measured 902.3912. CV (V vs NHE, ref to Fc/Fc+) -1.119, 1.321. N N N N Ph Ph Ph Si OSi SiO Et Et 1 2 3 4 5 6 Et Et Et 1a: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of triethylchlorosilane (40 mg, 0.37 mmol). The reaction was refluxed overnight and monitored by TLC. 1. General Methods Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.70. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1a as a purple solid (127 mg, 0.14 mmol, 95%). 1H NMR (400 MHz, CDCl3) d 8.83 (s, 8H, H1), 8.18 – 8.13 (m, 8H, H2), 7.79 – 7.70 (m, 12H, H3, H4), -1.36 (t, J = 7.9 Hz, 18H, H6), -2.68 (q, J = 8.0 Hz, 12H, H5). 13C NMR (101 MHz, CDCl3) d 143.9, 141.7, 134.1 130.5, 127.7, 126.8, 117.7, 4.49, 2.80. IR (cm-1) 3057, 2942, 2867, 2797, 2378, 2265, 2101, 2081, 1944, 1881, 1758, 1597, 1534, 1491, 1439, 1409, 1354, 1226, 1205, 1173, 1060, 1006, 882, 845, 801, 748. UV-Vis (CH2Cl2): λmax (nm) 421 (Soret band), 513, 552, 589, 625 (Q bands); Fluorescence (CH2Cl2, λex = 430): λem (nm) 605, 643. HRMS-ASAP m/z [M]+ calculated for C56H58N4O2Si3 902.3868, measured 902.3912. CV (V vs NHE, ref to Fc/Fc+) -1.119, 1.321. N N N N Ph Ph Ph Si OSi SiO Et Et 1 2 3 4 5 6 Et Et Et refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.70. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1a as a purple solid (127 mg, 0.14 mmol, 95%). 1H NMR (400 MHz, CDCl3) d 8.83 (s, 8H, H1), 8.18 – 8.13 (m, 8H, H2), 7.79 – 7.70 (m, 12H, H3, H4), -1.36 (t, J = 7.9 Hz, 18H, H6), -2.68 (q, J = 8.0 Hz, 12H, H5). 13C NMR (101 MHz, CDCl3) d 143.9, 141.7, 134.1 130.5, 127.7, 126.8, 117.7, 4.49, 2.80. IR (cm-1) 3057, 2942, 2867, 2797, 2378, 2265, 2101, 2081, 1944, 1881, 1758, 1597, 1534, 1491, 1439, 1409, 1354, 1226, 1205, 1173, 1060, 1006, 882, 845, 801, 748. UV-Vis (CH2Cl2): λmax (nm) 421 (Soret band), 513, 552, 589, 625 (Q bands); Fluorescence (CH2Cl2, λex = 430): λem (nm) 605, 643. HRMS-ASAP m/z [M]+ calculated for C56H58N4O2Si3 902.3868, measured 902.3912. 1. General Methods CV (V vs NHE, ref to Fc/Fc+) -1.119, 1.321. 5 5 1b: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of triisopropylchlorosilane (71 mg, 0.37 mmol). The reaction was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1b as a purple solid (132 mg, 0.14 mmol, 91%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 8.15 – 8.07 (m, 8H, H2), 7.81 – 7.69 (m, 12H, H3, H4), -1.31 (d, J = 7.5 Hz, 36H, H6), -2.26 (septet, J = 7.5 Hz, 6H, H5). 13C NMR (101 MHz, CDCl3) d 144.3, 141.6, 134.0, 130.8, 127.8, 126.8, 118.3, 15.5 11.0. IR (cm-1) 2918, 2857, 2168, 2116, 2078, 1883, 1817, 1597, 1533, 1491, 1354, 1232, 1205, 1172, 1076, 1033, 1007, 881, 836, 800, 747, 698. UV-Vis (CH2Cl2): λmax (nm) 422 (Soret band), 512, 550, 591, 623 (Q bands). Fluorescence (CH2Cl2, λex = 430 nm): λem (nm) 592, 627. MS (EI+) m/z [M]+ calculated for C62H70N4O2Si3 986.5, measured 986.5. CV (V vs NHE ref to Fc/Fc+) -1.218, 1.353. N N N N Ph Ph Ph Si OSi SiO iPr iPr 1 2 3 4 5 6 iPr iPr iPr 1b: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of triisopropylchlorosilane (71 mg, 0.37 mmol). The reaction was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. 1. General Methods 13C NMR (101 MHz, CDCl3) d 144.5, 141.9, 134.1, 127.8, 126.7, 118.4, 120.2, 23.2, 15.1, 7.5. IR (cm-1) 2920, 2848, 1597, 1537, 1491, 1440, 1355, 1240, 1207, 1062, 1008, 939, 829, 801, 749. UV-Vis (CH2Cl2): λmax (nm) 418 (Soret band), 512, 549, 586, 618 (Q bands); Fluorescence (CH2Cl2, λex = 427): λem (nm) 605, 646. MS (EI+) m/z [M]+ calculated for C56H58N4O2Si3 902.38, measured 902.39. CV (V vs NHE ref to Fc/Fc+) -1.144, 1.342. was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1c as a purple solid (115 mg, 0.13 mmol, 86%). 1H NMR (400 MHz, CDCl3) d 8.86 (s, 8H, H1), 8.20 – 8.14 (m, 8H, H2), 7.81 – 7.70 (m, 12H, H3, H4), -1.61 (s, 18H, H6), -3.34 (s, 12H, H5). 13C NMR (101 MHz, CDCl3) d 144.5, 141.9, 134.1, 127.8, 126.7, 118.4, 120.2, 23.2, 15.1, 7.5. IR (cm-1) 2920, 2848, 1597, 1537, 1491, 1440, 1355, 1240, 1207, 1062, 1008, 939, 829, 801, 749. UV-Vis (CH2Cl2): λmax (nm) 418 (Soret band), 512, 549, 586, 618 (Q bands); Fluorescence (CH2Cl2, λex = 427): λem (nm) 605, 646. MS (EI+) m/z [M]+ calculated for C56H58N4O2Si3 902.38, measured 902.39. CV (V vs NHE ref to Fc/Fc+) -1.144, 1.342. N N N N Ph Ph Ph Si OSi SiO Bu Bu 1 2 3 4 5 6 7 8 Bu Bu Bu 1d: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of tributylchlorosilane (87 mg, 0.37 mmol). The reaction mixture N N N N Ph Ph Ph Si OSi SiO Bu Bu 1 2 3 4 5 6 7 8 Bu Bu Bu 1d: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. 1. General Methods The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1b as a purple solid (132 mg, 0.14 mmol, 91%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 8.15 – 8.07 (m, 8H, H2), 7.81 – 7.69 (m, 12H, H3, H4), -1.31 (d, J = 7.5 Hz, 36H, H6), -2.26 (septet, J = 7.5 Hz, 6H, H5). 13C NMR (101 MHz, CDCl3) d 144.3, 141.6, 134.0, 130.8, 127.8, 126.8, 118.3, 15.5 11.0. IR (cm-1) 2918, 2857, 2168, 2116, 2078, 1883, 1817, 1597, 1533, 1491, 1354, 1232, 1205, 1172, 1076, 1033, 1007, 881, 836, 800, 747, 698. UV-Vis (CH2Cl2): λmax (nm) 422 (Soret band), 512, 550, 591, 623 (Q bands). Fluorescence (CH2Cl2, λex = 430 nm): λem (nm) 592, 627. MS (EI+) m/z [M]+ calculated for C62H70N4O2Si3 986.5, measured 986.5. CV (V vs NHE ref to Fc/Fc+) -1.218, 1.353. N N N N Ph Ph Ph Si OSi SiO iPr iPr 1 2 3 4 5 6 iPr iPr iPr N N N N Ph Ph Ph Si OSi SiO iPr iPr 1 2 3 4 5 6 iPr iPr iPr 1c: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of tert- butyldimethylchlorosilane (56 mg, 0.37 mmol). The reaction N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 tBu N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 tBu N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 tBu 6 was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the crude residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1c as a purple solid (115 mg, 0.13 mmol, 86%). 1H NMR (400 MHz, CDCl3) d 8.86 (s, 8H, H1), 8.20 – 8.14 (m, 8H, H2), 7.81 – 7.70 (m, 12H, H3, H4), -1.61 (s, 18H, H6), -3.34 (s, 12H, H5). 1. General Methods The reaction mixture was stirred for 5 min, followed by the addition of tributylchlorosilane (87 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1d as a purple solid (141 mg, 0.13 mmol, 89%). 1H NMR (400 MHz, CDCl3) d 8.83 (s, 8H, H1), 8.18 (d, J = 5.8 Hz, 8H, H2), 7.80 – 7.69 (m, 12H, H3, H4), 0.36 – 0.17 (m, 30H, H7, H8), N N N N Ph Ph Ph Si OSi SiO Bu Bu 1 2 3 4 5 6 7 8 Bu Bu Bu was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1d as a purple solid (141 mg, 0.13 mmol, 89%). 1H NMR (400 MHz, CDCl3) d 8.83 (s, 8H, H1), 8.18 (d, J = 5.8 Hz, 8H, H2), 7.80 – 7.69 (m, 12H, H3, H4), 0.36 – 0.17 (m, 30H, H7, H8), 7 7 -1.18 – -1.46 (m, 12H, H6), -2.54 – -2.95 (m, 12H, H5). 13C NMR (100 MHz, CDCl3) d 143.9, 141.8, 134.4, 130.6, 127.8, 126.8, 117.8, 26.1, 23.4, 13.5, 12.2. IR (cm-1) 2951, 2914, 2850, 1598, 1534, 1490, 1440, 1355, 1292, 1059, 1008, 883, 800, 752. UV-Vis (CH2Cl2): λmax (nm) 424 (Soret band), 514, 555, 596, 627 (Q bands); Fluorescence (CH2Cl2, λex = 424): λem (nm) 598, 645. MS (EI+) m/z [M]+ calculated for C68H82N4O2Si3 1070.5, measured 1070.5. CV (V vs NHE, ref to Fc/Fc+)) -1.184, 1.307. 1e: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2- dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of trihexylchlorosilane (105 mg, 0.37 mmol). 1. General Methods The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1e as a purple solid (178 mg, 0.15 mmol, 97%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 8.21 – 8.13 (m, 8H, H2), 7.80 – 7.67 (m, 12H, H3, H4), 1.02 – 0.85 (m, 18H, H10 ), 0.71 (t, J = 7.3 Hz, 12H, H9), 0.63 (tt, J = 9.4, 6.5 Hz, 12H, H8), 0.23 (tt, J = 7.5, 7.5 Hz, 12H, H7), -1.33 (m, 12H, H6), -2.67 – -2.82 (m, 12H, H5). 13C NMR (101 MHz, CDCl3) d 143.7, 134.3, 130.5, 141.6, 127.7, 126.6, 117.6, 32.9, 31.15,22.5, 21.0, 14.1, 12.3. IR (cm-1) 2953, 2916, 2849, 1597, 1526, 1490, 1440, 1356, 1207, 1176, 1068, 1010, 884, 799, 750. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 514, 555, 595, 627 (Q bands); Fluorescence N N N N Ph Ph Ph Si OSi SiO Hex Hex 1 2 3 4 5 6 7 8 9 10 Hex Hex Hex 1e: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2- dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of trihexylchlorosilane (105 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon N N N N Ph Ph Ph Si OSi SiO Hex Hex 1 2 3 4 5 6 7 8 9 10 Hex Hex Hex 1e: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2- dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of trihexylchlorosilane (105 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. 1. General Methods The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1e as a purple solid (178 mg, 0.15 mmol, 97%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 8.21 – 8.13 (m, 8H, H2), 7.80 – 7.67 (m, 12H, H3, H4), 1.02 – 0.85 (m, 18H, H10 ), 0.71 (t, J = 7.3 Hz, 12H, H9), 0.63 (tt, J = 9.4, 6.5 Hz, 12H, H8), 0.23 (tt, J = 7.5, 7.5 Hz, 12H, H7), -1.33 (m, 12H, H6), -2.67 – -2.82 (m, 12H, H5). 13C NMR (101 MHz, CDCl3) d 143.7, 134.3, 130.5, 141.6, 127.7, 126.6, 117.6, 32.9, 31.15,22.5, 21.0, 14.1, 12.3. IR (cm-1) 2953, 2916, 2849, 1597, 1526, 1490, 1440, 1356, 1207, 1176, 1068, 1010, 884, 799, 750. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 514, 555, 595, 627 (Q bands); Fluorescence N N N N Ph Ph Ph Si OSi SiO Hex Hex 1 2 3 4 5 6 7 8 9 10 Hex Hex Hex (105 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitore (105 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et3N, 9:1), Rf = 0.72. The filtrate was then triturated using cold MeCN until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1e as a purple solid (178 mg, 0.15 mmol, 97%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 8.21 – 8.13 (m, 8H, H2), 7.80 – 7.67 (m, 12H, H3, H4), 1.02 – 0.85 (m, 18H, H10 ), 0.71 (t, J = 7.3 Hz, 12H, H9), 0.63 (tt, J = 9.4, 6.5 Hz, 12H, H8), 0.23 (tt, J = 7.5, 7.5 Hz, 12H, H7), -1.33 (m, 12H, H6), -2.67 – -2.82 (m, 12H, H5). 13C NMR (101 MHz, CDCl3) d 143.7, 134.3, 130.5, 141.6, 127.7, 126.6, 117.6, 32.9, 31.15,22.5, 21.0, 14.1, 12.3. IR (cm-1) 2953, 2916, 2849, 1597, 1526, 1490, 1440, 1356, 1207, 1176, 1068, 1010, 884, 799, 750. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 514, 555, 595, 627 (Q bands); Fluorescence 8 8 (CH2Cl2, λex = 423): λem (nm) 602, 649. 1. General Methods HRMS-ESI m/z [M]+ calculated for C80H106N4O2Si3 1238.7653, measured 1238.7653. CV (V vs NHE, ref to Fc/Fc+)) -1.235, 1.317. 1f: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of dimethylphenylchlorosilane (63 mg, 0.37 mmol). ). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 7 8 Ph 1f: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of dimethylphenylchlorosilane (63 mg, 0.37 mmol). ). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1f as a purple solid (129 mg, 0.14 mmol, 92%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 7.98 – 7.91 (m, 8H, H2), 7.75 – 7.69 (m, 4H, H4), 7.69 – 7.63 (m, 8H, H3), 6.72 (tt, J = 7.4, 1.4 Hz, 2H, H8), 6.50 – 6.41 (m, 4H, H7), 4.52 (dd, J = 7.9, 1.4 Hz, 4H, H6), -2.89 (s, 12H, H5). 13C NMR (101 MHz, CDCl3) d 177.8, 143.6, 141.1, 140.2, 134.2, 131.1, 131.1, 127.9, 127.3, 126.9, 126.2, 2.8. IR (cm-1) 3051, 2946, 2552, 2377, 2104, 2077, 1884, 1814, 1761, 1597, 1533, 1490, 1440, 1399, 1355, 1239, 1206, 1174, 1115, 1067, 1007, 799, 743. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 514, 552, 591, 628 (Q bands). Fluorescence (CH2Cl2, λex = 427): λem (nm) 605, 649. HRMS-ASAP m/z [M+H]+ calculated for C60H51N4O2Si3 943.3320, measured 943.3338. 1. General Methods CV (V vs NHE, ref to Fc/Fc+) -1 099 1 349 N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 7 8 Ph N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 7 8 Ph residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1f as a purple solid (129 mg, 0.14 mmol, 92%). 1H NMR (400 MHz, CDCl3) d 8.82 (s, 8H, H1), 7.98 – 7.91 (m, 8H, H2), 7.75 – 7.69 (m, 4H, H4), 7.69 – 7.63 (m, 8H, H3), 6.72 (tt, J = 7.4, 1.4 Hz, 2H, H8), 6.50 – 6.41 (m, 4H, H7), 4.52 (dd, J = 7.9, 1.4 Hz, 4H, H6), -2.89 (s, 12H, H5). 13C NMR (101 MHz, CDCl3) d 177.8, 143.6, 141.1, 140.2, 134.2, 131.1, 131.1, 127.9, 127.3, 126.9, 126.2, 2.8. IR (cm-1) 3051, 2946, 2552, 2377, 2104, 2077, 1884, 1814, 1761, 1597, 1533, 1490, 1440, 1399, 1355, 1239, 1206, 1174, 1115, 1067, 1007, 799, 743. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 514, 552, 591, 628 (Q bands). Fluorescence (CH2Cl2, λex = 427): λem (nm) 605, 649. HRMS-ASAP m/z [M+H]+ calculated for C60H51N4O2Si3 943.3320, measured 943.3338. CV (V vs NHE, ref to Fc/Fc+) -1.099, 1.349. 9 N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 7 8 Ph Ph 1g: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of CH3Si(C6H5)2Cl (86 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1g as a purple solid (130 mg, 0.12 mmol, 82 %). 1. General Methods The reaction mixture was stirred for 5 min, followed by the addition of tert- butyldiphenylchlorosilane (101 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1h as a purple solid (154 mg, 0.13 mmol, 90%). 1H NMR (400 MHz, CDCl3) d 8.68 (s, 8H, H1), 7.83 (d, J = 6.7 Hz, 8H, H2), 7.80 – 7.72 (m, 4H, H4), 7.69 (t, J = 7.2 Hz, 8H, H3), 6.89 (t, J = 7.4 Hz, 4H, H8), 6.60 (t, J = 7.5 Hz, 8H, H7 ), 4.56 (d, J = 7.1 Hz, 8H, H6), -1.40 (s, 18H, H5). 13C NMR (101 MHz, CDCl3) d 144.6, 141.4, 134.3, 133.9, 133.4, 130.5, 127.6, 127.1, 126.6, 125.7, 118.5, 24.5, 16.7. IR (cm-1) 3053, 2923, 2849, 1597, 1540, 1424, 1355, 1259, 1206, 1174, 1102, 1042, 1008, 938, 799, 750. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 512, 550, 587, 628 (Q bands); Fluorescence (CH2Cl2, λex = 431): λem(nm) 607, 648. MS (EI+) m/z [M]+ calculated for C76H66N4O2Si3 1150.44, measured 1150.45. CV (V vs NHE, ref to Fc/Fc+) -1.105, 1.345. 1h: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of tert- butyldiphenylchlorosilane (101 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1h as a purple solid (154 mg, 0.13 mmol, 90%). 1. General Methods 1H NMR (400 MHz, CDCl3) δ 8.75 (s, 8H, H1), 7.75 – 7.66 (dd, 12H, H4, H2), 7.63 (t, J = 7.4 Hz, 8H, H3), 6.78 (tt, J = 7.7, 1.3 Hz, 4H, H8), 6.49 (t, J = 7.4 Hz, 8H, H7), 4.56 (dd, J = 7.9, 1.3 Hz, 8H, H6), - 2.65 (s, 6H, H5). 13C NMR (101 MHz, CDCl3) δ 143.8, 141.1, 137.7, 134.3, 131.7, 130.9, 127.8, 127.5, 126.7, 126.3, 118.1, - 5.4. IR (cm-1) 1596, 1541, 1484, 1354, 1247, 1205, 1173, 1110, 1059 (vs), 1008 (vs), 850 (s), 795 (s), 737 (s), 700 (vs). UV-Vis (CH2Cl2): λmax (nm) 422 (Soret band), 514, 553, 592, 628 (Q bands); Fluorescence (CH2Cl2, λex = 423): λem (nm) 600, 648. MS (EI+) m/z [M]+ calculated for C70H54N4O2Si3 1066.35, measured 1066.36. CV (V vs NHE, ref to Fc/Fc+) N N N N Ph Ph Ph Si OSi SiO 1 2 3 4 5 6 7 8 Ph Ph 8 the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1g as a purple solid (130 mg, 0.12 mmol, 82 %). 1H NMR (400 MHz, CDCl3) δ 8.75 (s, 8H, H1), 7.75 – 7.66 (dd, 12H, H4, H2), 7.63 (t, J = 7.4 Hz, 8H, H3), 6.78 (tt, J = 7.7, 1.3 Hz, 4H, H8), 6.49 (t, J = 7.4 Hz, 8H, H7), 4.56 (dd, J = 7.9, 1.3 Hz, 8H, H6), - 2.65 (s, 6H, H5). 13C NMR (101 MHz, CDCl3) δ 143.8, 141.1, 137.7, 134.3, 131.7, 130.9, 127.8, 127.5, 126.7, 126.3, 118.1, - 5.4. IR (cm-1) 1596, 1541, 1484, 1354, 1247, 1205, 1173, 1110, 1059 (vs), 1008 (vs), 850 (s), 795 (s), 737 (s), 700 (vs). UV-Vis (CH2Cl2): λmax (nm) 422 (Soret band), 514, 553, 592, 628 (Q bands); Fluorescence (CH2Cl2, λex = 423): λem (nm) 600, 648. MS (EI+) m/z [M]+ calculated for C70H54N4O2Si3 1066.35, measured 1066.36. CV (V vs NHE, ref to Fc/Fc+) -1.105, 1.352. 10 1h: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL, 0.37 mmol) was added. 1. General Methods 1H NMR (400 MHz, CDCl3) d 8.68 (s, 8H, H1), 7.83 (d, J = 6.7 Hz, 8H, H2), 7.80 – 7.72 (m, 4H, H4), 7.69 (t, J = 7.2 Hz, 8H, H3), 6.89 (t, J = 7.4 Hz, 4H, H8), 6.60 (t, J = 7.5 Hz, 8H, H7 ), 4.56 (d, J = 7.1 Hz, 8H, H6), -1.40 (s, 18H, H5). 13C NMR (101 MHz, CDCl3) d 144.6, 141.4, 134.3, 133.9, 133.4, 130.5, 127.6, 127.1, 126.6, 125.7, 118.5, 24.5, 16.7. IR (cm-1) 3053, 2923, 2849, 1597, 1540, 1424, 1355, 1259, 1206, 1174, 1102, 1042, 1008, 938, 799, 750. UV-Vis (CH2Cl2): λmax (nm) 423 (Soret band), 512, 550, 587, 628 (Q bands); Fluorescence (CH2Cl2, λex = 431): λem(nm) 607, 648. MS (EI+) m/z [M]+ calculated for C76H66N4O2Si3 1150.44, measured 1150.45. CV (V vs NHE, ref to Fc/Fc+) -1.105, 1.345. 11 1i: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL g, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of triphenylchlorosilane (109 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1i as a purple solid (150 mg, 0.13 mmol, 85%). 1H NMR (400 MHz, CDCl3) d 8.65 (s, 8H, H1), 7.68 – 7.63 (tt, J = 7.7, 1.3 Hz, 4H, H4), 7.55 (t, J = 7.6 Hz, 8H, H3), 7.39 (dd, J = 8.1, 1.2 Hz, 8H, H2), 6.84 (tt, J = 7.4, 1.4 Hz, 6H, H7), 6.53 (t, J = 7.6 Hz, 12H, H6), 4.67 – 4.59 (dd, J = 7.6, 1.2 Hz, 12H, H5). 13C NMR (101 MHz, CDCl3) d 117.9, 126.4, 126.5, 127.6, 127.8, 130.9, 132.9, 143.6, 140.8, 134.4, 132.9, 130.9, 127.8, 127.6, 126.5, 126.4, 117.9. IR (cm-1) 3049, 1596, 1490, 1355, 1207, 1104, 1060, 1009, 852, 798, 745, 697. UV-Vis (CH2Cl2): λmax (nm) 425 (Soret band), 515, 553, 592, 633 (Q bands); Fluorescence (CH2Cl2, λex = 427): λem (nm) 602, 645. HRMS-ASAP m/z [M+H]+ calculated for C80H59N4O2Si3 1191.3945, measured 1191.3917. 1. General Methods The reaction mixture was then refluxed for 1 h. The reaction mixture was allowed to cool to rt and then MeOH (40 mL) was poured into the flask. The mixture was stirred at rt overnight. The crude solid was isolated by vacuum filtration and was washed with hot MeOH yield 2 as a fine, purple solid (0.62 g, 1.00 mmol, 27%). 1H NMR (400 MHz, CDCl3) d 8.86 (s, 8H, H1), 8.23 (d, 8H, H2), 7.77 (m, 12H, H3, H4), -2.74 (s, 2H, H5). 13C NMR (101 MHz, CDCl3) d 142.4, 134.8, 131.4 (br) , 127.9, 126.9, 120.4. UV-Vis (CH2Cl2): λmax (nm) 417 (Soret band), 514, 549, 590, 646 (Q bands). Fluorescence (CH2Cl2): λem (nm) 650, 719. Spectroscopic data are consistent with those reported previously.1 1 h. The reaction mixture was allowed to cool to rt and then MeOH (40 mL) was poured into the flask. The mixture was stirred at rt overnight. The crude solid was isolated by vacuum filtration and was washed with hot MeOH yield 2 as a fine, purple solid (0.62 g, 1.00 mmol, 27%). 1H NMR (400 MHz, CDCl3) d 8.86 (s, 8H, H1), 8.23 (d, 8H, H2), 7.77 (m, 12H, H3, H4), -2.74 (s, 2H, H5). 13C NMR (101 MHz, CDCl3) d 142.4, 134.8, 131.4 (br) , 127.9, 126.9, 120.4. UV-Vis (CH2Cl2): λmax (nm) 417 (Soret band), 514, 549, 590, 646 (Q bands). Fluorescence (CH2Cl2): λem (nm) 650, 719. Spectroscopic data are consistent with those reported previously.1 3: In a 50 mL round-bottom flask with a magnetic stir bar, 4 (100 mg, 0.14 mmol) was dissolved in THF (15 ml) and distilled H2O (5 mL). The reaction mixture was refluxed for 2 h, monitoring the conversion of starting material by TLC. Upon completion, the reaction mixture was washed with a saturated aqueous solution of NaHCO3 (30 mL) followed by brine (30 mL). The organic layers were dried over MgSO4, filtered and concentrated in vacuo to yield 3 as a purple solid (95 mg, 0.14 mmol, 98%). 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 8H, H1), 8.23 (d, 8H, H2), 7.76 (m, 12H, H3, H4). 13C NMR (101MHz, CDCl3) δ 142.5, 134.9, 128.0, 127.0, 120.4. IR (cm-1) 2918, 2852, 2322, 2102, 2079, 1891, 1595, 1526, 1489, 1439, 1353, 1204, 1071, 1007, 828, 800, 750, 698. 1. General Methods CV (V vs NHE, ref to Fc/Fc+)) - 1 128 1 340 1i: In a 50 mL round-bottom flask with a magnetic stir bar, 3 (100 mg, 0.15 mmol) was dissolved in 1,2-dichloroethane (10 mL) to which Et3N (50 µL g, 0.37 mmol) was added. The reaction mixture was stirred for 5 min, followed by the addition of triphenylchlorosilane (109 mg, 0.37 mmol). The reaction mixture was refluxed overnight and monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo and the residue was purified using a short silica plug (petroleum ether:Et2O:Et3N, 5:2:1), Rf = 0.63. The filtrate was then triturated using cold EtOH until no further precipitation occurred. The solid was filtered through a sintered glass funnel which was then dried under vacuum to yield 1i as a purple solid (150 mg, 0.13 mmol, 85%). 1H NMR (400 MHz, CDCl3) d 8.65 (s, 8H, H1), 7.68 – 7.63 (tt, J = 7.7, 1.3 Hz, 4H, H4), 7.55 (t, J = 7.6 Hz, 8H, H3), 7.39 (dd, J = 8.1, 1.2 Hz, 8H, H2), 6.84 (tt, J = 7.4, 1.4 Hz, 6H, H7), 6.53 (t, J = 7.6 Hz, 12H, H6), 4.67 – 4.59 (dd, J = 7.6, 1.2 Hz, 12H, H5). 13C NMR (101 MHz, CDCl3) d 117.9, 126.4, 126.5, 127.6, 127.8, 130.9, 132.9, 143.6, 140.8, 134.4, 132.9, 130.9, 127.8, 127.6, 126.5, 126.4, 117.9. IR (cm-1) 3049, 1596, 1490, 1355, 1207, 1104, 1060, 1009, 852, 798, 745, 697. UV-Vis (CH2Cl2): λmax (nm) 425 (Soret band), 515, 553, 592, 633 (Q bands); Fluorescence (CH2Cl2, λex = 427): λem (nm) 602, 645. HRMS-ASAP m/z [M+H]+ calculated for C80H59N4O2Si3 1191.3945, measured 1191.3917. CV (V vs NHE, ref to Fc/Fc+)) - 1.128, 1.340. 12 2: In a 250 mL round-bottom flask with a magnetic stir bar, benzaldehyde (1.74 g, 16.4 mmol) was added into propionic acid (30 mL). The mixture was heated to reflux for 15 min. Pyrrole (1.00 g, 14.9 mmol) was added dropwise to the reaction mixture over a period of 5 min. The reaction mixture was then refluxed for 2: In a 250 mL round-bottom flask with a magnetic stir bar, benzaldehyde (1.74 g, 16.4 mmol) was added into propionic acid (30 mL). The mixture was heated to reflux for 15 min. Pyrrole (1.00 g, 14.9 mmol) was added dropwise to the reaction mixture over a period of 5 min. 1. General Methods UV-Vis (CH2Cl2): λmax (nm) 418 3: In a 50 mL round-bottom flask with a magnetic stir bar, 4 (100 mg, 0.14 mmol) was dissolved in THF (15 ml) and distilled H2O (5 mL). The reaction mixture was refluxed for 2 h, monitoring the conversion of starting material by TLC. Upon completion, the reaction mixture was washed with a saturated aqueous solution of NaHCO3 (30 mL) followed by brine (30 mL). The organic layers were dried over MgSO4, filtered and concentrated in vacuo to yield 3 as a purple solid (95 mg, 0.14 mmol, 98%). 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 8H, H1), 8.23 (d, 8H, H2), 7.76 (m, 12H, H3, H4). 13C NMR (101MHz, CDCl3) δ 142.5, 134.9, 128.0, 127.0, 120.4. IR (cm-1) 2918, 2852, 2322, 2102, 2079, 1891, 1595, 1526, 1489, 1439, 1353, 1204, 1071, 1007, 828, 800, 750, 698. UV-Vis (CH2Cl2): λmax (nm) 418 13 446, (Soret band), 446, 514, 550, 590, 649 (Q bands). Fluorescence (CH2Cl2): λem (nm) Spectroscopic data are consistent with those reported previously.2 TPP-SiCl2: In a 250 mL round-bottom flask with a magnetic stir bar, 2 (100 mg, 0.16 mmol) was dissolved in anhydrous CH2Cl2 (100 mL). Et3N (36.2 mg, 0.358 mmol) was added and the mixture was stirred for 5 min. Next, trichlorosilane (26 mg, 0.20 mmol) was added and the reaction was stirred at rt for 24 h. The mixture TPP-SiCl2: In a 250 mL round-bottom flask with a magnetic stir bar, 2 (100 mg, 0.16 mmol) was dissolved in anhydrous CH2Cl2 (100 mL). Et3N (36.2 mg, 0.358 mmol) was added and the mixture was stirred for 5 min. Next, trichlorosilane (26 mg, 0.20 mmol) was added and the reaction was stirred at rt for 24 h. The mixture was cooled to 0 °C and quenched by adding H2O (10 mL). The reaction mixture was washed with a saturated aqueous solution of NaHCO3 (2 × 50 mL) followed by brine (50 mL). The organic layer was dried over MgSO4, filtered and concentrated in vacuo to yield TPP-SiCl2 as a purple solid (99 mg, 0.14 mmol, 85%). 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 8H, H1), 8.14 (m, 8H, H2), 7.74 (m, 12H, H3, H4). 13C NMR (101MHz, CDCl3) δ 117.7, 127.2, 128.1, 131.8, 134.3, 140.6, 143.5. Spectroscopic data are consistent with those reported previously.3 was cooled to 0 °C and quenched by adding H2O (10 mL). 1. General Methods The reaction mixture was washed with a saturated aqueous solution of NaHCO3 (2 × 50 mL) followed by brine (50 mL). The organic layer was dried over MgSO4, filtered and concentrated in vacuo to yield TPP-SiCl2 as a purple solid (99 mg, 0.14 mmol, 85%). 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 8H, H1), 8.14 (m, 8H, H2), 7.74 (m, 12H, H3, H4). 13C NMR (101MHz, CDCl3) δ 117.7, 127.2, 128.1, 131.8, 134.3, 140.6, 143.5. Spectroscopic data are consistent with those reported previously.3 14 3. 1H, 13C, and Variable-Temperature NMR Spectroscopic Characterization of Synthesized Compounds Figure S1. 1H NMR spectrum of 1a (400 MHz, CDCl3, 298 K). 3. 1H, 13C, and Variable-Temperature NMR Spectroscopic Characterization of Synthesized Compounds re NMR Spectroscopic Characterization of Synthesized Compounds 3. 1H, 13C, and Variable-Temperature NMR Spectroscopic Characterization of Synthesized Compounds 3C, and Variable-Temperature NMR Spectroscopic Characterization o Figure S1. 1H NMR spectrum of 1a (400 MHz, CDCl3, 298 K). 15 15 Figure S2. 13C NMR spectrum of 1a (101 MHz, CDCl3, 298 K). Figure S2. 13C NMR spectrum of 1a (101 MHz, CDCl3, 298 K). 16 Figure S3. 1H NMR spectrum of 1b (400 MHz, CDCl3, 298 K). Figure S3. 1H NMR spectrum of 1b (400 MHz, CDCl3, 298 K). 17 17 Figure S4. 13C NMR spectrum of 1b (101MHz, CDCl3, 298 K). Figure S4. 13C NMR spectrum of 1b (101MHz, CDCl3, 298 K). 18 gure S5. 1H NMR spectrum of 1c (400 MHz, CDCl3, 298 K). Figure S5. 1H NMR spectrum of 1c (400 MHz, CDCl3, 298 K). 19 Figure S6. 13C NMR spectrum of 1c (101MHz, CDCl3, 298 K). Figure S6. 13C NMR spectrum of 1c (101MHz, CDCl3, 298 K). 20 igure S7. 1H NMR spectrum of 1d (400 MHz, CDCl3, 298 K). Figure S7. 1H NMR spectrum of 1d (400 MHz, CDCl3, 298 K). DCl3, 298 K). 21 21 Figure S8. 13C NMR spectrum of 1d (101 MHz, CDCl3, 298 K). Figure S8. 13C NMR spectrum of 1d (101 MHz, CDCl3, 298 K). 22 22 gure S9. 1H NMR spectrum of 1e (400 MHz, CDCl3, 298 K). Figure S9. 1H NMR spectrum of 1e (400 MHz, CDCl3, 298 K). 23 igure S10. 13C NMR spectrum of 1e (101 Hz, CDCl3, 298 K). Figure S10. 13C NMR spectrum of 1e (101 Hz, CDCl3, 298 K). 24 24 gure S11. 1H NMR spectrum of 1f (400 MHz, CDCl3, 298 K). Figure S11. 1. General Methods 1H NMR spectrum of 1f (400 MHz, CDCl3, 298 K). 25 Figure S12. 13C NMR spectrum of 1f (101MHz, CDCl3, 298 K). Figure S12. 13C NMR spectrum of 1f (101MHz, CDCl3, 298 K). 26 gure S13. 1H NMR spectrum of 1g (400 MHz, CDCl3, 298 K). ure S13. 1H NMR spectrum of 1g (400 MHz, CDCl3, 298 K). Figure S13. 1H NMR spectrum of 1g (400 MHz, CDCl3, 298 K). l3, 298 K). 27 27 ure S14. 13C NMR spectrum of 1g (101MHz, CDCl3, 298 K). Figure S14. 13C NMR spectrum of 1g (101MHz, CDCl3, 298 K). 28 gure S15. 1H NMR spectrum of 1h (400 MHz, CDCl3, 298 K). Figure S15. 1H NMR spectrum of 1h (400 MHz, CDCl3, 298 K). 29 e S16. 13C NMR spectrum of 1h (101 MHz, CDCl3, 298 K). Figure S16. 13C NMR spectrum of 1h (101 MHz, CDCl3, 298 K). 30 Figure S17. 1H NMR spectrum of 1i (400 MHz, CDCl3, 298 K). Figure S17. 1H NMR spectrum of 1i (400 MHz, CDCl3, 298 K). 31 S18. 13C NMR spectrum of 1i (101MHz, CDCl3, 298 K). Figure S18. 13C NMR spectrum of 1i (101MHz, CDCl3, 298 K). 32 32 3.1. Variable-Temperature (VT) NMR Spectroscopy (Metallo)porphyrins are known to exhibit a variety of conformational dynamics in solution, and the dynamic processes associated with porphyrin ligands include macrocyclic inversion meso- and b-substituent rotation.4 In order to investigate this phenomenon, we performed VT NMR measurements to determine conformational dynamics of these SOPS. A series of spectra were recorded for a solution (CDCl2) of 1f ranging from 193 K to 298 K. Figure S19. Partial 1H NMR spectra of 1f, recorded from 298 K to 193 K. Figure S19. Partial 1H NMR spectra of 1f, recorded from 298 K to 193 K. 33 4. UV-Vis Spectroscopy The spectra in Figure S20 shows the UV-Vis absorption features of the SOPS (1a-1i) in their characteristic Soret band and Q band regions. Spectra were measured in a 10 mm path-length cuvette at room temperature. 10 µM Sample concentrations in anhydrous CH2Cl2 were used for all measurements. The intensities were plotted as absorption (arbitrary units). Concentration effects on the Q band region for compounds (1a-1i) are plotted (Figure S21-29) and shows a linear concentration between absorbance and concentration for all compounds, demonstrating no aggregation occurs in solutions at the measured concentrations. Figure S20. UV-Vis spectra of the SOPS (1a-1i) a) in the Soret band region and b) in the Q band region. ctra of the SOPS (1a-1i) a) in the Soret band region and b) in the Q band region. -Vis spectra of the SOPS (1a-1i) a) in the Soret band region and b) in the Q band region. Figure S20. UV-Vis spectra of the SOPS (1a-1i) a) in the Soret band region and b) in the Q band region. Figure S20. UV-Vis spectra of the SOPS (1a-1i) a) in the Soret band region and b) in the Q 34 Figure S21. Plot of Q band lmax at different concentrations for compound 1a in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q Figure S21. Plot of Q band lmax at different concentrations for compound 1a in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S21. Plot of Q band lmax at different concentrations for compound 1a in CH2Cl2. a) 35 Figure S22. Plot of Q band lmax at different concentrations for compound 1b in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S22. Plot of Q band lmax at different concentrations for compound 1b in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. lmax at different concentrations for compound 1b in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. 36 Figure S23. Plot of Q band lmax at different concentrations for compound 1c in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S23. 4. UV-Vis Spectroscopy Plot of Q band lmax at different concentrations for compound 1c in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. 37 37 Figure S24. Plot of Q band lmax at different concentrations for compound 1d in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q Figure S24. Plot of Q band lmax at different concentrations for compound 1d in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S24. Plot of Q band lmax at different concentrations for compound 1d in CH2Cl2. a) 38 Figure S25. Plot of Q band lmax at different concentrations for compound 1e in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4 Figure S25. Plot of Q band lmax at different concentrations for compound 1e in CH2Cl2. a) Figure S25. Plot of Q band lmax at different concentrations for compound 1e in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. 39 Figure S26. Plot of Q band lmax at different concentrations for compound 1f in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q Figure S26. Plot of Q band lmax at different concentrations for compound 1f in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. f Q band lmax at different concentrations for compound 1f in CH2Cl2. a) lmax of Q1, b) lmax o Figure S26. Plot of Q band lmax at different concentrations for compound 1f in CH2Cl2. a) l tions for compound 1f in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. 40 Figure S27. Plot of Q band lmax at different concentrations for compound 1g in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q Figure S27. Plot of Q band lmax at different concentrations for compound 1g in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. 41 Figure S28. Plot of Q band lmax at different concentrations for compound 1h in CH2Cl2. 4. UV-Vis Spectroscopy a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S28. Plot of Q band lmax at different concentrations for compound 1h in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S28. Plot of Q band lmax at different concentrations for compound 1h in CH2Cl2. a) tions for compound 1h in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4 42 Figure S29. Plot of Q band lmax at different concentrations for compound 1i in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4 Figure S29. Plot of Q band lmax at different concentrations for compound 1i in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. Figure S29. Plot of Q band lmax at different concentrations for compound 1i in CH2Cl2. a) l tions for compound 1i in CH2Cl2. a) lmax of Q1, b) lmax of Q2, c) lmax of Q3, and d) lmax of Q4. 43 5. Photoluminescence Spectroscopy Samples for room temperature fluorescence spectroscopy (Figure S30) were prepared as 10 µM CH2Cl2 solutions. The solid-state photoluminescence of PorSils 1a, 1b, 1i and TPP-H2 were also measured at room temperature by preparing dispersions (1 wt% films) in an optically clear ZEONEX® Cyclo Olefin Polymer (COP) matrix or optically clear PMMA. Films were prepared by drop casting solutions of the polymer and SOPS. The results are shown in Figures S31-32. Sample 1i for VT fluorescence was measured as a 1 µM 2-MeTHF solution and as an optically clear ZEONEX Cyclo Olefin Polymer (COP) matrix. 2-Methyltetrahydrofuran (2-MeTHF) was used as the solvent system, because it forms a stable organic glass which is UV-Vis transparent at low temperatures. Both solution- and solid-state samples were kept at each temperature for at least 10 min to equilibrate prior to recording the spectra. The results of VT fluorescence are summarized in Figure S33. 44 44 Figure S30. Normalized emission profiles acquired for solutions of SOPS (1a-1i), parent 2, and 3 in CH2Cl2. Figure S30. Normalized emission profiles acquired for solutions of SOPS (1a-1i), parent 2, and 3 in CH2Cl2. 45 Figure S31. Normalized emission profiles of a) 1a (SiEt3) and b) 1d (SiiPr3) dispersed in optically clear amorphous polymer films (1 wt%) are contrasted with the emission in CH2Cl2 (10 μM). Figure S31. Normalized emission profiles of a) 1a (SiEt3) and b) 1d (SiiPr3) dispersed in optically clear amorphous polymer films (1 wt%) are contrasted with the emission in CH2Cl2 (10 μM). 46 Figure S32. Normalized emission profiles of a) 1i (SiPh3) and b) 2 (TPP-H2) dispersed in optically clear amorphous polymer films (1 wt%) are contrasted with the emission in CH2Cl2 (10 μM). Figure S32. Normalized emission profiles of a) 1i (SiPh3) and b) 2 (TPP-H2) dispersed in optically clear amorphous polymer films (1 wt%) are contrasted with the emission in CH2Cl2 (10 μM). 47 47 Figure S33. VT fluorescence spectra of 2-MeTHF solutions (l = 10 mm, T = 80–290 K) of 1i λex = 425 nm, c = 1 μM; Inset in panel (a): Legend for temperatures in all panels uorescence spectra of 2-MeTHF solutions (l = 10 mm, T = 80–290 K) of 1i λex = 425 nm, c = 1 μM; Inset in panel (a): Legend for all panels Figure S33. 5. Photoluminescence Spectroscopy VT fluorescence spectra of 2-MeTHF solutions (l = 10 mm, T = 80–290 K) of 1i λex = 425 nm, c = 1 μM; Inset in panel (a): Legend for temperatures in all panels 48 Table S1. Raw CV data seen in Figures S35-40, and values derived by calibration to oxidation of ferrocene. 6. Cyclic Voltammetry The cyclic voltammetry (CV) measurements were carried out with Gamry Interface 1000 potentiostat/galvanostat controlled by an external computer and utilizing a three-electrode configuration at 25 ℃. The working electrode was a Pt disc with a surface area of 0.071 cm2. A Pt wire served as the counter electrode. Saturated calomel electrode (SCE) was employed as the reference electrode and separated from the bulk of the solution by a double bridge. Electrochemical grade NBu4PF6 in anhydrous degassed CH2Cl2 was employed as the supporting electrolyte at a concentration of 0.1 M. Figure S34. Redox potentials of SOPS (1a–1i), 2 (TPP-H2) and 3. Data were collected using a 0.1 M solution of NBu4PF6 in CH2Cl2 at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. 2 has a second oxidation peak at 1.65 V. Figure S34. Redox potentials of SOPS (1a–1i), 2 (TPP-H2) and 3. Data were collected using a 0.1 M solution of NBu4PF6 in CH2Cl2 at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. 2 has a second oxidation peak at 1.65 V. x potentials of SOPS (1a–1i), 2 (TPP-H2) and 3. Data were collected using a 0.1 M solution of NBu4PF6 in CH2Cl2 at 100 mV s-1 to l standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. 2 has a second oxidation peak at 1.65 V. 49 Table S1. Raw CV data seen in Figures S35-40, and values derived by calibration to oxidation of ferrocene. Table S1. Raw CV data seen in Figures S35-40, and values derived by calibration to oxidation of ferrocene. 50 Figure S35. CV of a) 1a and b) 1b. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). a b b b a a Figure S35. CV of a) 1a and b) 1b. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). 51 51 b Figure S36. CV of a) 1c, and b) 1d. 6. Cyclic Voltammetry Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). a b b a a Figure S36. CV of a) 1c, and b) 1d. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). 52 Figure S37. CV of a) 1e, and b) 1f. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). a b b b a a Figure S37. CV of a) 1e, and b) 1f. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). 53 53 Figure S38. CV of a) 1g, and b) 1h. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2 . Reference oxidation peaks (ferrocene). a b b a Figure S38. CV of a) 1g, and b) 1h. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2 . Reference oxidation peaks (ferrocene). 54 Figure S39. CV of 1i. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). Figure S39. CV of 1i. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2. Reference oxidation peaks (ferrocene). 55 Figure S40. CV of a) 2, and b) 3. Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2 . Reference oxidation peaks (ferrocene). a b b b a a Figure S40. CV of a) 2, and b) 3. 6. Cyclic Voltammetry Data were collected using 0.1 MNBu4PF6 CH2Cl2 solutions at 100 mV s-1 to [Fc]/[Fc+] internal standard followed by conversion to NHE; [Fc]/[Fc+] = +765 mV vs. NHE in CH2Cl2 . *Reference oxidation peaks (ferrocene). 56 7. X-Ray Crystallographic Analysis Suitable single crystals of all four compounds 1f-i were mounted on a cryoloop with paratone oil and examined on a Bruker APEX-II CCD diffractometer equipped with a CCD area detector and an Oxford Cryoflex low temperature device. Data were collected at 150(2) K with Mo Kα radiation (λ = 0.71073 Å) using the APEX-II software.5 Cell refinement and data-reduction were carried out by SAINT. An absorption correction was performed by multi-scan method Suitable single crystals of all four compounds 1f-i were mounted on a cryoloop with paratone oil and examined on a Bruker APEX-II CCD diffractometer equipped with a CCD area detector and an Oxford Cryoflex low temperature device. Data were collected at 150(2) K with Mo Kα radiation (λ = 0.71073 Å) using the APEX-II software.5 Cell refinement and data-reduction were carried out by SAINT. An absorption correction was performed by multi-scan method implemented in SADABS.6 The structures of the complexes were solved by (SHELXT)7 and refined using SHELXL-2014 in the Bruker SHELXTL suite.8 Hydrogen atoms were added at calculated positions and refined with a riding model. In compound 1g, disordered solvent was removed using the SQUEEZE procedure in PLATON.9 Selected crystallographic data for the complexes are presented in Table S2. All four compounds have been deposited in the Cambridge Structural Database with numbers CCDC 1977452-1977455. 57 57 Table S2. Summary of select crystallographic data for compounds 1f-1i Compound 1f 1g 1h 1i Chemical formula C60H50N4O2Si3 C70H54N4O2Si3 C76H66N4O2Si3 C80H58N4O2Si3 Mr 943.31 1067.44 1151.59 1191.57 Crystal system, space group Monoclinic, P21/n Monoclinic, P21/n Tetragonal, I41/a Triclinic, P¯1 Temperature (K) 150 150 150 150 a, b, c (Å) 12.8165 (9), 22.7137 (14), 17.6229 (11) 11.6001 (6), 12.5673 (6), 21.2309 (11) 31.4939 (8), 12.3443 (3) 12.8070 (8), 14.8522 (9), 18.6822 (11) V (Å3) 5010.2 (6) 3066.6 (3) 12243.9 (7) 3122.9 (3) Z 4 2 8 2 Radiation type Mo Kα Mo Kα Mo Kα Mo Kα µ (mm−1) 0.14 0.13 0.13 0.13 Crystal size (mm) 0.15 × 0.1 × 0.05 0.23 × 0.10 × 0.04 0.39 × 0.27 × 0.27 0.12 × 0.06 × 0.03 Absorption correction Multi-scan Bruker SADABS Multi-scan Bruker SADABS Multi-scan Bruker SADABS Multi-scan Bruker SADABS Tmin, Tmax 0.640, 0.736 0.536, 0.786 0.945, 0.960 0.672, 0.739 No. 7. X-Ray Crystallographic Analysis of measured, independent, and observed [I > 2σ(I)] reflections 61068, 10069, 7509 38881, 5387, 4883 96129, 5701, 4924 83666, 10990, 9054 Rint 0.083 0.043 0.049 0.085 (sin θ/λ)max (Å−1) 0.630 0.594 0.667 0.595 R[F2 > 2σ(F2)], wR(F2), S 0.085, 0.165, 1.08 0.048, 0.154, 0.64 0.050, 0.166, 1.09 0.107, 0.184, 1.28 No. of reflections 10069 5387 5701 10990 No. of parameters 626 359 388 781 H-atom treatment H-atom parameters constrained H-atom parameters constrained H-atom parameters constrained H-atom parameters constrained Δρmax, Δρmin (e Å−3) 0.33, −0.47 0.36, −0.54 0.26, −0.28 0.58, −0.57 58 Figure S41. Molecular structure of 1f with appropriate labelling scheme. H-atoms are removed for clarity. Figure S41. Molecular structure of 1f with appropriate labelling scheme. H-atoms are removed for clarity. Figure S42. Solid-state superstructure of 1f viewed along the three unit cell axes. a - axis b - axis c - axis c - axis a - axis Figure S42. Solid-state superstructure of 1f viewed along the three unit cell axes. 59 Figure S43. Molecular structure of 1g with appropriate atomic labelling scheme. H-atoms are omitted for clarity. Figure S43. Molecular structure of 1g with appropriate atomic labelling scheme. H-atoms are omitted for clarity. Figure S44. Solid-state superstructure of 1g viewed along the three unit cell axes. a - axis b - axis c - axis a - axis a - axis c - axis c - axis Figure S44. Solid-state superstructure of 1g viewed along the three unit cell axes. 60 Figure S45. Molecular structure of 1h with appropriate atomic labelling scheme. H-atoms are omitted for clarity. Figure S45. Molecular structure of 1h with appropriate atomic labelling scheme. H-atoms are omitted for clarity. Figure S45. Molecular structure of 1h with appropriate atomic labelling scheme. H-atoms are omitted for clarity. Figure S46. Solid-state superstructure of 1h viewed along the three unit cell axes. a - axis b - axis c - axis c - axis a - axis c - axis Figure S46. Solid-state superstructure of 1h viewed along the three unit cell axes. 61 Figure S47. Molecular structure of the two crystallographically independent molecules of 1i with appropriate atomic labelling schemes. H-atoms are omitted for clarity. Figure S47. Molecular structure of the two crystallographically independent molecules of 1i with appropriate atomic labelling schemes. H-atoms are omitted for clarity. Figure S48. Solid-state superstructure of 1iʹ/1iʹʹ viewed along the three unit cell axes. a - axis b - axis c - axis s b - axis c - axis a - axis a - axis Figure S48. Solid-state superstructure of 1iʹ/1iʹʹ viewed along the three unit cell axes. 62 7.1 Intramolecular Contacts in Solid State The silyloxy caps can interact with the porphyrins by both C-H··· π and face to face aromatic interactions. Figures S49-53 illustrates the intramolecular contacts in the crystal structures of the aryl silyloxy cap 1f-1i are within or shorter than the van der Waals radius. 63 Figure S49. a) Structural formula of 1f where centroids of rings are colored to match crystal structure centroids. b), c), d), and e) Four views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S49. a) Structural formula of 1f where centroids of rings are colored to match crystal structure centroids. b), c), d), and e) Four views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). 64 Figure S50. a) Structural formula of 1g where centroids of rings are colored to match crystal structure centroids. b), c), and d) Four views of the solid-state structure showing intramolecular interactions of silyoxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S50. a) Structural formula of 1g where centroids of rings are colored to match crystal structure centroids. b), c), and d) Four views of the solid-state structure showing intramolecular interactions of silyoxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). 65 Figure S51. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. b), c), d), e), and f) Five views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S51. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. b), c), d), e), and f) Five views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S51. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. 7.1 Intramolecular Contacts in Solid State b), c), d), e), and f) Five views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S51. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. b), c), d), e), and f) Five views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). 66 Figure S52. a) Structural formula of 1iʹ where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Three views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S52. a) Structural formula of 1iʹ where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Three views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). 67 Figure S53. a) Structural formula of 1iʹʹ where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Three views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S53. a) Structural formula of 1iʹʹ where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Three views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). Figure S53. a) Structural formula of 1iʹʹ where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Three views of the solid-state structure showing intramolecular interactions of silyloxy cap with the porphyrin ring within/shorter than the sum of the van der Waals radii (≤ 3.8 Å). 68 8. In Silico Modelling X-ray diffraction analysis shows that the porphyrin rings of 1f–1i adopt a range of distorted conformations in solid state from ruffled to planar geometries. To determine the more stable conformation of these SOPS in solution, density functional theory (DFT) modelling was used to gain better insight into their conformational energetics. Select SOPs were optimized in Gaussian 09, Revision A.02,10 to minima (confirmed by frequency calculations on the same level of theory having no imaginary frequency) using the M06–2X functional, 6-31G basis set, and a C-PCM solvent model for CH2Cl2 (chosen as it is the solvent used for the majority of the physicochemical analysis). The calculations reveal (Figures S54–55) that the minimum energy geometries for all the alkyl (1a, 1c, 1e) and aryl (1f, 1g) SOPS we modelled include a ruffled porphyrin core. The steric and electronic interactions between the silyloxy cap and the porphyrin ring system change the preferred porphyrin geometry from planar to nonplanar. 69 Figure S54. The DFT minimized structures in CH2Cl2 compared to the X-Ray structure of a) 1f, b) 1g at M06–2X, using 6-31G basis set and a C-PCM solvent model for CH2Cl2. Figure S54. The DFT minimized structures in CH2Cl2 compared to the X-Ray structure of a) 1f, b) 1g at M06–2X, using 6-31G basis set and a C-PCM solvent model for CH2Cl2. Figure S55. The DFT minimized structures in CH2Cl2 of a) 1a, b) 1e, and c) 1c at M06–2X, using 6-31G basis set and a C-PCM solvent model for CH2Cl2. Figure S55. The DFT minimized structures in CH2Cl2 of a) 1a, b) 1e, and c) 1c at M06–2X, using 6-31G basis set and a C-PCM solvent model for CH2Cl2. Figure S55. The DFT minimized structures in CH2Cl2 of a) 1a, b) 1e, and c) 1c at M06–2X, using 6-31G basis set and a C-PCM solvent model for CH2Cl2. 70 9. Hirshfeld Surface Analysis of Solid-State Structures Hirshfeld surfaces11 give insights into the interactions between molecules in the crystal lattices. We calculated Hirshfeld surfaces for the crystal packing structures of 1f-1i′/1i′′ in CrystalExplorer1712 using an isovalue of 0.5 and mapping the normalized contact distance, dnorm. The surfaces highlight (Figures S56–61) in red any regions in which the molecular surfaces meet at distances shorter than the sum of van der Waals radii, while white and blue illustrate regions where they meet at distances that are the sum of the van der Waals radii or longer, respectively. Figure S56. Hirshfeld surface of 1f, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S56. Hirshfeld surface of 1f, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. 71 Figure S57. Hirshfeld surface of 1g, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S57. Hirshfeld surface of 1g, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. 72 Figure S58. Hirshfeld surface of 1h, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S58. Hirshfeld surface of 1h, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. 73 Figure S59. Hirshfeld surface of 1iʹ, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S59. Hirshfeld surface of 1iʹ, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S59. Hirshfeld surface of 1iʹ, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. 74 Figure S60. 9. Hirshfeld Surface Analysis of Solid-State Structures Hirshfeld surface of 1iʹʹ, showing the most significant close contacts, as well as views of t surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S60. Hirshfeld surface of 1iʹʹ, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. Figure S60. Hirshfeld surface of 1iʹʹ, showing the most significant close contacts, as well as views of the surface along the crystallographic a) and b) a-axis, and c) and d) b-axis. 75 Figure S61. Calculated Hirshfeld fingerprints for the X-ray crystal structures of the aryl SOPS. 1iʹʹ 1iʹ Figure S61. Calculated Hirshfeld fingerprints for the X-ray crystal structures of the aryl SOPS. 1iʹʹ 1iʹ 1iʹʹ 1iʹ Figure S61. Calculated Hirshfeld fingerprints for the X-ray crystal structures of the aryl SOPS. 76 Figure S62. a) Structural formula of 1f where centroids of rings are colored to match crystal structure centroids. b), c), d), and e) Intermolecular interactions of 1f within/shorter than van der Waals radius (< 3.8 Å). The 1f structure determinant pair was found using crystal explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of he isolated molecule. Figure S62. a) Structural formula of 1f where centroids of rings are colored to match crystal structure centroids. b), c), d), and e) Intermolecular interactions of 1f within/shorter than van der Waals radius (< 3.8 Å). The 1f structure determinant pair was found using crystal explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. 77 Figure S63. a) Structural formula of 1g where centroids of rings are colored to match crystal stru centroids. b), c), and d) Intermolecular interactions of 1g within/shorter than Vander Waals radius ( Å). The 1g structure determinant pair was found using Crystal Explorer using the Hirshfeld surface most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surfa th i l t d l l Figure S63. a) Structural formula of 1g where centroids of rings are colored to match crystal struc centroids. b), c), and d) Intermolecular interactions of 1g within/shorter than Vander Waals radius (< Å). The 1g structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. 9. Hirshfeld Surface Analysis of Solid-State Structures most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surfac the isolated molecule. Figure S63. a) Structural formula of 1g where centroids of rings are colored to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1g within/shorter than Vander Waals radius (< 3.8 Å). The 1g structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. Figure S63. a) Structural formula of 1g where centroids of rings are colored to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1g within/shorter than Vander Waals radius (< 3.8 Å). The 1g structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. 78 Figure S64. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1h within/shorter than Vander Waals radius (< 3.8 Å). The 1h structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. Figure S64. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1h within/shorter than Vander Waals radius (< 3.8 Å). The 1h structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. Figure S64. a) Structural formula of 1h where centroids of rings are colored coded to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1h within/shorter than Vander Waals radius (< 3.8 Å). The 1h structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. 79 Figure S65. a) Structural formula of 1i where centroids of rings are colored to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1i/1iʹ within/shorter than van der Waals radius (< 3.8 Å). 9. Hirshfeld Surface Analysis of Solid-State Structures The 1iʹ/1iʹʹ structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. Figure S65. a) Structural formula of 1i where centroids of rings are colored to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1i/1iʹ within/shorter than van der Waals radius (< 3.8 Å). The 1iʹ/1iʹʹ structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. Figure S65. a) Structural formula of 1i where centroids of rings are colored to match crystal structure centroids. b), c), and d) Intermolecular interactions of 1i/1iʹ within/shorter than van der Waals radius (< 3.8 Å). The 1iʹ/1iʹʹ structure determinant pair was found using Crystal Explorer using the Hirshfeld surface. The most dominant structure determinant pair is shown using Crystal Explorer with the Hirshfeld surface of the isolated molecule. 80 10. Calculation of Intermolecular Interaction Energies in the Solid State We performed DFT calculations to quantify the interaction energies between neighboring molecules in the solid state using the CE-HF [HF/3-21G] theory and CE-B3LYP [B3LYP/6- 31G(d,p)] energy model in CrystalExplorer17. Based on the X-ray crystal structure coordinates, a cluster was generated around a central molecule, extending to molecules that come within 3.8 Å of the central molecule at any point. As each of the crystal structures of 1f-h have only one unique molecule in the unit cell (Zʹ = 1), all of the surrounding molecules are related to the central molecule by a symmetry operation (labelled ‘Symmetry Op.’ in Tables S3–12). For the B3LYP [B3LYp/6-31G(d,p)] energy calculation, only the two most dominant structure determinant pairs were selected due to computational cost. Analysis was performed individually for the 1i′/1i′′ conformers present in the unit cell of the triphenyl derivative, generating a cluster of molecules around one of the conformers to model its local interactions. These energy calculations allow us to elucidate the total interaction energies (Etot, in kJ·mol−1) between neighboring molecules, based on the individual components for electrostatic (Eele), polarization (Epol), dispersion (Edis), and repulsion (Erep) energies. In each of the tables below, N indicates the number of molecules of a particular symmetry operation included in the cluster. R is the distance between the molecular centroids (mean atomic position) of the central molecule and the molecule generated by the given symmetry operation. A color code is given for each table entry, which matches the coloring of the relevant molecules in the corresponding cluster diagrams, Figures S66–70. 81 Table S3. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1f at CE-HF-321G theory. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x, y, z 12.82 -13.1 -2.4 -47.8 26.1 -36.9 2 x+1/2, -y+1/2, z+1/2 9.81 -4.9 -5.2 -98.8 39.6 -65.2 2 x+1/2, -y+1/2, z+1/2 12.04 -8.5 -4.8 -75.5 30.8 -54.9 2 -x+1/2, y+1/2, -z+1/2 13.07 -9.2 -2.7 -43.1 24.0 -30.5 2 -x+1/2, y+1/2, -z+1/2 15.46 -4.5 -1.2 -18.9 6.1 -17.4 1 -x, -y, -z 13.18 -11.2 -1.8 -37.0 15.4 -33.4 1 -x, -y, -z 10.56 -7.0 -2.1 -69.0 22.4 -52.5 1 -x, -y, -z 17.18 -2.6 -0.4 -8.6 2.7 -8.5 Table S4. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1f at CE-B3LYP-631G (d,p) theory. Table S4. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1f at CE-B3LYP-631G (d,p) theory. 10. Calculation of Intermolecular Interaction Energies in the Solid State Figure S66. The cluster of molecules used to model intermolecular interaction energies of 1f listed in Table S2 and Table S3. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S3 and Table S4. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x+1/2, -y+1/2, z+1/2 9.81 -9.6 -2.5 -98.8 50.6 -66.8 2 x+1/2, -y+1/2, z+1/2 12.04 -9.7 -2.4 -75.5 39.4 -53.5 Figure S66. The cluster of molecules used to model intermolecular interaction energies of 1f listed in Table S2 and Table S3. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S3 and Table S4. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x+1/2, -y+1/2, z+1/2 9.81 -9.6 -2.5 -98.8 50.6 -66.8 2 x+1/2, -y+1/2, z+1/2 12.04 -9.7 -2.4 -75.5 39.4 -53.5 Figure S66. The cluster of molecules used to model intermolecular interaction energies of 1f listed in Table S2 and Table S3. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S3 and Table S4. 82 Table S5. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1g at CE-HF-321G theory. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x, y, z 11.60 -9.2 -6.7 -109.0 47.1 -73.8 4 -x+1/2, y+1/2, -z+1/2 13.01 -10.6 -3.6 -51.3 20.2 -43.0 4 -x+1/2, y+1/2, -z+1/2 14.23 -0.2 -1.0 -19.1 6.8 -12.5 2 x, y, z 12.57 -24.6 -7.3 -79.5 40.5 -68.6 Table S6. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1g at CE-B3LYP-631G (d,p) theory. Table S6. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1g at CE-B3LYP-631G (d,p) theory. Table S6. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1g at CE-B3LYP-631G (d,p) theory. g ( ) 1g at CE-B3LYP-631G (d,p) theory. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x, y, z 11.60 -13.9 -3.3 -109.0 60.2 -74.9 4 -x+1/2, y+1/2, -z+1/2 13.01 -8.7 -1.7 -51.3 25.7 -39.3 4 -x+1/2, y+1/2, -z+1/2 14.23 -1.9 -0.5 -19.1 9.2 -13.3 2 x, y, z 12.57 -20.5 -3.5 -79.5 51.4 -61.7 Figure S67. The cluster of molecules used to model intermolecular interaction energies of 1g listed in Table S4 and Table S5. 10. Calculation of Intermolecular Interaction Energies in the Solid State The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S5 and Table S6. Figure S67. The cluster of molecules used to model intermolecular interaction energies of 1g listed in Table S4 and Table S5. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S5 and Table S6. 83 Table S7. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1h at CE-HF-321G theory N Symmetry Op. R/Å Eele Epol Edis Erep Etot 4 -y+3/4, x+1/4, z+1/4 14.48 -6.2 -1.6 -31.5 15.0 -23.6 2 x, y, z 12.34 -11.1 -3.0 -74.0 31.7 -54.2 4 y+3/4, -x+3/4, z+3/4 11.55 -10.9 -4.7 - 105.1 44.2 -73.0 4 -x+1/2, -y, z+1/2 16.91 -3.1 -0.4 -10.5 5.0 -8.7 2 -x, -y+1/2, z 15.75 -1.2 -0.3 -9.9 2.1 -8.6 Table S8. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1h at CE-B3LYP-631G (d,p) theory Table S7. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1h at CE-HF-321G theory y N Symmetry Op. R/Å Eele Epol Edis Erep Etot 4 -y+3/4, x+1/4, z+1/4 14.48 -6.2 -1.6 -31.5 15.0 -23.6 2 x, y, z 12.34 -11.1 -3.0 -74.0 31.7 -54.2 4 y+3/4, -x+3/4, z+3/4 11.55 -10.9 -4.7 - 105.1 44.2 -73.0 4 -x+1/2, -y, z+1/2 16.91 -3.1 -0.4 -10.5 5.0 -8.7 2 -x, -y+1/2, z 15.75 -1.2 -0.3 -9.9 2.1 -8.6 2 -x, -y+1/2, z 15.75 -1.2 -0.3 -9.9 2.1 -8.6 Table S8. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1h at CE-B3LYP-631G (d,p) theory Figure S68. The cluster of molecules used to model intermolecular interaction energies of 1h listed in Table S6 and Table S7. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S7 and Table S8. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x, y, z 12.34 -10.7 -1.4 -74.0 39.2 -52.6 4 y+3/4, -x+3/4, z+3/4 11.55 -13.5 -2.2 -105.1 55.5 -73.2 Table S8. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1h at CE-B3LYP-631G (d,p) theory Figure S68. The cluster of molecules used to model intermolecular interaction energies of 1h listed in Table S6 and Table S7. 10. Calculation of Intermolecular Interaction Energies in the Solid State The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S7 and Table S8. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 x, y, z 12.34 -10.7 -1.4 -74.0 39.2 -52.6 4 y+3/4, -x+3/4, z+3/4 11.55 -13.5 -2.2 -105.1 55.5 -73.2 Table S8. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1h at CE-B3LYP-631G (d,p) theory Figure S68. The cluster of molecules used to model intermolecular interaction energies of 1h listed in Table S6 and Table S7. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S7 and Table S8. Figure S68. The cluster of molecules used to model intermolecular interaction energies of 1h listed in Table S6 and Table S7. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S7 and Table S8. 84 Table S9. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹ at CE-HF-321G theory Table S9. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹ at CE-HF-321G theory Table S10. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹ at CE-B3LYP-631G (d,p) theory N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 - 11.70 -25.6 -5.5 -107.3 52.1 -84.2 2 - 17.02 -1.4 -0.5 -12.7 5.9 -8.4 2 x, y, z 12.81 -22.2 -8.5 -100.5 43.5 -83.4 2 - 17.66 -0.2 -0.2 -5.3 0.4 -4.8 2 x, y, z 15.24 -8.6 -1.9 -32.9 16.7 -26.1 2 x, y, z 14.85 -2.0 -2.4 -23.0 6.3 -19.2 2 - 12.16 -12.8 -8.0 -74.9 28.7 -62.5 Table S10. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹ at CE-B3LYP-631G (d,p) theory Table S10. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹ at CE-B3LYP-631G (d,p) theory N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 - 11.70 -22.5 -2.6 -107.3 65.4 -78.7 2 x, y, z 12.81 -19.6 -4.1 -100.5 56.0 -76.7 Figure S69. The cluster of molecules used to model intermolecular interaction energies for 1i listed in Table S8 and Table S9. 10. Calculation of Intermolecular Interaction Energies in the Solid State The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S9 and Table S10. Figure S69. The cluster of molecules used to model intermolecular interaction energies for 1i listed in Table S8 and Table S9. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S9 and Table S10. 85 Table S11. Calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹʹ at CE-HF-321G theory . Table S12. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹʹ at CE-B3LYP-631G (d,p) theory. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 - 11.70 -25.6 -5.5 -107.3 52.1 -84.2 2 - 17.02 -1.4 -0.5 -12.7 5.9 -8.4 2 - 17.66 -0.2 -0.2 -5.3 0.4 -4.8 2 - 13.06 -6.7 -3.4 -57.5 19.7 -44.9 2 - 12.16 -12.8 -8.0 -74.9 28.7 -62.5 2 x, y, z 12.81 -13.0 -3.2 -86.5 44.8 -57.0 2 x, y, z 15.24 1.5 -0.5 -15.9 4.9 -9.1 Table S12. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹʹ at CE-B3LYP-631G (d,p) theory. Table S12. Select calculated intermolecular interaction energies (in kJ·mol–1) for the solid-state structure of 1iʹʹ at CE-B3LYP-631G (d,p) theory. of 1iʹʹ at CE-B3LYP-631G (d,p) theory. Figure S70. The cluster of molecules used to model intermolecular interaction energies for 1iʹ listed in Table S10 and Table S11. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S11 and Table S12. N Symmetry Op. R/Å Eele Epol Edis Erep Etot 2 - 11.70 -22.5 -2.6 -107.3 65.4 -78.7 2 x, y, z 12.81 -14.9 -1.5 -86.5 55.8 -57.8 Figure S70. The cluster of molecules used to model intermolecular interaction energies for 1iʹ listed in Table S10 and Table S11. The central molecule is colored according to Hirshfeld surface and surrounding molecules are colored to match the color code in Table S11 and Table S12. 86 11. Compound Identity Validation by Low Resolution Mass Spectra In some cases we were unable to obtain high resolution mass spectra. For these compounds, we further characterize them by comparing zoomed isotopic distributions of the M+ peaks. These low resolution mass spectrometry (MS) results were acquired by AIMS Mass Spectrometry Laboratory at University of Toronto on a Waters GCT Premier instrument. The spectra shown were run in EI+ mode using the heated solids probe. The isotopic abundance pattern was computed using the Waters MassLynx software. Figure S71. Characterization of 1b using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. a b a b b Figure S71. Characterization of 1b using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. 87 igure S72. Characterization of 1c using isotopic distribution in low resolution mass spectrometry. a redicted isotopic distribution and b) observed isotopic distribution. a b a a Figure S72. Characterization of 1c using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. b b b Figure S72. Characterization of 1c using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. 88 gure S73. Characterization of 1d using isotopic distribution in low resolution mass spectrometry. a redicted isotopic distribution and b) observed isotopic distribution. a b a a b b Figure S73. Characterization of 1d using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. 89 igure S74. Characterization of 1g using isotopic distribution in low resolution mass spectrometry. redicted isotopic distribution and b) observed isotopic distribution. a b a b b Figure S74. Characterization of 1g using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. 90 gure S75. Characterization of 1h using isotopic distribution in low resolution mass spectrometry. a redicted isotopic distribution and b) observed isotopic distribution. a b a b Figure S75. Characterization of 1h using isotopic distribution in low resolution mass spectrometry. a) Predicted isotopic distribution and b) observed isotopic distribution. 91 12. References 12. References (1) (1) a) Marsh, D.F.; Falvo, R.E.; Mink, L.M. Microscale synthesis and 1H NMR analysis of tetraphenylporphyrins. J. Chem. Ed. 1999, 76(2), 237-239. b) Mohajer, D.; Zakavi, S.; Rayati, S.; Zahedi, M.; Safari, N.; Khavasi, H.R.; Shahbazian, S. Unique 1∶2 adduct formation of meso-tetraarylporphyrins and meso- tetraalkylporphyrins with BF3: a spectroscopic and ab initio study. New J. Chem. 2004, 28(12), 1600-1607. (2) (2) Ishida, S.; Yoshimura, K.; Matsumoto, H.; Kyushin, S. Selective Si–C Bond Cleavage on a Diorganosilicon Porphyrin Complex Bearing Different Axial Ligands Chem. Lett. 2009, 38(4), 362-363. (3) (3) a) Kane, K.M.; Lorenz, C.R.; Heilman, D.M.; Lemke, F.R. Substituent Effects on the Spectroscopic Properties and Reactivity of Hexacoordinate Silicon(IV) Porphyrin Complexes Inorg. Chem. 1998, 37, 669-673 b) Liu, J.; Yang, X.; Sun, L. Axial anchoring designed silicon–porphyrin sensitizers for efficient dye-sensitized solar cells Chem. Commun. 2013, 49(100), 11785-11787. (4) Lam, T. L.; Tong, K. C.; Yang, C.; Kwong, W. L.; Guan, X.; Li, M. De; Kar-Yan Lo, V.; Lai-Fung Chan, S.; Lee Phillips, D.; Lok, C. N.; Che, C. M. Luminescent Ruffled Iridium(Iii) Porphyrin Complexes Containing N-Heterocyclic Carbene Ligands: Structures, Spectroscopies and Potent Antitumor Activities under Dark and Light Irradiation Conditions. Chem. Sci. 2019, 10(1), 293-309. (4) (5) Bruker AXS Inc., Madison, Wisconsin, USA. (5) (6) Sheldrick, G.M. SADABS. 1996, University of Göttingen, Germany. (7) Sheldrick, G.M. (2014) SHELXT–Integrated space-group and crystal-structure determination Acta Cryst. 2014, A71, 3-8. (7) Sheldrick, G.M. (2014) SHELXT–Integrated space-group and crystal-structure determination Acta Cryst. 2014, A71, 3-8. (8) Sheldrick, G. M. SHELXTL Version 2014/7 (9) Spek, A.L. PLATON SQUEEZE: a tool for the calculation of the disordered solven contribution to the calculated structure factors Acta Cryst. 2015, C71, 9-18. (9) Spek, A.L. PLATON SQUEEZE: a tool for the calculation of the disordered solvent contribution to the calculated structure factors Acta Cryst. 2015, C71, 9-18. er, M. J.; McKinnon, J. J.; Wolff, S. K.; Grimwood, D. J.; Spackman, P. R.; (12) Turner, M. J.; McKinnon, J. J.; Wolff, S. K.; Grimwood, D. J.; Spackman, P. R.; Jayatilaka, D.; Spackman, M. A. CrystalExplorer17 (2017). University of Western Australia. 12. References (10) Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G.A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.P.; Izmaylov, A.F.; Bloino, J.; Zheng, G.; Sonnenberg, J.L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr., J.A.; Peralta, J.E.; Ogliaro, F.; Bearpark, M.; Heyd, J.J.; 92 Brothers, E.; Kudin, K.N.; Staroverov, V.N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J.C.; Iyengar, S.S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J.M.; Klene, M.; Knox, J.E.; Cross, J.B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.E.; Yazyev, O.; Austin, A.J.; Cammi, R.; Pomelli, C.; Ochterski, J.W.; Martin, R.L.; Morokuma, K.; Zakrzewski, V.G.; Voth, G.A.; Salvador, P.; Dannenberg, J.J.; Dapprich, S.; Daniels, A.D.; Farkas, O.; Foresman, J.B.; Ortiz, J.V.; Cioslowski, J.; Fox, D.J. 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https://openalex.org/W4393604549	https://revistaincasciences.ingemmet.gob.pe/index.php/incasciences/article/download/32/28	es		Evidencias de tectónica transpresiva en la cordillera occidental del Centro del Perú (11° - 12°30’ S)	Deleted Journal	2,024	cc-by	3,494	Mamani & Jacay / Incasciences 1 (2): 34 - 44, 2023 doi:10.47347/incasciences.v1i2.32 Articulo Original Recibido: octubre 2023 Aceptado: noviembre 2023 INCASCIENCES Geociencias para el mundo Evidencias de tectónica transpresiva en la cordillera occidental del Centro del Perú (11° - 12°30’ S) Evidence of Transpressional Tectonics in the Western Cordillera of Central Peru (11° - 12°30' S) Yuly Mamani 1 Javier Jacay 2 1Instituto Geológico Minero y Metalúrgico, Lima. Perú 2Universidad Nacional Mayor de San Marcos (UNMSM) Av. Venezuela cuadra 34 s/n, Ciudad Universitaria, Lima, Perú. (jjacayh@unmsm.edu.pe) Resumen Los Andes del Perú Central, ubicados entre los paralelos 11° y 12°30’ de latitud sur, muestran un registro geológico que evidencia sucesivas etapas de deformación que afectaron al orógeno desde el Campaniano hasta el Mastrichiano y el Eoceno. Durante el Mesozoico, la sedimentación estuvo controlada por la tectónica extensional, que posteriormente, en el Cenozoico, estas secuencias fueron deformadas debido a la inversión tectónica de fallas antiguas. Esto dio lugar a un importante sistema transcurrente que desempeñó un papel decisivo en la configuración estructural del orógeno Andino, compuesto por rocas volcánicas y volcanosedimentarias. Los eventos tectónicos se manifiestan en forma de escamas tectónicas, pliegues y estructuras de tipo flor positiva que han dado forma a esta parte de la Cordillera Occidental. Palabras clave: Cordillera Occidental, transpresivo, tectónica, sistemas transcurretes. Abstract The Andes of Central Peru, located between latitudes 11° and 12°30’ south, exhibit a geological record that reveals successive stages of deformation that affected the orogeny from the Campanian to the Maastrichtian and Eocene. During the Mesozoic, sedimentation was controlled by extensional tectonics, but in the Cenozoic, these sequences were deformed due to the tectonic inversion of ancient faults. This resulted in a significant transcurrent system that played a decisive role in shaping the structural configuration of the Andean orogeny, composed of volcanic and volcanosedimentary rocks. The tectonic events are manifested in the form of tectonic scales, folds, and positive flower structures that have shaped this part of the Western Cordillera. Keywords: Western Cordillera, transpressional, tectonics, strike-slip systems. Esta obra está publicada bajo la licencia CC BY 4.0 Correspondencia Yuly Mamani ymamanip@ingemmet.gob.pe Mamani & Jacay / Incasciences 1 (2): 34 - 44, 2023 35 INTRODUCCIÓN La estructuración de las cadenas de montaña de los Andes, se caracteriza por presentar sistemas de fallas inversas paralelas a la orientación andina (sureste-noroeste), que han desempeñado un papel preponderante en la configuración del orógeno andino. Una sección geológica esquemática elaborada en sentido transversal al eje de los Andes muestra la configuración tectónica de la cordillera y sus principales unidades morfoestructurales (Figura 1B). En el área ubicada entre la altiplanicie disectada y el piedemonte de la Cordillera Occidental, se encuentran una serie de fallas transcurrentes e inversas de orientación andina (sureste-noroeste). Este trabajo resume la evolución estructural de la cordillera occidental comprendida entre los 11° y 12°30’ de latitud, analizando trabajos previos y utilizando secciones estructurales locales. Estas fallas, ubicadas en el sector occidental, se extienden a lo largo de varias decenas de kilómetros (ver Figura 1A). Figura 1. Mapa de ubicación de la zona de estudio, se muestran las principales fallas que afectan el territorio (modificado de INGEMMET, 2016). B) Corte transversal de los Andes del Perú central sonde se muestran los estilos tectónicos característicos. Evidencias de tectónica transpresiva MARCO GEOLÓGICO REGIONAL En los Andes centrales, las unidades litoestratigráficas que lo conforman han sido reconocidas y descritas por los trabajos pioneros de McLaughlin (1924), Steinmann (1930), McKinstry (1932), Terrones (1949), seguido por los estudios de Megard (1978), Salazar (1983), Zimmerninck (1985). 36 Posterior al Santoniano, sobre las unidades litoestratigráficas anteriormente descritas y según su ubicación, se depositan las capas rojas de la Formación Casapalca (Campaniano – Paleoceno), que yace sobre una superficie de erosión en los sectores occidentales, o concordantemente de manera transicional sobre la Formación Celendín en los sectores Estas unidades litoestratigráficas abarcan el inmediatamente por delante de las estructuras intervalo cronoestratigráfico entre el Jurásico de inversión. y el Cuaternario, siendo las principales unidades la Formación Oyón (Titoniano), En el Paleógeno – Neógeno, sobre una Grupo Goyllarisquizga con las formaciones importante superficie de erosión y sellando a Chimú, Santa, Carhuaz y Farrat (Valanginiano- las unidades sedimentarias, se encuentran los Barremiano), formaciones Pariahuanca afloramientos de las unidades volcánicas y (Aptiano – Albiano inferior), Chúlec (Albiano volcanosedimentarias descritas como los grupos inferior – Albiano medio), Pariatambo (Albiano Calipuy y Rímac, además de las formaciones medio), Jumasha (Albiano superior – Turoniano) Colqui, Millotingo, Carlos Francisco, y Celendín (Coniaciano – Santoniano). Huarochiri, Catrovirreyna, entre otros (Figura 2). Mamani & Jacay / Incasciences 1 (2): 34 - 44, 2023 37 Figura 2. Mapa geológico regional donde se muestran la distribución de las estructuras estudiadas (modificado de Cobbing, 1973 y Salazar 1983) Evidencias de tectónica transpresiva GEOLOGÍA ESTRUCTURAL Los sistemas de cabalgamientos presentes en la Cordillera Occidental del Perú central han sido tratados por diferentes autores especialmente por Szekely (1967 y 1969), quien presenta una serie de cortes estructurales suroeste-noreste, localizados entre el lago Huallacocha y el anticlinal de Chumpe, resaltando las estructuras imbricadas para la zona más occidental. 38 con diferente estilo estructural que correspondería a hemigrabens invertidos, calculando igualmente un acortamiento entre 40% y 50%, haciendo énfasis de que los niveles de despegue se sitúan en las formaciones Oyón y Pariatambo. Ángeles (1987) documentó la relación entre las mesoestructuras y las microestructuras que fueron descritas como “napas” inferior y superior expuestas entre los ríos Alis y Sinhua en Así mismo, menciona que los niveles de la parte alta del valle del rio Cañete, obteniendo despegue de las imbricaciones, son las lutitas un acortamiento entre 50% a 60% y, donde los negras de la Formación Pariatambo. niveles de despegue también se ubican en las formaciones Oyón y Pariatambo. Wilson (1963) y Harrison (1960) describieron un patrón estructural similar a lo descrito para Los autores antes citados interpretaron un la Formación Oyón expuesto en la parte alta del sistema imbricado o de escamas para la valle del rio Huaura. Cordillera Occidental, desarrollando un sistema de cabalgamientos con vergencia al este, en el Megard (1978) evidenció la complejidad que destacan una serie de fallas inversas que estructural presente en la quebrada Huachuga estructuran una faja plegada. – sector de Huampar y en la parte media a superior del valle del rio Rímac, describiendo RESULTADOS las estructuras bajo la terminología de una Las observaciones de campo realizadas en “zone imbriquée” relacionado a una zona de el presente estudio, han permitido reconocer cizallamiento con rumbo andino, que fueron dos unidades tectónicas competentes, Unidad producto de las fallas inversas reactivadas de Tectónica inferior conformado por el Grupo antiguas fallas normales de edad mesozoica. Goyllarisquizga (formaciones Chimú, Santa, Carhuaz y Farrat) y las formaciones Pariahuanca, Lepry (1981) realizó importantes observaciones Chúlec, y la Unidad Tectónica superior, sobre el estilo microtectónico de las rocas conformado por la Formación Jumasha; como paleozoicas y mesozoicas y su relación con unidades incompetentes son descritas las las estructuras mayores, presentando cortes formaciones Oyón, Pariatambo y Celendín, estructurales típicos de una faja plegada siendo estas ultimas unidades las que juegan un calculando un porcentaje de acortamiento de rol importante como niveles de despegue para 20% para la región de Yauli, demostrando la estructuración del sistema de cabalgamientos que la Formación Pariatambo es el nivel de de la Cordillera Occidental. despegue en la estructuración de las secuencias de cabalgamientos, Romani (1982) realizó un Del mismo modo, las observaciones de campo, estudio detallado de la estratigrafía y un esbozo la interpretación de las imágenes satelitales y la de la sedimentología de las rocas cretácicas de elaboración de las cartas geológicas, documentado Uchucchacua, que expone cabalgamientos en por la orientación predominante de los sistemas zonas estructurales y compartimientos cada uno de pliegues relacionado a su vergencia con la Mamani & Jacay / Incasciences 1 (2): 34 - 44, 2023 39 dirección de inclinación de la charnela y grado de la Formación Jumasha cabalga mediante una inclinación de su plano axial. falla inversa a la Formación Carlos Francisco (Figura 3B), así mismo en el sector occidental Su relación con los sistemas de fallas inversas, de la laguna Punrun (SE del cuadrángulo de indican un sistema de propagación típico de una Oyón) la Formación Jumasha se sobreyace faja plegada con vergencia al este. a la Formación Casapalca siendo el nivel de despegue la Formación Pariatambo. Las fallas presentan trazos de baja sinuosidad (vista en planta), han configurado pliegues con Los anticlinales y sinclinales que afectan a plano axial vertical o ligeramente inclinado, la Formación Casapalca se han estructurado esta geometría estaría relacionada a una rampa mediante fallas ciegas (blind thrust fault) Estas tectónica de alto ángulo, evidenciando en superficie estructuras tienen su nivel de despegue en las pliegues apretados en la Formación Jumasha que margas lutáceas de la Formación Celendín. están relacionados a fallas inversas con probable despegue en la Formación Pariatambo. Las observaciones de campo documentan los cambios de los niveles de despegue, mientras que, En el frente de los cabalgamientos, se observa la Formación Oyón es la unidad despegue para los una fuerte esquistosidad tectónica con rumbos sectores más occidentales, la Formación Pariatambo entre N 40°O y N 20° O, paralela a las fallas lo es para el sector central y la Formación Celendín inversas con vergencia al este. es la unidad de despegue en los sectores más orientales de la Cordillera Occidental, además los Estas afectan a los niveles superiores de las niveles de despegue migran desde niveles inferiores formaciones Jumasha y Casapalca (ver Figura hacia los niveles más superiores. 3a), visibles en el sector de Ticlio, abra de cerro Yuncan (entre Santader y Chungar), En los niveles superiores, las principales abra de Antaccasa, laguna Jurpay, abra Calera estructuras desarrolladas han conformado (Puagjanca), entre otros. valles lineales, como en río Blanco, donde afloran pliegues (anticlinales y sinclinales) Se interpretan como producto de la inversión en flor positiva, asociados a la progresión de tectónica.” fallas inversas en los cerros Tatajayco, Paccha, Amargura y Jupay Grande, en San Mateo. El sistema tectónico de la Cordillera Occidental (paralelos 11° - 12°30’ S) tiene como nivel de Entre los paralelos 11° - 12°30’S, se observan despegue a la Formación Oyón (Titoniano), estructuras compresivas con vergencia al oeste, que permite sobreyacer a la Formación como los pliegues anticlinales y sinclinales del Chimú (Valanginiano) sobre las unidades cerro San Cristóbal - Carhua (Canta). litoestratigráficas más jóvenes, como ocurre Picoyanque (sector occidental del cuadrángulo Estos pliegues presentan un plano axial vertical de Ondores) donde la Formación Chimú a ligeramente inclinado, relacionado con la sobreyace a la Formación Yantac (Eoceno) propagación de una falla inversa de alto ángulo que pertenece a base del Grupo Calipuy; con vergencia al oeste. mientras que en la laguna Chuspicocha y Yuracmayo (SE del cuadrángulo de Matucana), Evidencias de tectónica transpresiva 40 El nivel de despegue estaría a la base de la Formación Arahuay (Albiano medio Turoniano), que se sobrepone a los niveles volcánicos paleógenos. En las zonas de ‘valles encañonados’, como el cañón del Infiernillo (río Rímac) y Pampamayu (río Baños), es posible observar estructuras tectónicas asociadas al nivel medio inferior de sistemas transpresivos. En el cerro Araro, parte alta del valle del río Huaral (Santa Cruz de Andamarca), el sistema de pliegues con plano axial vertical afecta al Grupo Calipuy (Eoceno – Mioceno inferior), en contacto con la Formación Carhuaz mediante una falla inversa de alto ángulo, componente de un sistema transpresivo, conformando una estructura de inversión con vergencia al oeste (Figura 3C). En el cañón del Infiernillo (río Rímac), se pueden observar desde cizallamientos anastomosados hasta lineales con buzamiento de alto ángulo, que afectan a unidades como la Formación Chimú. En la parte alta del valle de Lincha, en las lomas del cerro Huayllapucro – Tintac (Yauyos), se observa que la secuencia volcánica del Grupo Sacsaquero (Oligoceno) y las formaciones Castrovirreyna (Oligoceno – Mioceno inferior) y Caudalosa (Mioceno superior), se encuentran plegadas y falladas, con pliegues asimétricos y con estructuras de empuje con vergencia occidental. Por otro lado, en la zona del cañón de Pampamayu (río Baños), se pueden observar estructuras de inversión tectónica con fuerte cizallamiento y con plano axial subvertical con vergencia al oeste. Entre Santiago de Anchucaya, Matucana y Huanza se extiende un sistema de fallas de NO-SE que afectan a secuencias cenozoicas moderadamente plegadas con vergencia al este (Figuras 3D); además se observan, estratos de crecimiento en la Formación Huarochirí (Mioceno medio – superior) en el poblado de Jicamarca (11°44’21’’ S – 76°42’16’’ O) que se han desarrollado en los niveles de tobas y volcanoclásticos basculados como respuesta a fallas extensionales que afectan a las rocas volcánicas del Grupo Rímac. Las fallas inversas son responsables de invertir las sucesiones sedimentarias del sustrato precenozoico, como la Formación Chimú. Esta unidad se presenta intensamente cizallada, con fracturas paralelas a la estratificación, asociadas a la zona de la falla río Blanco. DISCUSIONES En este trabajo, argumentamos que la estructuración de la Cordillera Occidental En el sector noreste del cuadrángulo de Chosica (paralelos 11° - 12°30’ S) está constituida por un (al este del poblado de Lachaqui) las secuencias sistema de plegamiento con orientación andina. volcanosedimentarias de la Formación Colqui se muestran deformadas, exhibiendo pliegues Se evidencia que los niveles de despegue asociados a fallas ciegas (falla cuyo plano de la tectónicos en el sector occidental se ubican falla termina antes de llegar a la superficie) con en la Formación Oyón y migran a los niveles vergencia al este y con rumbo andino (Figura superiores donde se localizan las formaciones 3C) asociadas a fallas dextrales de rumbo E-O. Pariatambo y Celendín. El trazo de la falla sobre la carta geológica presenta una baja sinuosidad y está asociado a un fuerte cizallamiento tectónico. Esto se debe principalmente a la estructuración en rampas tectónicas de alto ángulo con vergencia al este. También se observan en ciertas áreas el desarrollo Mamani & Jacay / Incasciences 1 (2): 34 - 44, 2023 41 de estructuras compresivas con vergencia al oeste. oeste están cubiertas por las rocas volcánicas del Eoceno-Oligoceno. Estos sistemas de fallas de vergencia pacífica han sido previamente señalados en la Cordillera Se interpreta que este periodo de tiempo es relevante Occidental en algunos cortes estructurales para la estructuración de esas fallas fuera de secuencia. realizados por Romani (1982) en la región de Los depósitos del Paleoceno superior a Uchucchacua y por Angeles (1987) en el área de Oligoceno están deformados y afectados por Huacuypacha (alto Cañete). fallas inversas que han exhumado las rocas del Cretácico inferior, lo que ha resultado en las El cizallamiento tectónico asociado a las deformaciones sintectónicas presentes en los estructuras de pliegues y fallas menores (sistemas depósitos del Mioceno (Formación Huarochirí). Riedel) presentes en los niveles superiores, así como el asociado a los sistemas de fallas inversas, Por lo tanto, se puede sostener que la edad han ocurrido después de la inversión tectónica. de deformación de estas estructuras es del Mioceno, como había sido reconocido por Están vinculados localmente a un sistema Megard (1984). transcurrente (Figura 3A). Igualmente, Muñoz y Sepúlveda (1992), Muñoz Ademas, los estratos de crecimiento presentes y Charrier (1996), Drozdzewski y Mon (1999), en rocas del Mioceno se interpretan como Victor (2004), Farías et al. (2005), Carrera et respuesta a un sistema extensional o transtesivo, al. (2006), Lebinson et al. (2020), DeCelles et de manera paralela en el sector externo. al. (2015), entre otros, describen estructuras similares para los Andes chilenos y argentinos. Las estructuras post y sin tectónicas han ocurrido en varios períodos de la historia geológica de la Estos investigadores concluyen la importancia región andina. de la inversión tectónica en un sistema transpresivo para la estructuración de la Las estructuras compresivas con vergencia al Cordillera Occidental en estas regiones. Figura 3. A) Vista panorámica al sur de cerro Panapashatran (cuadrángulo de Matucana) donde una serie de fallas inversas afectan a las Formaciones Jumasha y Casapalca conformando escamas tectónicas que generan pliegues de arrastre e intensa esquistosidad tectónica. B) Mapa satelital del valle de rio Blanco, donde la Formación Jumasha (Ju) sobreyace mediante una falla inversa a la Formación Carlos Francisco (CF), un sistema de fallas transcurrentes a lo largo de la cual se intruyen diversos cuerpos dacíticos (DA). C) Afloramientos del Grupo Calipuy afectado por una falla fuera de secuencia (back thrust), cerro Araro en la parte alta del valle de Huaral, vista al sur. D) Falla inversa ciega que afecta a secuencias volcanoclásticos de la Formación Colqui y genera un pliegue por propagación de falla, al este de Lachaqui. E) Vista de la margen derecha de quebrada. Huallapampa al sur de Matucana, donde la falla inversa que afecta a secuencias de lavas del Grupo Rímac, pliega ligeramente a las tobas de la Formación Huarochirí además esta es aprovechada por un dique feeder de un cuerpo traquiandesitico. Evidencias de tectónica transpresiva 42 Mamani & Jacay / Incasciences 1 (2): 34 - 44, 2023 43 Conclusiones A partir de los trabajos de cartografiado y Cobbing, J (1973). Geología de los cuadrángulos observaciones estructurales en esta región de Barranca, Ámbar, Oyón, Huacho, Huaral del Perú central, se evidencia que los niveles y Canta. Boletín INGEMMET. Serie A-26: de despegue son las unidades incompetentes Carta Geológica Nacional, 172p. (Oyón, Pariatambo y Celendín), las cuales invierten las sucesiones estratigráficas desde DeCelles P.G., Zandt G., Beck S.L., Currie la base del Cretácico, estructurándolo en un C.A., Ducea M.N., Kapp P., Gehrels G.E., sistema de plegamiento de vergencia al este. Carrapa B., Quade J. & Schoenbohm L.M. (2015). Cyclical orogenic processes in the Los anticlinales y sinclinales que se presentan Cenozoic central Andes. The Geological con un plano axial vertical y la presencia de una Society of America Memoir 212, p: 459-490. foliación y esquistosidad tectónica asociada a las fallas son manifestaciones relacionadas con Drozdzewski G. & Mon R. (1999). Oppositelylos frentes de inversión tectónica. verging thrusting structures in the North Argentine Andes compared with the German El conjunto de estructuras tectónicas, como Variscides. Acta Geologica Hispanica, v. 34, el rumbo casi recto del trazo de las fallas nº 2-3, p. 185-196. inversas en superficie, los planos axiales de los pliegues verticales y la presencia de estructuras Farías M., Charrier R., Comte D., Martinod J., compresivas con vergencia al este y al oeste, nos & Gérard Hérail G. (2005). Late Cenozoic sugieren la existencia de un sistema transpresivo deformation and uplift of the western para la Cordillera Occidental. flank of the Altiplano: Evidence from the depositional, tectonic, and geomorphologic El desarrollo de estratos de crecimiento en evolution and shallow seismic activity unidades superiores no es simplemente una (northern Chile at 19°30’ S). Tectonics, Vol. manifestación de una tectónica distensiva o 24, TC4001, DOI: 10.1029/2004TC001667. transtensiva, sino que requerirá futuros estudios para su resolución. Harrison, J. (1960). Structural doubts about the Andes of Peru. Internat. Geol. Congr., 21st Referencias Sess., Proc., pt. 18, p. 7-13. Ángeles, C. (1987). Les chevauchements de la Cordillère Occidentale par 12°15’ (Andes du Lebinson F., Turienzo M., Sánchez N., Pérou Central). These 3° cycle Univ.S.T.L., Cristallini E., Araujo V. & Dimieri L. Montpellier, 184 p. (2020). Kinematics of a backthrust system in the Agrio fold and thrust belt, Argentina: Carrera N., Muñoz J.A., Sabat F., R. Mon Insights from structural analysis and R.& Roca E. (2006). The role of inversion analogue models. Journal of South American tectonics in the structure of the Cordillera Earth Sciences. 100, 7-4, p: 1-18. Oriental (NW Argentinean Andes). Journal of Structural Geology 28 1921-1932. Evidencias de tectónica transpresiva 44 Megard, F. (1984). The andean orogenic period Victor, P.; Oncken, O.& Glodny, J. (2004). and its major structures in central and Uplift of the western Altiplano plateau: northerh Peru. Journal of the Geological Evidence from the Precordillera between Society, London, 141: 893-900. 20° and 21°S (northern Chile). Tectonics, 23: TC400. DOI: 10.1029/2003TC001519. Muñoz, N., & R. Charrier (1996). Uplift of the western border of the Altiplano on a Wilson, J. (1963). Cretaceous stratigraphy of west-vergent thrust system, northern Chile. central Andes of Peru. American Association of Journal of South American Earth Sciences, Petroleum Geologists Bulletin., V. 47, p. 1-34. 9, 171 – 181. Zimmernink, W. (1985). Geology and Muñoz, N., & Sepúlveda P. (1992). Estructuras mineralogy of Felicidad and Santander: compresivas con vergencia al oeste en el two Peruvian skarn-type deposits in borde oriental de la Depresión Central, norte Cretaceous (sedimentary) host rock (with de Chile (19°15’S). Revista Geológica de a case study of various garnet types). Chile, 19, 241-247. Doktorwurde der Naturwissenschaftlich Mathematisch, Ruprecht-Karls Universität, Romani, M. (1982). Géologie de la région Heidelberg:1985,170 p. minière Uchucchacua Hacienda Otuto, Pérou. Thèse 3eme cycle, Grenoble, 176 pp. Salazar, H. (1983). Geología de los cuadrángulos de Matucana y Huarochirí. Boletín INGEMMET, N° 36, Serie A: Carta Geológica Nacional, 71p. Szekely, T. (1967). Geology near Huallacocha lakes, central high Andes, Peru. American Association of Petroleum Geologists Bulletin, V. 51 (7), p: 1346-1353. Szekely, T. (1969). Structural geology, Cochas to Yauricocha, central High Andes, Peru. American Association of Petroleum Geologists Bulletin 53(3): 553-567. Terrones, A. (1949). La estratigrafía del distrito minero de Morococha. Boletín de la Sociedad Geológica del Perú, v. 2 (8), pp. 1-15.
W4392630179.txt	https://hal.science/hal-00632016/document	en		FERO: Finding extreme relativistic objects. I. Statistics of relativistic Fe K<SUB>alpha</SUB> lines in radio-quiet Type 1 AGN	null	2,010	cc-by	19,263	FERO: Finding extreme relativistic objects. I. Statistics of relativistic Fe Kalpha lines in radio-quiet Type 1 AGN I. de La Calle Pérez, A. L. Longinotti, M. Guainazzi, S. Bianchi, M. Dovciak, M. Cappi, G. Matt, G. Miniutti, P. O. Petrucci, E. Piconcelli, et al. To cite this version: I. de La Calle Pérez, A. L. Longinotti, M. Guainazzi, S. Bianchi, M. Dovciak, et al.. FERO: Finding extreme relativistic objects. I. Statistics of relativistic Fe Kalpha lines in radio-quiet Type 1 AGN. Astronomy and Astrophysics - A&A, 2010, 524, pp.A50. �hal-00632016� HAL Id: hal-00632016 https://hal.science/hal-00632016 Submitted on 30 Nov 2021 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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. Distributed under a Creative Commons Attribution 4.0 International License Astronomy & Astrophysics A&A 524, A50 (2010) DOI: 10.1051/0004-6361/200913798 c ESO 2010 FERO: Finding extreme relativistic objects I. Statistics of relativistic Fe Kα lines in radio-quiet Type 1 AGN I. de la Calle Pérez1 , A. L. Longinotti2,1 , M. Guainazzi1 , S. Bianchi3 , M. Dovčiak4 , M. Cappi11 , G. Matt3 , G. Miniutti5 , P. O. Petrucci6 , E. Piconcelli7 , G. Ponti8,9 , D. Porquet10 , and M. Santos-Lleó1 1 2 3 4 5 6 7 8 9 10 11 European Space Astronomy Centre of ESA, Apartado 50727, 28080 Madrid, Spain e-mail: icalle@sciops.esa.int MIT Kavli Institute for Astrophysics and Space Research, Cambridge, USA Dipartimento di Fisica, Università degli Studi Roma Tre, via della Vasca Navale 84, 00146 Roma, Italy Astronomical Institute AS CR, Boční II 1401/1a, 14131 Praha 4, Czech Republic Centro de Astrobiología (CSIC-INTA); LAEFF, PO Box 78, Villanueva de la Cañada, Madrid 28691, Spain Laboratoire d’Astrophysique, UMR5571 Université J. Fourier/CNRS, Observatoire de Grenoble BP53, 38041 Grenoble Cedex 9, France Osservatorio Astronomico di Roma, via Frascati 33, 00040 Monteporzio Catone, Italy School of Physics and Astronomy, University of Southampton, Highfield, Southampton SO17 1BJ, UK APC, Université Paris 7 Denis Diderot, 75205 Paris, France Observatoire Astronomique de Strasbourg, Université Louis-Pasteur, CNRS, INSU, 11 rue de l’Université, 67000 Strasbourg, France INAF-IASF Bologna, via Gobetti 101, 40129 Bologna, Italy Received 30 November 2009 / Accepted 27 July 2010 ABSTRACT Context. Accretion models predict that fluorescence lines broadened by relativistic eﬀects should arise from reflection of X-ray emission onto the inner region of the accretion disc surrounding the central black hole of active galactic nuclei (AGN). The theory behind the origin of relativistic lines is well established, and observational evidence from a moderate number of sources seems to support the existence of these lines. Aims. The aim of this work is to establish the fraction of AGN with relativistic Fe Kα lines, and study possible correlations with source physical properties. Methods. An XMM-Newton collection of 149 radio-quiet Type 1 AGN has been systematically and uniformly analysed in order to search for evidence of a relativistically broadened Fe Kα line. To enable statistical studies, an almost complete, flux-limited subsample of 31 sources has been defined by selecting the FERO sources observed by the RXTE all-sky Slew Survey with a count rate in the 3– 8 keV energy band greater than 1 cts/sec. The 2–10 keV spectra of the FERO sources where compared with a complex model including most of the physical components observed in the X-ray spectra of Seyfert galaxies: a power law primary continuum modified by nonrelativistic Compton reflection and warm absorption, plus a series of narrow Fe line reflection features. Results. The observed fraction of sources in the flux-limited sample that show strong evidence of a relativistic Fe Kα line is 36%. This number can be interpreted as a lower limit to the fraction of sources that present a relativistic broad Fe Kα line in the wider AGN population. The average line equivalent width (EW) is of the order of 100 eV. The outcome of the fit yields an average disc inclination angle of 28 ± 5◦ and an average power-law index of the radial disc emissivity law of 2.4 ± 0.4. The spin value is well constrained only in 2 cases (MCG-6-30-15 and MRK 509); in the rest of the cases, whenever a constraint can be placed, it always implies the rejection of the static black hole solution. The Fe Kα line EW does not correlate with disc parameters or with system physical properties, such as black hole mass, accretion rate, and hard X-ray luminosity. Key words. quasars: emission lines – galaxies: nuclei – galaxies: active – X-ray: galaxies 1. Introduction The standard scenario for the X-ray emission in active galactic nuclei (AGN) assumes that the observed X-ray power-law continuum originates in the inner regions of the AGN closest to the central super-massive back hole, via inverse Compton scattering of soft-energy photons in a corona of relativistic electrons located somewhere above the accretion disc (e.g. Haardt & Maraschi 1993). The X-ray illumination of optically thick cold matter, such as the molecular torus and/or the accretion disc, gives rise to a Compton reflection spectral component containing Appendices are only available in electronic form at http://www.aanda.org a series of fluorescent lines from Kα transitions in metal atoms (e.g. George & Fabian 1991). From a combination of fluorescence yield (proportional to the fourth power of the atomic number) and cosmic abundance, the most prominent is the Kα line emitted by neutral iron at 6.4 keV. When the Compton reflection originates in distant material like the molecular torus envisaged in unification models (Antonucci 1993), the profile of the Fe Kα emission line is narrow and unresolved by present X-ray detectors. In contrast, the reflection component originating in the inner accretion disc is affected by the black hole’s strong gravitational field, which modifies the line profile. The numerous studies of the eﬀect of gravity on the narrow emission line all agree by describing the resulting Article published by EDP Sciences Page 1 of 22 A&A 524, A50 (2010) relativistic profile as skewed and asymmetric because of a combination of kinematic and relativistic eﬀects (e.g. Fabian et al. 2000), with a red wing extending towards low X-ray energies due to gravitational redshift and transverse Doppler redshift (e.g. Fabian et al. 1989). Since relativistic lines originate within a few gravitational radii from the central object, the study of their shape and intensity may represent a potential probe of the physical processes taking place in the innermost regions of the AGN. The line profile is in fact very sensitive to the accretion disc properties, such as the radial extension and dependence of the line emissivity, the ionisation state of the material, the observer’s inclination angle relative to the disc axis, and the spin of the black hole (for a review see Reynolds & Nowak 2003; Fabian & Miniutti 2009; Matt 2006, and references therein). The first unambiguous observational evidence of relativistic Fe Kα lines in the X-ray spectra of AGN was found in the spectrum of MCG-6-30-15 obtained by the ASCA satellite (Tanaka et al. 1995) and lately, in several other Seyfert Galaxies (Nandra et al. 1997) to the point where the broad line was considered a common feature of AGN. Nevertheless, the presence of relativistic broad Fe Kα lines is nowadays more controversial than ever since only a handful of sources seem to possess a truly relativistically broadened Fe Kα line, e.g. MCG-6-30-15 (Fabian et al. 2002) or NGC 3516 (Markowitz et al. 2008). When studies based on sizable samples of sources are considered, the average fraction of sources with relativistic broad Fe Kα lines is never higher than 40% (Porquet et al. 2004; Jiménez-Bailón et al. 2005; Guainazzi et al. 2006; Nandra et al. 2007). In the past decade the advent of Chandra, XMM-Newton and Suzaku has undoubtedly provided deeper knowledge and more thorough understanding of AGN X-ray spectral properties by means of superior spectral resolution and higher throughput. For instance, it has been confirmed that about half of quasars and Seyfert galaxies show the so-called warm absorbers, i.e., ionised material outflowing along the line of sight, which is revealed as a series of absorption features imprinted mainly in the soft X-ray spectral band (Piconcelli et al. 2005; Blustin et al. 2005, and references therein). Emission lines from highly ionised atoms, including iron, can also be observed (see Bianchi et al. 2005). The presence of this absorbing/emitting gas may introduce further complexities into the Fe K band: it has been shown that warm absorbers with suﬃciently high column density and ionisation state may distort the continuum underlying the iron line, mimicking a relativistic broad red wing (e.g. NGC 3783, Reeves et al. 2004, and MCG-6-30-15, Miller et al. 2008; see also Turner & Miller 2009 for a review). Also, a blend of emission lines from helium and hydrogen-like iron at 6.7–6.97 keV can be mistaken for a broad disk line observed at high inclination (NGC 7314, Yaqoob et al. 2003, and MRK 590, Longinotti et al. 2007). While the latter scenario is fully taken into account in our baseline model, only a simple parameterization of the warm absorber was adopted, because an unknown number of a priori absorbing systems do not allow a systematic analysis to be performed (see Sect. 3.3 for details). The FERO (finding extreme relativistic objects) project is part of a wider investigation on AGN carried out on archival XMM-Newton data (see Sect. 2). It was designed to address two fundamental questions on the relativistic broad Fe Kα line: i) how common is relativistic broadening in AGN, and ii) does the presence of a broad line depend on other source’s physical properties ? Page 2 of 22 To try to answer these questions, all the sources in our sample were fitted with one and the same model, which includes all absorption and emission components known to be potentially present in AGN (see Sect. 3.3) and able to aﬀect the emission in the iron K band. This paper reports the results of the spectral analysis on the individual sources of the sample. A companion paper (Longinotti et al., in prep.) is devoted to the analysis of stacked spectra. The structure of this paper is as follows. Section 2 describes the selection of the sample. Section 3 describes the analysis procedure, including the spectral analysis and the spectral model used to describe our data. Section 4 summarizes our results to be discussed in Sect. 5. Section 6 closes the paper with the conclusions drawn from the study presented here. 2. The sample The FERO AGN collection proceeds from the CAIXA catalogue of AGN recently published by Bianchi et al. (2009a,b). CAIXA is the largest catalogue of high signal-to-noise X-ray spectra of AGN which consists of all the radio-quiet X-ray unobscured (NH < 2 × 1022 cm−2 ) AGN observed by XMM-Newton in targeted observations, whose data are public as of March 2007. The sample, through a complete and homogeneous spectral analysis, is characterized in terms of the parameters adopted by the best-fit models. CAIXA includes a total of 77 quasars and 79 Seyfert galaxies. The redshift distribution spans from z = 0.002 to z = 4.520 (almost 90% within z < 1) and the distribution of the hard X-ray luminosities covers a range between L2−10 keV = 2.0 × 1041−3.9 × 1046 erg s−1 . Since the source selection criteria used in this work are by large the same described there, the reader is deferred to these works for more details. Here, those general aspects that are more relevant to the relativistic line analysis carried out in this work are recalled. 2.1. The FERO AGN collection The starting sample consisted of 161 radio-quiet X-ray unobscured sources targeted by XMM-Newton with public data up to April 2008. Only sources with local column density from cold gas lower than NH ≤ 2 × 1022 cm−2 are included in the sample to avoid heavily absorbed spectra in the 2 to 10 keV spectral region. With respect to CAIXA (which sums up 156 sources), the following 5 AGNs were added: ESO 511-G030, IRAS 05078+1626, MRK 704, NGC 3227, NGC 526A. The first three sources were specifically proposed and granted in the XMM-Newton AO6 for the purpose of the FERO project; the other two objects became publicly available in the data archive during the development of the FERO analysis. NGC 4151 fullfills the required conditions to be in our sample, but it was excluded (both from FERO and CAIXA) based on its complex X-ray spectra and extreme spectral variability attributed to several absorbing systems (Puccetti et al. 2007). Based on a signal-to-noise criterion, as reasoned in Sect. 3.2, 12 sources are dropped from the starting sample, leaving what constitutes the FERO sample with a total of 149 sources. Out of the 149 sources, 67 are classified as quasars (RQQs) and 82 classified as Seyfert 1s (Sy) according to the value of the absolute optical magnitude MB (QSO MB < −23; Sy MB > −23) as defined in the Véron-Cetty and Véron catalogue 2006 (VéronCetty & Véron 2006). Radio Loud objects are excluded from the sample according to the value of the radio-loudness parameter (R; Stocke et al. 1992). For QSO the condition log(R) > 1 is applied to reject a source, while in the case of Seyferts, in addition I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN to log(R) > 2.4, sources are also excluded if log(RX ) > −2.755 (Panessa et al. 2007), where RX is the X-ray radio-loudness parameter (Terashima & Wilson 2003) (see Bianchi et al. 2009a for details). No redshift restriction has been imposed in our sample, which contains mostly local sources with 90% of the sources at a redshift ≤0.5 and 60% at a redshift ≤0.1. A distinction has been made between Broad Line (BL; 40% of full sample) and Narrow Line (NL; 20% of full sample) sources, using as threshold the value of the Full Width Half Maximum (FWHM) of the Hβ line whenever available (65% of the sources in our sample): Hβ ≥ 2000 km s−1 for BL and Hβ < 2000 km s−1 for NL (Osterbrock & Pogge 1987). This distinction between NL and BL refers only to this standard limit on Hβ , while no optical Type 2 objects are present in the sample. When needed, and not derived in this work, source properties are extracted from the CAIXA catalogue. 2.2. The Flux-limited sample The selection criteria with which CAIXA and FERO were assembled do not provide a complete sample in the sense that the sources are not selected according to a physical property: the main point of the FERO project is to include as many objects as possible and this of course depends predominantly on the availability of public observations. Because of the nature of the FERO project, one of the fundamental requirements was to identify a complete, unbiased subsample of sources with high signal to noise in order to derive meaningful constraints on the properties of the (unknown) parent population of local radio-quiet AGN. Hence, sources from the RXTE all-sky Slew Survey (XSS, Revnivtsev et al. 2004) having a count rate in the 3–8 keV energy band greater than 1 cts/sec and fulfilling the FERO source selection criteria were identified. This defines a flux-limited sample of 33 sources. The XSS is nearly 80% complete at the selected flux level for sources with Galactic latitude greater than 10◦ . For two of them, UGC 10683 and ESO 0141-G055, no XMM-Newton data were available as of April 2008. This leaves the number of XSS-selected bright sources in the FERO sample to 31 (listed in Table A.1 in Appendix A). Throughout the paper, these 31 sources will be referred to as the flux-limited sample. 3. Analysis The data corresponding to the 149 observations have been uniformly analysed using SASv6.5 (Gabriel et al. 2004) and the latest calibration files available. Event lists were obtained for the EPIC-pn camera following standard SAS data reduction procedures. Due to its larger eﬀective area, only EPIC-pn (Strüder et al. 2001) data has been considered in this work. The filtering of event lists for periods of high background activity was performed by maximizing the source signal to noise as in Piconcelli et al. (2004). The typical source extraction regions are circular and of the order of 50 in radii for small window mode, while for full frame and large window modes, the source extraction regions range from 20 to 40 in radii. Background spectra were extracted from circular source-free regions, except for observations taken in small window mode where blank-field event lists were used as described in Read & Ponman (2003), of a 50 in radii and close to the target. Spectra aﬀected by a pileup larger than 1% were rejected. Full details of the data reduction and spectra accumulation are reported in Bianchi et al. (2009a). 3.1. XMM-Newton observations The data of the observations presented here were public as of April 2008. All the observations are target observations with exposure times ranging between 1 ks and 400 ks, with 90% of the observations below 100 ks. 3.1.1. Multiple observations When multiple observations of the same source were available, the individual spectra were combined and treated, for all purposes, as a single observation. The FTOOLS tasks mathpha, adrmf, and addarf were used correspondingly to sum the source and background counts spectra and the response matrix and ancillary files, using the exposure times of the individual observations to be summed as weights in these last two cases. However, spectra were only combined if the observations were taken with the same observing mode and if the source was in similar flux state, i.e., if the total flux and power-law spectral index in the 2–10 keV energy band were consistent within the statistical errors. If these criteria were not met, then the observation with the longest exposure time was chosen to prevent introducing sourcerelated bias in the selection. This procedure ensures that mixing diﬀerent observations from a given source does not aﬀect the results derived from our data set. A total of 22 sources in our sample have multiple observations. Table 1 lists on a source-by-source basis XMM-Newton observation IDs that have been combined. For completeness, Table B.1 in Appendix B lists those sources for which multiple observations are available, but only one was considered; so no spectra were combined. This approach is diﬀerent from the one taken in CAIXA, where for sources with multiple observations, the one with the longest exposure was selected systematically as independent of the flux state. The importance of achieving high signal-to-noise spectra for the FERO project, meant that it was preferred to co-add multiple observations of the same source, when available. 3.2. Spectral analysis The time-averaged spectra were re-binned in order not to oversample the intrinsic energy resolution of the EPIC-pn camera (150 eV at 6 keV) by a factor larger than 3, while making sure that each spectral channel contains at least 25 backgroundsubtracted counts to ensure the applicability of the χ2 goodnessof-fit test. Fits were performed in the 2–10 keV energy range. To ensure good quality spectral fits, only those source spectra with at least 17 d.o.f. in this energy range were considered in the analysis. (12 out of the 161 sources in the initial sample did not meet this criterion.) For the spectral analysis and fitting, XSPEC v12.3.0 (Arnaud 1996) was used. Throughout the analysis, solar abundances were assumed after Anders & Ebihara (1982). 3.3. Spectral model All spectra were fitted with the following baseline model: e−σNH · W(Γ, NHi , ξ) · A[E −Γ + C(Γ, R) + 5 Gi + Ky(θ, β, a)] (1) i=1 where the diﬀerent model components are • Galactic absorption (e−σNH ), where NH was fixed to the galactic column density and σ is the photoelectric cross section of the process according to Morrison & McCammon (1983); • Power law (N(E) ∝ E−Γ ), where Γ is the photon index of the primary power-law X-ray source spectrum; Page 3 of 22 A&A 524, A50 (2010) Table 1. List of sources within the FERO sample where multiple observations are available. Source name PG 1440+356 AKN 564 MCG-6-30-15 NGC 5548 MRK 876 ESO15-IG011 MCG-01-13-025 NGC 3516 REJ2248-511 H0557-385 TONS 180 NGC 7469 NGC 3783 List of XMM-Newton observation IDs combined Observation date 0005010101 0005010201 0005010301 0107660201 0006810101 0006810301 0206400101 0029740101 0029740701 0029740801 0111570101 0111570201 0089960301 0109960101 0089960401 0102040601 0102041301 0103861701 0103862001 0103863001 0103861401 0107460601 0107460701 0109070401 0109070601 0109130501 0109131001 0110890401 0110890701 0112170101 0112170301 0207090101 0207090201 0112210101 0112210201 0112210501 2003-01-01 2003-01-04 2003-01-07 2001-12-23 2000-06-17 2001-06-09 2005-01-05 2001-07-31 2001-08-02 2001-08-04 2000-07-11 2000-07-11 2001-07-09 2000-12-24 2001-07-12 2001-04-13 2001-08-29 2000-09-29 2001-10-31 2002-08-28 2000-08-30 2001-04-10 2001-11-09 2000-10-26 2001-10-31 2002-04-03 2002-09-17 2000-12-14 2002-06-30 2000-12-26 2000-12-26 2004-11-30 2004-12-03 2000-12-28 2001-12-17 2001-12-19 Observation exposure time (ks) 17.2 10.6 18.1 24.2 7.4 7.5 69.2 55.8 85.7 86.8 28.8 37.9 58.7 16.0 19.8 2.6 2.4 5.0 4.5 4.3 1.5 64.3 88.7 10.1 9.8 4.0 6.5 20.3 12.6 12.3 16.1 59.2 55.0 26.1 51.2 93.2 Source name 1H0419-577 PG1115+080 IRAS17020+4544 MRK 507 MRK 279 MRK 766 SDSSJ135724.51+652505.9 LBQS1228+1116 MRK 509 List of XMM-Newton observation IDs combined Observation date 0148000401 0148000501 0148000601 0148000201 0148000301 0148000701 0112600401 0203560201 0203560401 0082340101 0206860101 0206860201 0300910401 0300910701 0302480401 0302480501 0302480601 0109141301 0304030301 0304030401 0304030501 0304030601 0304030701 0096020101 0304030101 0305920301 0305920601 0306630101 0306630201 0306090201 0306090301 0306090401 0130720201 0130720101 2003-03-30 2003-06-25 2003-09-16 2002-09-25 2002-12-27 2003-11-15 2000-12-04 2004-06-10 2004-06-26 2001-11-25 2004-08-30 2004-09-05 2005-06-17 2005-09-01 2005-11-15 2005-11-17 2005-11-19 2001-05-20 2005-05-25 2005-05-27 2005-05-29 2005-05-31 2005-06-03 2000-05-20 2005-05-23 2005-04-04 2005-06-23 2005-12-13 2005-12-17 2005-10-18 2005-10-20 2006-04-25 2001-04-20 2000-10-25 Observation exposure time (ks) 11.1 10.7 11.3 11.8 7.6 1.1 5.7 65.5 71.2 53.8 13.4 12.3 15.8 11.4 41.4 40.7 22.8 89.6 68.9 65.8 64.9 63.1 20.4 25.7 66.2 21.1 12.0 60.4 83.2 59.8 32.4 48.6 27.8 20.7 Notes. Multiple observations were combined where available and treated, for all purposes, as a single observation. Observation IDs in bold correspond to those observations that were combined, while observation IDs not marked in bold were discarded. • Compton reflection from neutral material (C(Γ, R)), where R is the reflection factor as compared to material emitting over 2π in solid angle (see however Murphy & Yaqoob 2009 for some caveats about this interpretation). The high-energy cutoﬀ of the primary spectrum was fixed to 100 keV. The viewing angle of the reflecting material was fixed to 18◦ , while the reflection normalization (R) was left free during the fitting procedure. The model used was the XSPEC PEXRAV component (Magdziarz & Zdziarski 1995); • Ionised absorption (W(Γ, NHi , ξ)) from warm gas in the local AGN environment was introduced through the XSPEC model ABSORI. Although more accurate and complex models exist for warm absorption, they would overfit the lowerquality data in our sample when uniformly applied to the whole sample. Both the intrinsic column density (NHi ) and ionisation parameter (ξ) were left free during the fitting procedure, while the continuum slope (Γ) was tied to the photon index of the primary power-law continuum. The temperature of the absorber was fixed to 2×105 K (∼17 eV), Page 4 of 22 representative of the temperature found in warm absorbers (see e.g. Krongold et al. 2007); • Narrow lines (Gi ): 4 zero-width Gaussian lines were included with centroid energies fixed at 6.4 keV (neutral Fe I Kα ), 6.7 keV (ionised Fe XXV), 6.96 keV (ionised Fe XXVI), and 7.06 keV (neutral Fe I Kβ ). The normalization of the Fe I Kβ line was limited to less than 16% that of the Fe I Kα line flux (Molendi et al. 2003). The two ionised lines were included in the baseline model as several studies show that they represent an important spectral component in AGN. Among the possible physical interpretations of these features are an origin in the accretion disc (e.g. Reeves et al. 2001; Pounds et al. 2001; Miller et al. 2007) or produced in photoionised circumnuclear matter (e.g. Bianchi & Matt 2002; Bianchi et al. 2005); • Fe I 6.4 keV Compton shoulder (Gi ). The Fe I Kα Compton shoulder of the narrow component of the Fe I fluorescent line was considered by using a Gaussian line at 6.3 keV, with 0.05 keV as the fixed width and free normalization allowed I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN to be no more than ∼20% that of the 6.4 keV Fe I Kα line, following Matt (2002); • Relativistic line (Ky(θ, β, a), Kyrline model). To model the eﬀect of the strong gravitational field on the emission regions of the disc closest to the central black hole, the Ky set of models within XSPEC was used (Dovčiak et al. 2004). The model considers accretion disc emission around a rotating black hole with a given spin, a. In contrast to models such as diskline or laor, Kyrline allows the spin to be fitted as a free parameter. For the line emission, a fixed centroid energy of 6.4 keV (in the rest frame of the source), is assumed, while the emission between the innermost stable orbit and 400 gravitational radii is integrated, keeping these limits fixed during the fitting procedure. The radial dependence of the disc emission is modelled with a power law of index β (∝ r−β ), while Laor’s limb darkening law was adopted to characterize the angular dependence. Here, the radial disc emissivity is referred to as β. The disc inclination angle (θ), β, and a are free parameters during the fitting procedure. In summary, the model that was uniformly fitted to our data consists of 9 components with a total of 14 free independently-fitted parameters. For those cases in which the value of the FWHM of the Hβ line is available, the model has 15 free parameters (see Sect. 3.3.1). The fitting procedure was carried out over diﬀerent steps. First, the power law with neutral reflection and absorption were fitted to the data, followed by the addition of the narrow line components one by one (in the order listed above). The last model component added to the fit was the relativistic line. After each addition of a new component, a fit was performed, and no model component discarded regardless of the goodness-of-fit. This implies that the χ2 values obtained from the fits correspond to the best-fit given by this model. Its important to stress that no attempt has been made as a whole or on a source-by-source basis to find the best-fit model yielding the lowest possible χ2 value. For reference, Table C.1 gives, for those sources belonging to the flux-limited sample, the most relevant parameters of the best fit for the model considered. 3.3.1. Limitations to model parameters During the fitting procedure, some limitations are introduced for the following parameters of the baseline model: ◦ The disc inclination angle in the Kyrline model was limited to a maximum value of 60◦ . In the Seyfert unification scenario (Antonucci 1993), Type 1 objects are not expected to have large viewing angles. It has been found that relaxing the restriction on the inclination angle yields a number of detections of broad and highly skewed lines with equivalent widths of the order of 1 keV. This eﬀect is simply because in sources with poor statistics above 7 keV, the broad line model fits the continuum, also reflected by required large disc-inclination angles (>80◦). ◦ The disc emissivity in the Kyrline model was limited to a maximum value of 6 given that steeper profiles are not expected, even taking strong general relativistic eﬀects into account on the primary emission such as light bending (see Miniutti & Fabian 2004). ◦ To possibly constrain the parameter space, limitations to the spin parameter in the Kyrline model were tested. Three fitting runs were produced with black hole spin fixed to 0.998 (Kerr), fixed to 0 (Schwarzschild), and free to vary between 0 and 0.998. In the first two cases, no systematic diﬀerence was found when comparing the results in terms of χ2 improvement, leading us to conclude that none of the two extreme spin values can be assumed a priori. Therefore, the results presented are extracted from the run where the black hole spin is left free to vary during the fitting procedure. ◦ The power-law index (Γ) and the reflection fraction (R) were limited following the work by Dadina (2008) on a BeppoSAX sample of Type 1 Seyfert galaxies. Given the distributions of Γ and R derived by Dadina (2008), a limit of three times the 1σ standard deviation of the average values is chosen for Γ and R (Γ ∈ [0.45:3.33]; R ∈ [0.:3.33]). ◦ When available in the literature, the value of the FWHM of the optical Hβ line was used to set an upper limit to the width of the narrow component of the 6.4 keV neutral Fe I Kα line, leaving the width of the line free. This choice is consistent with the possibility that for some sources, the Fe I Kα line may be produced in the optical broad line region (see e.g. Bianchi et al. 2008 and references therein). When the Hβ FWHM was not available, the Fe I Kα line width was assumed to be unresolved (σ ≡ 0). 3.3.2. Test of spectral model components During the process of selecting the baseline model, several tests were performed with diﬀerent model components. The most important ones are summarised below. ◦ To contemplate the hypothesis that the reprocessed AGN continuum in Type 1 objects is made by contribution of both torus and disc reflection, the presence of two reflection components at the same time was tested. The second PEXRAV disc component was relativistically blurred using the convolution model kdblur, to take possible relativistic eﬀects into account. After close inspection of the contribution from each physical system, it was concluded that the sensitivity band of XMM-Newton does not allow disentangling the reflection component from the torus from that of the disc. Since the presence of torus reflection is very well supported observationally by the almost ubiquitous narrow Fe I Kα component (Bianchi et al. 2004; Yaqoob & Padmanabhan 2004), only this PEXRAV component has been included in our baseline model. ◦ The eﬀect of reflection from an ionised accretion disc was tested by replacing the torus reflection with the model component PEXRIV (Magdziarz & Zdziarski 1995): Eq. (1) is thus modified to include the disc ionisation parameter ξ in the function C(Γ, R), which is left free during the fitting procedure. The centroid energy in the Kyrline model was consistently set to 6.7 keV. The results from this test are discussed in Sects. 4 and 5. 4. Results The EW of the Kyrline component was used as a proxy for establishing significant detections of relativistic Fe I Kα lines in the sample. The line EW is defined in this work as the ratio of the flux of the Kyrline component to the flux of the continuum, as defined in Sect. 3.3, integrated over the energy range between ±5% around the line rest-frame energy (6.4 keV). The error in the normalization of the Kyrline component is used to estimate the uncertainty in the corresponding line EW. The threshold for significant detections is set to 5σ confidence level (c.l.). This is a somewhat more conservative approach than normally taken when reporting relativistic line detections in individual objects, Page 5 of 22 A&A 524, A50 (2010) Fig. 1. Equivalent width of the relativistic broad 6.4 keV Fe Kα line vs. hard X-ray counts (2– 10 keV) for the FERO sources. Filled circles indicate line detections at ≥5σ confidence level (where error bars indicate the 90% confidence level intervals), while filled squares indicate line upper limits at the 90% confidence level. White stars indicate sources belonging to the flux-limited sample (see Sect. 2.2, for details). but it has to be kept in mind that the FERO project is a statistical study of a large sample that includes spectra of very diﬀerent levels of statistical quality. For those sources with no significant line detection, the upper limits to the line EW at the 90% c.l. are provided. In the following, errors, upper and lower limits, on the relevant parameters are given at the 90% c.l. for one interesting parameter (Δχ2 = 2.71). 4.1. Sources with detected broad Fe lines. Figure 1 shows the EW of the broad Fe Kα line at 6.4 keV as a function of source counts in the 2–10 keV band. This plot shows two things. First, significant broad Fe Kα line detections are concentrated in the portion of the plot dominated by spectra 5 with good statistical quality (roughly with > ∼1.5×10 hard X-ray counts). Second, overall, the distribution of line EW upper limits versus hard X-ray counts follows the expected trend; i.e., in the absence of a significant broad line, √ the sensitivity (the ability to detect a broad line) goes as (1/ (cts2−10 keV ). The dispersion of EW upper limits around this direction, for a given range of hard X-ray counts, could be explained in terms of scattering of the model parameters. Table 2 provides the detailed list of the significant relativistic broad Fe Kα line detections with the best-fit parameters of the Kyrline model. From now on, this table is referred to as the list of detected relativistic Fe Kα lines in the FERO sample. In the fitting procedure, relativistic eﬀects from neutral and ionised reflection were tested as explained in Sect. 3.3.2. The test for a neutral Fe Kα broad line yields in total 11 significant detections at ≥5σ c.l.. The fit with the model with an ionised Fe Kα line yields a detection in all the 11 sources, except IC4329A, 1H0707-495, and MCG-5-23-16, which only provide detection in the neutral case. In the 8 sources where a relativistic broad Fe Kα line is equally detected by means of neutral and ionised Page 6 of 22 reflection models, the best-fit models were carefully examined and the model with the lowest χ2 preferred. MRK 509 is the only source for which the ionised reflection provides a significant better-fit statistic (with the probability of the improvement being by chance of 2.2 × 10−7 ). MRK 766 and ARK 120 on the contrary, show significant broad Fe lines only in the ionised case. At the end, neutral and ionised runs were merged, which yields 13 sources with strong evidence of a broad Fe line. Last, it is worth pointing out that the only source where a relativistic broad Fe Kα line is claimed for the first time in this work is ESO511-G030, with an EW of 57+30 −11 eV. Two sources, PG1211+143 and 1H0707-495, are characterised by extremely high EW of the Fe Kα line (Figure 1, see also Table 2). The data included in our sample for both sources have similar spectral shapes, showing deep spectral features at ∼7 keV and spectral curvature in the Fe energy band. Pounds et al. (2003a) argue that PG1211+143 presents a high column density (5 × 1023 cm−2 ) of highly ionised matter along the line of sight partially covering the central hard X-ray source and that the apparent presence of a relativistic Fe Kα emission line could be an artifact of absorption. However, the relativistic broad line interpretation is not discarded, and the same authors find that a broad line with a large EW, of the order of 600–900 eV, and a combination of absorption features, provide a good interpretation of the data. This value of the Fe Kα line EW is in good agreement with the value found in this work (635+341 −247 eV). Nevertheless, whether there is a relativistically broadened Fe Kα line in this source is debatable, as new results from a more recent analysis on data from 2007 indicate (Pounds et al. 2009). 1H0707-495 also shows a prominent flux drop around 7 keV, which can be modelled with an absorption edge assuming large Fe overabundance (Gallo et al. 2004). Fabian et al. (2004) interpret the drop around 7 keV in terms of relativistically blurred ionised reflection from the accretion disc, and derive an EW for I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN Table 2. Sources within the FERO sample where a relativistic Fe Kα line is detected with a significance ≥5σ. Source Type(1) keV L2−10 X (1042 erg s−1 ) Cts2−10 keV (105 cts) EW (eV) θ (◦ ) a β χ2 /d.o.f. IC 4329A MCG-5-23-16 ESO511-G030 MCG-6-30-15 NGC 4051 NGC 3516 NGC 3783 NGC 3227 MRK 509 () MRK 766 () ARK 120 () 1H0707-495 PG 1211+143 BLSY NCSY NCSY BLSY NLSY BLSY BLSY BLSY BLQ NLSY BLSY NLSY NLQ 56.4 14.2 22.4 5.4 0.3 3.6 11.4 1.2 103.5 6.1 90.5 46.8 49.4 9.123 ± 0.009 8.853 ± 0.009 1.740 ± 0.004 13.927 ± 0.012 2.040 ± 0.004 2.810 ± 0.005 9.440 ± 0.010 3.541 ± 0.006 7.471 ± 0.009 7.816 ± 0.008 3.473 ± 0.006 0.110 ± 0.001 0.171 ± 0.001 39+14 −6 36+10 −6 57+30 −11 260+19 −18 260+56 −77 227+41 −78 88+11 −11 66+25 −11 170+26 −19 234+23 −49 140+31 −41 1775+511 −594 +341 635−247 28+6 −11 21+8 −3 18+7 −7 40+1 −3 22+6 −6 27+2 −3 <8 23+4 −4 53+1 −1 20+3 −2 >59 54+3 −3 52+4 −7 ≥0.0 ≥0.0 ≥0.0 +0.01 0.86−0.02 >0.46 >0.48 >0.16 ≥0.0 +0.03 0.78−0.04 >0.47 ≥0.0 ≥0.93 ≥0.92 <1.3 <1.6 <1.1 +0.2 4.1−0.2 +0.3 2.9−0.4 +0.2 2.8−0.3 +0.1 2.7−0.2 +0.6 1.9−0.5 >3.8 +0.2 2.7−0.1 +0.6 2.2−0.3 +1.22 4.13−0.65 >4.37 238.1/161 271.4/162 198.2/161 227.6/161 198.8/162 223.1/161 304.5/161 230.9/162 277.3/158 300.3/160 212.3/158 107.6/116 160.7/139 References(2) (1), (2), (3), (4) (5), (6) (7), (8), (9), (10) (11), (12), (13) (14), (15), (16) (17), (18) (19), (20) (21), (22) (23), (24) (25) (26), (27), (28) (29), (30) Notes. () 6.7 keV Kα line is preferred; * Sources for which there is a significant detection of a relativistic ionised 6.7 keV Kα line (see text for details). The first 11 sources listed in the table belong to the flux-limited sample. θ, a, and β are the disc inclination angle with respect to the observer, the black hole spin and disc emissivity, respectively, of the best-fit parameters of the Kyrline model. Except for the hard X-ray counts where 1σ errors are given, errors in any given parameter are given at the 90% confidence level. Also, upper and lower limits in any relevant paremeter are given at the 90% confidence level. (1) SY: Seyfert, Q: quasar, NL: narrow line, BL: broad line, NC: not classified. (2) References where evidence for a relativistic Fe Kα line for a particular object is reported (references marked in bold) as well as those where alternative scenarios are provided. The list of references is not meant to be complete. References. (1) Done et al. (2000); (2) McKernan & Yaqoob (2004); (3) Steenbrugge et al. (2005); (4) Gondoin et al. (2001); (5) Reeves et al. (2007); (6) Braito et al. (2007); (7) Wilms et al. (2001); (8) Fabian et al. (2002); (9) Vaughan & Fabian (2004); (10) Miller et al. (2008); (11) Ponti et al. (2006); (12) Uttley et al. (2004); (13) Pounds et al. (2004); (14) Iwasawa et al. (2004); (15) Nandra et al. (1999); (16) Turner et al. (2008); (17) Tombesi et al. (2007); (18) Reeves et al. (2004); (19) Markowitz et al. (2009); (20) Gondoin et al. (2003); (21) Ponti et al. (2009); (22) Page et al. (2003); (23) Pounds et al. (2003b); (24) Miller et al. (2007); (25) Vaughan et al. (2004); (26) Fabian et al. (2009); (27) Fabian et al. (2004); (28) Gallo et al. (2004); (29) Pounds et al. (2003a); (30) Pounds et al. (2009). the broad Fe Kα line of 1800 eV, in agreement with the value found in the FERO analysis (1775+511 −594 eV). Fabian et al. (2004) proposed that the X-ray spectrum of this source is disc reflection dominated, and as the high value of the line EW implies, high element abundances were required, in a similar way to absorptiondominated models (Gallo et al. 2004). Recently, Fabian et al. (2009) have presented new timing-based arguments in favour of the relativistic nature of the broad features in this source with the simultaneous detection of broad Fe L and K lines1 . However, Miller et al. (2010) has recently claimed that the Fabian et al. (2009) interpretation of the timing properties of the source is not unique and that solutions that do not invoke relativistic reprocessing might still be viable. Figure 2 shows the ratios of the data to the continuum and data to the best-fit model in the 2–10 keV energy range for the sources with a relativistic 6.4 keV Fe Kα broad line detection. For MRK509, MRK766 and ARK120, the figures were produced with the model including the ionised continuum and 6.7 keV Fe Kα line. The latter set of plots indicates how well the baseline model used in this work describes the data. The deviation of the best-fit model and the data is limited below ∼10% in the whole 2–10 keV energy range used for fitting, and below ∼5% if one excludes the last energy bins (>8 keV) with poorer statistics. Despite the fact that by selection the sources in FERO are unobscured (NH < 2 × 1022 cm−2 ) for two sources, MCG-5-23-16 and IC4329A, a downturn in the ratio plot below 3 keV is noticeable and could indicate extra cold absorption, which in principle would not have been taken into account 1 We point out that the XMM-Newton spectra of 1H0707-495 included in the FERO sample correspond to the ones used by Fabian et al. (2004) and that Fabian et al. (2009) used a diﬀerent dataset. by our baseline model. However, for the particular case of these two sources, it is believed that this downturn cannot be attributed to extra cold absorption since the parameters derived from the warm absorption model (ABSORI) used yield values compatible with those extracted from CAIXA (as seen in Table C.1), 22 −2 +0.04 22 −2 NH (z = 0) (1.20+0.13 −0.02 ) × 10 cm , and (0.33−0.02 ) × 10 cm for MCG-5-23-16 and IC4329A, respectively, and in both cases, compatible with neutral absorption (ξ < 0.02 erg cm−1 s−1 for MCG-5-23-16 and ξ < 0.47 erg cm−1 s−1 for IC4329A). For these two sources, any extra neutral absorption that is present should have been taken into account by the model component ABSORI. Table 2 lists in the last column the χ2 and corresponding number of degrees of freedom for those sources with evidence of a relativistic Fe Kα line. 4.2. Fraction of relativistic Fe lines 4.2.1. Detection fraction in FERO The fraction of relativistic Fe lines detected in the FERO sample is 9% (13/149). Considering only the sources in the fluxlimited sample, the detection fraction rises to 36% (11/31). These two numbers do not provide specific information on the intrinsic fraction of AGN that have broad Fe lines. They are limited by FERO being made of spectra of disparate quality and by the unavailability of a well-defined complete AGN sample. Nevertheless, the observed detection fraction can be considered as a lower limit for the intrinsic number of AGN that would show a broad Fe line if, for example, all sources were observed with the same signal-to-noise. Page 7 of 22 A&A 524, A50 (2010) 1.4 1.2 0.8 1.4 1.2 1 Data/model ratio 0.8 1.4 1.2 1 Data/model ratio 0.8 1.4 1.2 1 Data/model ratio 0.8 5 Energy (keV) 1.4 1.2 1 Data/model ratio 0.8 2 5 Energy (keV) 0.8 1 Data/model ratio 1.2 1.4 Full Relativistic − ARK120 (0147190101) 1.4 1.2 Data/model ratio 0.8 5 Energy (keV) 2 5 Energy (keV) 2 5 Energy (keV) 1.2 1 0.8 0.8 1 Data/model ratio 1.2 1.4 Full Relativistic − MRK509 (0000000022) 1.4 Full Relativistic − MRK509 (0000000022) Data/model ratio 5 Energy (keV) 1 1.4 1.2 Data/model ratio 2 1 Data/model ratio 1.2 1 1.4 1.2 Data/model ratio 1.4 1.2 1.4 1.2 Data/model ratio 1.4 1.2 Data/model ratio 2 Full Relativistic − ARK120 (0147190101) 0.8 1 1.4 1.2 1 2 Full Relativistic − 1H0707−495 (0148010301) 0.8 5 Energy (keV) 5 Energy (keV) 5 Energy (keV) 1 1.4 1.2 Data/model ratio 0.8 2 5 Energy (keV) Full Relativistic − ESO511−G030 (0502090201) Full Relativistic − 1H0707−495 (0148010301) Full Relativistic − MRK766 (0000000019) 0.8 2 2 0.8 2 1 1.4 1.2 1 5 Energy (keV) 5 Energy (keV) Full Relativistic − MCG−5−23−16 (0302850201) 5 Energy (keV) 1 1.4 Data/model ratio 1.2 5 Energy (keV) Full Relativistic − PG1211+143 (0112610101) Full Relativistic − MRK766 (0000000019) 2 Full Relativistic − ESO511−G030 (0502090201) 0.8 2 0.8 2 1 Data/model ratio 2 1 1.4 1.2 1 0.8 5 Energy (keV) 5 Energy (keV) 0.8 5 Energy (keV) Full Relativistic − NGC3227 (0400270101) 5 Energy (keV) Full Relativistic − NGC4051 (0109141401) Full Relativistic − MCG−5−23−16 (0302850201) 1.4 1.2 1 Data/model ratio 2 Full Relativistic − PG1211+143 (0112610101) 2 0.8 2 0.8 5 Energy (keV) Full Relativistic − NGC3227 (0400270101) 2 1 1.4 Data/model ratio 1.2 5 Energy (keV) Full Relativistic − IC4329A (0147440101) 1.4 1.2 1 2 5 Energy (keV) Full Relativistic − NGC4051 (0109141401) 0.8 2 0.8 Data/model ratio 2 1 1.4 1.2 Data/model ratio 1 5 Energy (keV) Full Relativistic − IC4329A (0147440101) Data/model ratio 5 Energy (keV) Full Relativistic − NGC3783 (0000000013) 0.8 2 Full Relativistic − NGC3516 (0000000008) 0.8 2 5 Energy (keV) Full Relativistic − NGC3783 (0000000013) Data/model ratio Data/model ratio 1.2 Data/model ratio 0.8 1 1.4 1.2 1 Data/model ratio 0.8 2 Data/model ratio Full Relativistic − NGC3516 (0000000008) 1.4 Full Relativistic − MCG−6−30−15 (0000000003) 1.4 Full Relativistic − MCG−6−30−15 (0000000003) 2 5 Energy (keV) 2 5 Energy (keV) Fig. 2. Set of two figures for those sources with a relativistic 6.4 keV (6.7 keV) Fe Kα line EW detection ≥5σ confidence level: data to the best-fit continuum ratio (left set column), and data to the best-fit model ratio (right set column). The vertical dashed line is placed for reference at 6.4 keV (6.7 keV) in the rest frame of the source. On top of each figure the model used for the ratio, the corresponding source name, and the observation ID are given. For those sources with summed observations a dummy observation ID is given. Page 8 of 22 I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN It is possible to estimate statistical errors on the detection fractions to reflect the uncertainty introduced by considering a limited number of sources. Detection fractions are calculated as val1 /val2 , where val1 and val2 indicate a given number of sources used to work out a given detection fraction. For the errors of val1,2 , Poisson statistics are assumed, where the errors are calculated as the average of the upper (ΔUL ) and lower limit (ΔLL ) of the 84% c.l. (which correspondto the commonly used ΔUL = 1 + val1,2 + 0.75 and ΔLL = val1,2 − 0.25, according to Gehrels 1986), both of which are good approximations for this moderate confidence level and are accurate to better than 5% even for low values of val1,2 (see Gehrels 1986, for more information). The error of val1 /val2 is propagated quadratically. In this way, the detections fractions and their associated errors are 9 ± 3% and 36 ± 14% for the FERO sample and for the fluxlimited sample, respectively. A broader understanding of the incidence of the relativistic line in our work can be achieved by considering the upper limits on the line intensity that characterize the majority of the sources. Within the FERO sample, ∼16 ± 4% of broad line EW upper limits are below 100 eV. This value corresponds to the EW of a neutral disc with an inclination of 60◦ , for an illuminating power law of Γ = 2 and solar abundances (Matt et al. 1992). If the analysis is restricted to the 20 sources with no significant broad line within the flux-limited sample, the fraction of sources in which the line EW is limited below 100 eV is 50% (10/20) (for reference, for 75, 50 and 25 eV, the fractions are respectively 25%, 20%, and <13%). However, 5 sources within the flux-limited sample have measured EWs lower than and inconsistent with this threshold, implying deviations from the standard scenario either in terms of very high disc inclination (unlikely given the relatively unobscured nature of these AGN), element abundances lower than solar or a non-standard accretion disc geometry (George & Fabian 1991). Last, Table D.1 in Appendix D lists the 20 sources with broad line upper limits in the flux-limited sample. In particular, four sources (MRK 279, NGC 5548, ESO 198-G24 and MRK 590) exhibit upper limits below 40 eV, arbitrarily chosen to be comparable to the lowest line EW detected. Explaining line EWs below this value in the framework of standard accretion theories is not straightforward, and it requires the use of non-standard system properties. Assuming the above threshold, the non-detection fraction of broad lines in the flux-limited sample can then be calculated as 13% (4/31). Otherwise stated, the presence of a broad line cannot be excluded a priori in the remaining 87% of the sources in the flux-limited sample (although the line is formally detected only in 36% of the sources at a 5σ level). This fraction is interpreted here as the upper limit to the fraction of AGN exhibiting relativistically broadened Fe lines in the parent population. 4.2.2. Detection fraction and warm absorber In some cases, the use of the model component ABSORI could be regarded as too simplistic, however necessary for the approach considered here. Nevertheless, the influence of the selected warm absorption component for the baseline model in determining the detection fraction was tested by running a consistency check over the 31 sources belonging to the flux-limited sample. The test replaces the model component ABSORI by the more complex and complete ZXIPCF model and looks for a significant change in the fraction of detected broad lines and the average broad line EW. ZXIPCF uses a grid of XSTAR photoionised absorption models and allows defining a covering fraction, which has been fixed to 1, and column density and ionisation parameters of the absorbing material, which have been left free during the fitting procedure. The results of this test show that the introduction of the ZXIPCF model component does not significantly provide a better fit, and yield a detection fraction of broad iron Kα lines of 39 ± 15% (12/31). This result is fully consistent with the 36 ± 14% (11/31) derived when using ABSORI for the warm absorption, and concludes that the detection fraction is not significantly aﬀected by the approach taken in this work to model the warm absorber. Out of the sources from the flux-limited sample listed in Table 2, only ESO511-G030 does not show evidence of a broad line at the 5 sigma level when using ZXIPCF. In turn, MRK704 and NGC4593 show a broad line at the 5.5σ and 5.3σ levels, respectively, both at the limit of our detection threshold of 5 σ. The average line EW derived from the detected lines is EW = 154 ± 112 eV ( EWw = 73 ± 3 eV), again both consistent with the results derived when using the absorption model ABSORI (see Sect. 4.3.1). 4.2.3. Detection fraction and X-ray luminosity Eleven out of the 13 lines detected correspond to sources classified as Seyfert galaxies, while only 2 come from a source classified as quasar. In a similar manner to the previous section, this corresponds to a detection fraction of 13 ± 5% and ≤6% for Seyfert galaxies and quasars respectively. If the exercise is restricted to sources in the flux-limited sample, the detection fractions are 36 ± 15% and ≤92% for Seyfert galaxies and quasars, respectively. Owing to the low number of lines detected in quasars and the low number of quasars present in the flux-limited sample, only upper limits to the detection fraction can be derived. Figure 3 shows the distribution of the 2-10 keV luminosity for the FERO sample, highlighting those sources for which a significant relativistic broad Fe Kα line detection has been found. All broad Fe line detections correspond to objects with luminosities below ∼1044 erg s−1 . To investigate a possible dependence of relativistic broadening on the source luminosity, the following three luminosity bins are defined: L1: LX < 0.25 × 1044 erg s−1 ; L2: 0.25 × 1044 < LX < 1.20 × 1044 erg s−1 ; L3: LX > 1.20 × 1044 erg s−1 . Considering the FERO sample, each bin was chosen to contain the same number of sources (∼50), with no further distinctions (see Table 3). No significant diﬀerence is found between the number of sources with a relativistic line when comparing the lowest luminosity bin L1 to the intermediate luminosity bin L2, either using the sources from the FERO (0.7σ) or flux-limited (0.5σ) sample. The highest luminosity bin L3 contains no detections. However, with the uncertainties in the detection fraction in the L3 luminosity bin (see Table 3), it is possible to estimate the diﬀerence in detection fraction between the L1 and L3 luminosity bins, at the 2.2σ and 0.9σ levels for the FERO and flux-limited samples, respectively. To remove the dependency with luminosity and check whether an intrinsic diﬀerence exist between Seyfert galaxies, and quasars in terms of detection fraction, a luminosity range containing the same number of Seyfert galaxies, and quasars is selected (L: 0.15 × 1044 < LX < 2.69 × 1044 erg s−1 , Table 3). The detection fractions are 10 ± 7% (4/40) and ≤10% (2/40) for Seyfert galaxies and quasars, respectively, for sources in the FERO sample, and 18 ± 14% (3/17) and ≤92% (1/3) for sources in the flux-limited sample. The low number of detections prevents any statistically significant results from being drawn. Table 3 summarizes the diﬀerent detection fraction for the diﬀerent source Page 9 of 22 A&A 524, A50 (2010) Table 4. Weighted mean ( EWw ) and 1σw standard deviation, and mean ( EW) and 1σ standard deviation, of the Fe Kα line EW distribution. Source Class SY+QSO (11) SY (10) QSO (1) EWw σw EW σ (eV) 76 ± 3 72 ± 3 170 ± 22 (eV) 39 37 – (eV) 143 ± 27 141 ± 30 170 ± 22 (eV) 91 95 – Notes. Only sources with an Fe Kα broad line detection within the fluxlimited sample were used to compute these values. A distinction has been made according to source class. The number of sources used in each case to derive these values is in parenthesis). 4.3. Average properties of the relativistic broad Fe Kα line within the flux-limited sample Fig. 3. X-ray luminosity distribution in the 2–10 keV energy band for the FERO sample. This sample has been split into Seyfert galaxies (Sy; 82 sources), quasars (QSO; 67 sources), narrow line (NL; 30 sources) and broad line (BL; 60 sources). The shaded area corresponds to the luminosity of those sources within the flux-limited sample for which a significant relativistic broad Fe Kα line detection has been found. The numbers shown on the right in each panel are the median values of the correspondent distribution, also marked by the vertical dashed line. Table 3. Fraction of sources in the FERO sample with a significant relativistic broad Fe Kα line EW detection for diﬀerent source type and luminosity class. Obj. Class/ Luminosity Sy+QSO Sy QSO NL BL L1 (Sy+QSO) L2 (Sy+QSO) L3 (Sy+QSO) L Sy L QSO Sample FERO Flux Limited (%) (%) 9 ± 3 (13/149) 36 ± 14 (11/31) 13 ± 5 (11/82) 36 ± 15 (10/28) ≤6 (2/67) ≤92 (1/3) 13±9 (4/30) ≤100 (2/4) 12±6 (7/60) 35 ± 18 (7/20) 16 ± 7 (8/50) 44 ± 22 (8/18) 10 ± 6 (5/50) 27 ± 23 (3/11) ≤2 (0/49) ≤47 (0/2) 10 ± 7 (4/40) 18 ± 14 (3/17) ≤10 (2/40) ≤92 (1/3) Notes. Sy: Seyfert galaxy, QSO: quasar; NL: narrow line, BL: broad line; L1: LX < 0.25 × 1044 erg s−1 ; L2: 0.25 × 1044 < LX < 1.20 × 1044 erg s−1 ; L3: LX > 1.20 × 1044 erg s−1 , where L1, L2 and L3 were chosen to contain the same number of sources (∼50); L: 0.15 × 1044 < LX < 2.69 × 1044 erg s−1 , L was chosen to contain the same number of Seyfert galaxy and quasars (40) (see text for details). and luminosity class within the whole FERO and flux-limited samples. The correlation between the Fe Kα line EW and the hard X-ray luminosity based on individual source detections and within the flux-limited sample is investigated in Sect. 4.4. This analysis is extended to the remainder of the FERO sample in the companion paper by Longinotti et al. Page 10 of 22 In the next two sections, the average properties of the relativistic broad Fe Kα line are discussed, first, in terms of the line equivalent width and second, in terms of the disc parameters derived from the Kyrline model. To derive meaningful results, only the 31 sources in the flux-limited sample are considered; therefore, information from the 11 sources (the first 11 sources listed in Table 2) with a broad Fe Kα line detection and the 20 sources with an upper limit of the broad Fe Kα line are used (see Table D.1). 4.3.1. Line equivalent width For each source with a significant relativistic broad line detection, the measured value of the broad Fe Kα line EW is always below 300 eV (see Table 2). Considering all the detected lines, the mean value of the distribution of broad Fe Kα line EW is 143 ± 27 eV, with a 1σ standard deviation of 91 eV. It has to be kept in mind that the derived value for the mean is likely to overestimate the true mean of the distribution. The explanation can be found in the fact that the parent distribution of line EW is truncated by the ability to detect weak lines, which can only be found in sources with very a high signal-to-noise spectrum (see Fig. 1). To derive a more representative value of the mean EW, the weighted mean of the Fe Kα broad line EW distribution was calculated using the 1σ statistical errors on the line EW for the individual weights. In this way, a weighted mean value of 76 ± 3 eV with 1σ standard deviation of 39 eV is obtained. This value is lower than the mean value derived above, and consistent with it to within ∼1.3σ. There are two important caveats regarding the weighted mean. One, the calculation of the weighted mean assumes that the individual values of the line EW share the same underlying distribution and that such distribution is Gaussian, which does not necessarily have to be the case. Second, the use of the inverse square of the individual errors in the calculation of the weighted mean implies that, for a set of measurements with the same relative errors, the weighted mean is biased towards the lower values in the set. With a few exceptions, the relative errors in the line EW are comparable regardless of the value of the line EW, which implies that lower values of the line EW contribute more to the weighted mean. As a result, one could consider these two values, i.e. the mean and the weighted mean, one biased towards higher EW values and the other towards lower EW values, as the limits where the true mean of the distribution of relativistic broad Fe Kα line EW in AGN would be expected. Table 4 summarizes these results. I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN Table 5. Mean and 1σ standard deviation of the best-fit Kyrline model parameters for sources with a relativistic broad Fe Kα line detection. Line Parameter (Kyrline) θ(◦ ) β Mean σ 28 ± 1 2.4 ± 0.1 5 0.4 Notes. With only two spin measurements, the mean black hole spin is omitted. Fig. 4. Normalised distribution (shaded area) for the line EW for sources with a significant broad Fe Kα line detection within the flux-limited sample. The 1-dimensional histograms of this distribution is overlaid (white histogram). Figure 4 shows the normalised distribution of the line EW for sources with a significant broad Fe Kα line detection within the flux-limited sample. This distribution is generated by combining one Gaussian distribution per measurement, where each Gaussian takes the mean and standard deviation of the corresponding best-fit-parameter value and its error at the 1σ level (upper or lower limits are not present in this plot). Overlaid on top of the Gaussian distribution is the corresponding 1-dimensional histogram, highlighting the contribution of each individual measurement to the overall distribution. Both distributions are normalised to their respective peak values. 4.3.2. Disc parameters The Kyrline model parameters θ, β, and a respectively correspond to the disc inclination angle with respect to the observer, the power-law index of the radial disc emissivity law, and the black hole spin, and together they provide important information on the properties and conditions of the accretion disc. These values are reported in Table 2 for the sources with a significant broad Fe Kα line detection. For some sources, these parameters could not be constrained in the spectral fit and in these cases lower or upper limits are reported. Using only the parameters from the 11 line detections within the flux-limited sample, the mean and 1σ standard deviation values of θ, β and a are derived via the censored mean method described in Bianchi et al. (2009a). While these authors include measurements and upper limits, lower limits are also considered here. Table 5 lists these mean values and the 1σ standard deviation obtained using this method. (With only two spin measurements, the mean black hole spin is omitted.) Figure 5 shows normalised distributions for θ and β. As for the case of the distribution of the line EW in Sect. 4.3.1, these distributions are generated by combining one Gaussian distribution per measurement. The black hole spin is constrained well only in two cases, MCG-6-30-15 and MRK509, +0.03 with a best-fit value of 0.86 (+0.01 −0.02 ) and 0.78 (−0.04 ), respectively. The mean value of β is about 2.4, but with a wide spread of values. Among the 11 sources, 6 have values of β higher than and inconsistent with 2, which means that emission is dominated by the innermost accretion disc regions. In three sources β is definitely lower than 2, and the emission is therefore dominated by the outermost regions. It must be recalled that a power law is often a poor approximation of the real emissivity law, which depends on the (unknown) geometry of the emitting corona. (See for instance Martocchia et al. 2000; Martocchia et al. 2002, for the emissivity laws in the lamp-post geometry.) It is, however, worth noting that in two sources, MCG-6-30-15 and MRK 509, β is about 4, or larger, which, under the lamp-post assumption, would imply an emitting source located at only a few gravitational radii from the black hole (Martocchia et al. 2002). In addition and for the case of MCG-6-30-15, Wilms et al. (2001) argue that this is not suﬃcient to explain the high β value and conclude that it is necessary to invoke magnetic extraction of the black hole spin energy to explain it (Blandford & Znajek 1977). Not much can be said about the spin distribution. In five cases this parameter is totally unconstrained, since any permitted value of a is acceptable, and in another case it is rather poorly constrained (a > 0.16). In three cases, lower limits of about 0.5 can be placed, while in the remaining two sources, the measurements imply high values of the spin, but inconsistent with maximally rotating. It is interesting to note that, whenever a constraint can be placed, it always implies the rejection of the static black hole solution. However, this is a likely observational bias since non rotating black holes produce narrower lines, which are easier to detect with respect to the underlying continuum than lines produced by maximally spinning black holes, where the red tail of these lines can be confused with the underlying continuum. A maximally spinning black hole solution is statistically favoured than a static black hole solution. Given how few sources there are, the approximations made (see for instance the above discussions on β) and the subtle interplay between the various disc parameters, it is however premature to draw any firm conclusion in this respect. The relation of the relativistic broad Fe Kα line intensity with θ, and β is also plotted in Fig. 6. As for black hole spin, constraints are too few (two sources only) to allow for discussion of any possible correlation of the line EW with a. These two distributions only show two things. First, the distribution of Fe Kα line intensity with θ is consistent with the fact that relativistic lines arising in accretion discs seen at high inclination angles rather than from face-on discs are more diﬃcult to detect for a given value of the line EW (as noted by Nandra et al. 2007, see also Matt et al. 1992). Second, the apparent lack of detections of small EW lines with steep emissivity profiles is most likely an observational bias, and any real correlation between the two physical quantities can not be claimed. 4.4. Correlation studies within the flux-limited sample Given all the upper limits derived for the line parameters, the censored fit approach was adopted. This procedure for linear Page 11 of 22 A&A 524, A50 (2010) Fig. 5. Normalised distributions (shaded area) for the disc inclination angle (left) and disc emissivity (right) for sources with a significant broad Fe Kα line detection within the flux-limited sample. The 1-dimensional histograms of these distributions are overlaid (white histograms). Fig. 6. Dependence of disc inclination angle and emissivity with the relativistic broad Fe Kα line EW. Filled circles indicate line detections at ≥5σ confidence level (where error bars indicate the 90% confidence level intervals), line upper limits are given at the 90% confidence level. Kyrline parameters upper and lower limits are given at the 90% confidence level. fitting is applicable to data containing both measurements and upper and/or lower limits, and it is based on the method used by Schmitt (1985) and Isobe et al. (1986). Here it was applied in the same way as in Bianchi et al. (2009b). For each correlation analysis, the mean Spearman’s rank correlation coeﬃcient and null hypothesis probability were calculated. Only correlations with a null hypothesis probability lower than 10−3 (99.9% c.l.) will be considered statistically significant. The correlations investigated in this work involve, on one hand, the broad Fe Kα line EW and the disc emissivity (β), and on the other, some source physical properties such as the black hole mass (MBH ), the Eddington luminosity (LEdd ), and the acbol ). Within the censored fit cretion rate (expressed in terms of LLEdd method, the errors used for the line EW are 1σ errors, while the upper limits are at the 90% c.l.. In those correlations involving β, the censored fit approach described in Bianchi et al. (2009b) was slightly modified to account for the lower limits to Page 12 of 22 the β parameter. The lower limit to β can be interpreted simply as consistent with 6 at the 90% c.l.. This value of 6 is the maximum value allowed for β in our model (see Sect. 3.3.1). The values of the black hole mass were taken from the CAIXA catalogue (Bianchi et al. 2009a), and they are available for 24/31 of the objects in the flux-limited sample. (The reader is deferred to this paper for the references from which these values have been extracted.) Using the black hole mass (MBH ), the Eddington luminosity was derived in this work according to the usual definition: LEdd = (4πGMmp c) / σt = 1.26 × 1038 (MBH /M ) erg s−1 . The accretion rate is considered as the ratio of the bolometric luminosity to the Eddington Luminosity: Lbol LEdd , where the bolometric luminosity has been derived by applying the following bolometric correction: Lbol = kb × Lx , where kb is the correction factor dependent on the 2–10 keV X-ray luminosity (Lx) following Marconi et al. (2004). The hard X-ray luminosities derived in this work for those sources with a broad I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN Table 6. Source physical properties used in this work for those sources with a relativistic 6.4 keV Fe Kα line detection within the flux-limited sample. Source name IC4329A MCG-5-23-16 ESO511-G030 MCG-6-30-15 NGC4051 NGC3516 NGC3783 NGC3227 MRK509 MRK766 ARK120 LX2−10 keV Log10 (BH) Lbol LEddington Lbol /LEddington (1044 erg s−1 ) 0.56 0.14 0.22 0.05 0.003 0.03 0.11 0.01 1.04 0.06 0.90 (M ) 8.08 7.85 – 6.19 6.13 7.36 6.94 – 7.86 6.28 8.27 (1044 erg s−1 ) 15.97 2.72 4.87 0.82 0.02 0.49 2.05 0.12 35.37 0.93 29.58 (1044 erg s−1 ) 7.42 89.20 – 1.95 1.70 28.86 10.97 – 91.28 2.40 234.6 0.46 0.03 – 0.42 0.01 0.02 0.19 – 0.39 0.39 0.13 Table 7. Results of the correlation studies within the flux-limited sample. Broad Fe Kα Line EW (Y) vs. Black hole mass (X) Accretion rate (X) X-ray luminosity (X) Beta (Y) vs. Black hole mass (X) Accretion rate (X) X-ray luminosity (X) a 1.64 ± 0.12 1.82 ± 0.15 7.62 ± 5.16 b −0.20 ± 0.11 0.08 ± 0.13 −0.14 ± 0.12 Mean spearman rho −0.34 0.15 −0.13 Null hypothesis probability 0.14 0.50 0.51 0.36 ± 0.11 0.38 ± 0.12 −0.32 ± 4.19 −0.02 ± 0.09 0.01 ± 0.10 0.01 ± 0.10 0.05 0.12 0.17 0.75 0.59 0.39 Notes. Mean intercept (a) and slope (b) for the linear fits of a log (Y) = a + b × log (X) function described in the text. Errors in these two quantities are the statistical one standard deviation. Fe Kα line detection are reported in Table 6, and they are in good agreement with those derived in the CAIXA catalogue. Table 7 and Fig. 7 summarize the results of the correlation studies carried out. Table 7 lists the mean value of the Spearman’s rank coeﬃcient for the correlation of the broad Fe Kα line EW and β with the three physical parameters of interest, together with the corresponding null hypothesis probability. A negative value of Spearman rho indicates anti-correlation between the two parameters of interest. On the basis of these results, it is concluded that no statistically significant correlation is found with any of the considered physical properties of the AGNs. Last, the relativistic Fe Kα line EW derived in this work and all the diﬀerent parameters included in the CAIXA catalogue were correlated (see Table 1 in Bianchi et al. 2009a). No statistically significant correlation has been found with any of the parameters, including the FWHM of the Hβ line and the narrow Fe 6.4 keV Kα line EW. However, the low statistics prevents us from drawing any robust physical conclusions from the lack of such correlations. 5. Discussion Despite the fact that the first relativistically distorted line in an AGN was observed about 15 years ago by ASCA (Tanaka et al. 1995; Nandra et al. 1997), and despite strong eﬀorts to study them with more recent observatories, like XMM-Newton and Suzaku, there are still large uncertainties on the relative number of AGNs that possess such lines. The FERO project has been designed to address the fundamental question of how often these lines appear in AGN by estimating the fraction of objects where relativistic eﬀects are detected on a well-defined sample. This number is crucial to establish if and how the standard AGN paradigm needs to be modified. A collection of 149 radio-quiet Type 1 AGN, all targeted by XMM-Newton, has been assembled (the largest ever assembled for this kind of study) in order to tackle the question posed above. One fundamental requirement of the FERO project was to identify a complete and unbiased subsample of sources with high signal-to-noise spectra to be able to derive meaningful constraints on the physical properties of the sources. Only then it would be possible to extrapolate the results derived from this subsample to the unknown parent population of local radio-quiet AGN. A flux-limited sample of 31 objects was defined by selecting the XSS sources (see Sect. 2.2) with count rates in the 3–8 keV energy band greater than 1 cts/sec that fulfill the FERO selection criteria (i.e. local Type 1 radio-quiet X-ray unobscured sources). In this section the statistical properties of the fluxlimited sample are discussed. The baseline model used in FERO aims at providing the most accurate representation of the physical ingredients that are typically found in an average Type 1 AGN, so this model includes a Compton reflection component and absorption by line-of-sight ionised gas for modelling the continuum, a series of narrow Fe lines (5), and a relativistic Fe Kα line component fitted by the Kyrline model. The spin of the black hole, the radial emissivity profile of the accretion disc, and the disc inclination angle to the observer are free parameters in the fit of the line profile. To achieve a uniform analysis over the whole FERO sample, no attempt has been made to find the best-fit model on a sourceby-source basis, nor has any attempt been made to find a best-fit model by using the FERO sample as a whole. To reduce the parameter space during the fitting procedure, several model parameters were investigated to see whether they could be constrained due either to physical, geometrical, or observational considerations. Tests were also carried out on including diﬀerent model components in the baseline model, and the results have served either to consider them in the final baseline model or rule them Page 13 of 22 A&A 524, A50 (2010) Fig. 7. From top to bottom, relativistic broad Fe Kα line equivalent width and β vs. black hole mass, accretion rate and hard X-ray luminosity. Filled circles indicate broad Fe Kα line detections at ≥5σ confidence level (where error bars indicate the 90% confidence level intervals). The dashed vertical lines in the bottom plots indicate the L1, L2, and L3 luminosity bins as described in the text (see Sect. 4.2.3). out. The most important of these tests was the replacement in the baseline model of the neutral reflection component by an ionised reflection component, with the consequent change in the relativistic line from neutral iron to ionised iron. In fact, the results from these two scenarios were merged and presented as if coming from a single one. Last, a consistency test was carried out by using the hard X-ray spectra of all the sources in the flux-limited sample, for which a significant detection of the relativistic line can be claimed (those listed in Table 2), by describing it with the most self-consistent reflection model envisaged, however keeping the model as simple as possible. The comparison of the results of Page 14 of 22 this more complex and self-consistent model for the reflection components indicates that it does not significantly aﬀect the results derived from the more phenomenological approach chosen for the baseline model adopted throughout the paper (refer to Appendix E for more details). 5.1. Fraction of relativistic lines The main result of the FERO analysis is the relativistic broad line detection fraction, 36±14% in the flux-limited FERO subsample. The observed detection fraction is regarded as a lower limit to the intrinsic number of AGN that would show a broad Fe Kα I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN line if, for example, all sources were observed with the same, and suﬃcient, signal-to-noise ratio. It is important to stress once more that the observed detection fraction comes as a result of adopting a rather conservative detection threshold of 5σ confidence level, which is necessary since FERO does not focus on reporting individual broad line detections in particular objects. Instead, it focuses on statistical studies of the sample as a whole, which includes spectra of very diﬀerent statistical quality. Nevertheless, as a reference, by relaxing this requirement to a 3σ confidence level, the above detection fraction rises to 61 ± 20% (19/31). Upper limits on the line EW can also provide important information. For example, the fraction of sources in the flux-limited sample in which the broad Fe Kα line EW is limited below 100 eV is of the order of 50% (where 100% of line EW UL are below 360 eV). This result points to a much lower value of the average line intensity derived for local Type 1 AGN compared to the one derived from the mean spectra of high redshift (z > ∼ 1) Type 1 AGN ( EW ∼ 560 eV; Streblyanska et al. 2005), although there should be some caution in adopting this result since the high mean EW reported could come as a result of the method adopted for co-adding the spectra (Yaqoob 2006). The result derived in this work is also consistent with the upper limit of <400 eV derived by Corral et al. (2008). Using results on individual sources, the FERO analysis has provided upper limits below 40 eV in four sources (MRK 279, NGC 5548, ESO 198-G24, and MRK 590), which are constraining enough to have to stretch disc-accretion theories by requiring the use of non-standard system properties, such as very high disc inclination angles or element abundances significantly lower than solar (George & Fabian 1991). The non-detection fraction of broad lines in the flux-limited sample is 13% (4/31). Inverting this argument, the presence of a broad Kα line cannot be excluded a priori in the remainder 87% (although the line is formally detected in 36% (11/31) of the sources). The value of 87% can be regarded as the upper limit to the intrinsic fraction of AGN where a relativistic Fe Kα line with EW > 40 eV could be present in the parent sample. Nandra et al. (2007) have fully explored the range of eﬀects (geometry, ionisation, and element abundances) that would explain why a broad line is seen in some objects but not in others, so they are not repeated here. A direct comparison of the results extracted from FERO with previous works reveals that the detection fraction of 36% derived for the flux-limited sample is consistent with previous results, such as those reported in Nandra et al. (2007) where a detection fraction of 35–40% was obtained. When compared to similar studies on sizable XMM-Newton samples (e.g. Nandra et al. 2007; Brenneman & Reynolds 2009), our sample diﬀers in that our sample is very well-defined and homogeneously selected. Moderately X-ray obscured sources and radio-loud sources are both excluded from the FERO sample. However, the most significant diﬀerences are the model used to fit the relativistic profile, the fitting procedure, and the treatment of the sources with multiple observations. For instance, Brenneman & Reynolds (2009) fitted the continuum model on the entire XMM-Newton energy band, but they selected only one particular observation for each source, whereas Nandra et al. (2007) include multiple observations of the same target without combining them. 5.2. Disc parameters The second goal of the FERO project was to derive average line and system properties and look into the question of whether a relativistic broad line can be associated to any source physical properties and, if so, investigate this dependence in more detail. The average broad line EW is of the order of 100 eV, however, a wide range of broad line EW (40–260 eV) are observed. As a reference, a neutral reflector covering half of the sky, as seen from the X-ray source (Ω/2π ∼ 1) and irradiated by a power-law spectrum with Γ ∼ 1.9, should produce an Fe Kα line with equivalent width from around 100 to 150 eV in the inclination range 0◦ –60◦ and for solar abundances (George & Fabian 1991). Therefore, line EWs measured outside that range are likely to be produced mainly because i) the Fe abundance is diﬀerent from solar, and/or ii) the reflector (disc) subtends a nonstandard (smaller) solid angle as seen from the X-ray source. One physical scenario where broad line EWs > ∼ 150 eV are plausible, involves reprocess emission from a photoionised accretion disc (Matt et al. 1993) or under a geometrical scenario which involves anisotropic emission. The shape of the broad lines observed in the X-ray spectra of AGN are a potential carrier of information concerning the black hole spin. The issue of placing constraints to the black hole spin has been pursued by several authors (e.g. Nandra et al. 2007; Brenneman & Reynolds 2009). Using diﬀerent approaches, both studies reached similar conclusions. While some objects show evidence of a spinning black hole, with the present data it is very diﬃcult to reject the static solution. In this work the approach is that of modelling the line with a code capable of including the spin as a free parameter. The Kyrline model explicitly depends on the black hole spin. Kyrline is part of a suit of routines called KY (Dovčiak et al. 2004), a code that properly takes relativistic boosting, light bending, and time delays into account, assuming an axysymetric accretion disc geometry surrounding a black hole. As a general statement, the black hole spin is poorly constrained by the FERO analysis, except for MCG-6+0.03 30-15 (0.86+0.01 −0.02) and MRK 509 (0.78−0.04) where the Kerr black hole solution is favoured. In five other sources, the static solution cannot be rejected, while in a further five, the constraint implies values of the black hole spin above ∼0.5. Another aspect of the present study is to try to establish the physical drivers behind the relativistic broad line by looking into any correlation between the presence and/or properties of the broad line EW and sources physical properties, such as black hole mass, X-ray luminosity, or Eddington ratio. However, using only sources from the flux-limited sample, no significant correlation has been observed between the broad Fe Kα line EW and these source properties. As previous works on large samples have done (e.g. Iwasawa & Taniguchi 1993; Bianchi et al. 2007; Corral et al. 2008), the FERO analysis has looked for inverse correlation between Fe Kα line emission and X-ray luminosity, the so-called IT eﬀect (previously known a X-ray Baldwin eﬀect), where the EW of the narrow Fe Kα emission line is inversely proportional to the 2–10 keV X-ray luminosity. Earlier results on a smaller sample (∼100 AGN) using the spectra stacked residuals technique suggested that relativistic broad profiles follow a similar trend, where broad profiles are more common in lowluminosity AGN (Guainazzi et al. 2006). With information from individual source line profiles, the FERO analysis is unable to confirm such results, finding no statistically significant indication for the IT eﬀect on broad Fe lines. However, it is worth pointing out that the lack of sources in the flux-limited sample with luminosities above 1044 erg s−1 prevents any firm conclusions. The IT eﬀect will be investigated using the spectra stacked residuals technique using those sources in the FERO sample with an upper limit to the relativistic Fe Kα line EW (Longinotti et al., in prep.). Page 15 of 22 A&A 524, A50 (2010) 5.3. FERO in a wider context 6. Conclusions An important remark should be made about the relativistic line in sources that are characterized by other phenomena. A recent paper by De Marco et al. (2009) carried out a systematic search for the presence of red- or blue-shifted components in the timeaveraged spectra of the sources in the flux-limited sample. The study also looks into the spectral variability of these spectral components. A direct comparison with their results shows that 10 out of the 11 sources (∼90%) within the flux-limited sample (all except ESO 511-G030) with a relativistic broad line indicate either red- or blue-shifted components, or both. In 6 out of these 10 sources (IC4329A, NGC 3783, NGC 3516, MRK 509, MCG-6-30-15, and MRK 766) there is evidence of significant variability (>90%) in the excess map (5.4–7.2 keV). From the literature on the warm absorber phenomenon, we note that 9 out of 11 sources with line detections have warm absorbers: NGC 3783, NGC 3516, MRK 509, MCG-630-15, MRK 766, NGC 4051 (Blustin et al. 2005; McKernan et al. 2007), IC4329A (Steenbrugge et al. 2005), NGC 3227 (Markowitz et al. 2009). In the case of ESO 511-G030 the presence of line-of-sight ionised absorption has not been ascertained yet (Longinotti, private communication). The only 2 sources in the flux-limited sample with no warm absorber and no transient features are Ark 120 and MCG-5-23-16, so it is tempting to deem these two sources as the most robust detections, due to the lack of an alternative scenario to the relativistic line. That sources with significant relativistic lines simultaneously show Fe K band variability and a warm absorber is probably twofold. There might be a physical connection between, e.g., the presence of the relativistic line and of the transient features, with some variability associated to it. But, it could well be the result of an observational bias, because these sources were extensively (and repeatedly) observed because they were known to be interesting a priori, so they ended up with the best signal-tonoise data and this explains the coincidence of the broad Fe line, warm absorber, and transient features in the same sources. To conclude, it goes without saying that the FERO project would benefit from an expansion in several directions. The most obvious would be to complete the observations of the fluxlimited sources such that the number of X-ray counts in the 2– 10 keV fall above 1.5 × 105 cts. The expansion of the number of sources with 2–10 keV X-ray luminosities above 1044 erg s−1 would also allow studies like the IT eﬀect to be carried out at a significant level and, of course, to investigate other correlations where the X-ray luminosity plays an important role. Another crucial aspect to consider in future studies regarding relativistic disc lines is broadband coverage. This would allow exploration of the spectral regime above 10 keV and the opportunity to discern whether the spectral contribution at these energies can be attributed to a reflection component oﬀ distant matter and/or the accretion disc, analogous to the case of MCG-5-23-16 (Reeves et al. 2007). The importance of broadband coverage is also highlighted by, for example, the recent detection of a broad Fe Kα line in Fairall 9 in Suzaku observations, which have allowed a tight constraint on the black hole spin (Schmoll et al. 2009). The FERO project has highlighted the importance of the availability of a complete and well-exposed AGN sample to shed some light on the origin and properties of the relativistic broad Fe lines in AGN, the environment, and conditions in which they are produced, and to provide better understanding of the scientific drivers needed for future high-throughput X-ray missions, such as IXO. The main conclusions that are drawn from the results of the systematic and homogeneous spectral analysis of the large number of sources in FERO can be summarised as follows. Page 16 of 22 • The observed fraction of sources in the FERO sample that present strong evidence (≥5σ significance) of a relativistic broad Fe Kα line is of the order of 9% (13/149). Considering only sources from the flux-limited sample, the detection fraction rises to around 36% (11/31). This number can be interpreted as a lower limit to the intrinsic fraction of AGN with relativistic Fe line broadening. The sources with a significant Fe Kα line detection mostly belong to sources with good statistical quality spectra (1.5×105 cts), so it is not surprising that the majority belong to the flux-limited sample. Considering only sources from the flux-limited sample, a broad Fe Kα line at the level of 40 eV can be rejected in 4 objects, 87%, which can be regarded as an upper limit to the intrinsic fraction of AGN with relativistic Fe Kα line broadening. • There is no significant diﬀerence between Seyferts and quasars or narrow line and broad line objects in terms of detection fraction. • All sources with a broad Fe Kα line detection have a hard X-ray luminosity in the 2–10 keV energy band below ∼1 × 1044 erg s−1 . The diﬀerence in the detection fraction between the highest and lowest luminosity bins as defined in this work is only 2σ. • From those sources with a significant Fe Kα line detection within the flux-limited sample, it is found that: ◦ The average relativistic Fe Kα line EW is of the order of 100 eV (never higher than 300 eV for any given source). ◦ The average system properties and 1σ standard deviation inferred from the Kyrline model can be summarised as: θ = 28 ± 5◦ , consistent with an intrinsic random distribution of inclination angles. β = 2.4 ± 0.4, with a wide spread of values. The spin value a is poorly constrained, except for +0.01 MCG-6-30-15 (0.86(−0.02 )) and MRK509 (0.78(+0.03 −0.04)) where the Kerr black hole solution is favoured. ◦ No significant trend is found between the Fe Kα line EW and any of the line parameters investigated (θ, β, and a). ◦ No significant correlation has been found between the Fe Kα line EW or disc emissivity (β) and the source physical properties investigated, such as, black hole mass, accretion rate, and hard X-ray luminosity. Acknowledgements. This work is based on observations by XMM-Newton, an ESA science mission with instruments and contributions directly funded by ESA member states and NASA. I. de la Calle would like to acknowledge support by the Torres Quevedo fellowship from the Ministerio de Ciencia e Innovación Español and INSA. A.L. Longinotti acknowledges travel support provided by NASA through the Smithsonian Astrophysical Observatory (SAO) contract SV373016 to MIT for Support of the Chandra X-Ray Center, operated by SAO for and on behalf of NASA under contract NAS8-03060. We acknowledge financial support from the Faculty of the European space Astronomy Centre (ESAC). G. Miniutti thanks the Spanish Ministerio de Ciencia e Innovación Español and CSIC for support through a Ramón y Cajal contract. P.O. Petrucci acknowledges support by the PCHE (Phénomènes Cosmiques des Hauutes Energies) French working group. S. Bianchi, G. Matt and E. Piconcelli acknowledge financial support from Italian Space Agency, under grant ASI I/088/06/0. G. Ponti thanks ANR for support (ANR-06-JCJC-0047). We thank the referee for a careful reading of the manuscript and the useful comments that helped to improve it. 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C., et al. 2001, MNRAS, 328, 27 Yaqoob, T. 2006, in Conf. Proc. 230th Symp. IAU, Dublin (Ireland), ed. E. J. A. Meurs, & G. Fabbiano (Cambridge: Cambridge University Press), 461 Yaqoob, T., & Padmanabhan, U. 2004, ApJ, 604, 63 Yaqoob, T., George, I. M., Kallman, T. R., et al. 2003, ApJ, 596, 85 Pages 18 to 22 are available in the electronic edition of the journal at http://www.aanda.org Page 17 of 22 A&A 524, A50 (2010) Appendix A: Sources from the flux-limited sample List of 33 sources in the flux-limited sample. These sources have been selected from the RXTE all-sky Slew Survey (XSS, Revnivtsev et al. 2004) with a count rate in the 3–8 keV energy band greater than 1 cts/sec and fulfilling the FERO source selection criteria. The XSS is nearly 80% complete at the selected flux level for sources with Galactic latitude greater than 10◦ . For two of them, UGC 10683 and ESO 0141-G055, no XMM-Newton data were available as of April 2008. This leaves the number of XSS-selected bright sources in the FERO sample at 31. Appendix B: List of sources with multiple observations that have not been summed List of sources within the FERO sample where multiple observations are available but only one has been used (the one with longest exposure time). Appendix C: Best-fit-model parameters for sources belonging to the flux-limited sample Table C.1 gives some relevant best-fit-model parameters (see Sect. 3.3) for the 31 sources belonging to the flux-limited sample. Appendix D: Relativistic Fe Kα line EW upper limits for the sources in the flux-limited sample List of 20 sources within the flux-limited sample with an upper limit to the relativistic Fe Kα line EW. Appendix E: A final self-consistent test on the detections within the flux-limited sample As a final test, the hard X-ray spectra of all the sources in the flux-limited sample for which a significant detection of the relativistic line can be claimed has been described with the most selfconsistent reflection model envisaged, but keeping the model as simple as possible. The baseline model comprises one layer of ionised absorption (the ZXIPCF model), reflection oﬀ cold distant matter including the most important emission lines and the associated self-consistent reflection continuum (the PEXMON model), and reflection from the accretion disc (the Ross & Fabian REFLION model, convolved with the KYRLINE kernel). Two ionised emission lines with energies fixed at 6.7 keV and 6.96 keV are also included as in the previous phenomenological models used throughout the paper, although they are not statistically required in all cases. The PEXMON model is described in detail in Nandra et al. (2007) and describes the reflection spectrum from a cold slab of gas including both the reflection continuum and the most relevant emission lines, which are computed as self-consistently as possible according to the work by George & Fabian (1991). The metal abundances are fixed to the solar value (except for the case of MCG–6-30-15, see below) and the reflector inclination with respect to the line of sight to 60 degrees, which is appropriate for torus-like reflection in Seyfert 1 galaxies. The illuminating continuum is a power law with the same photon index as the power-law component of our spectral model. The REFLION model describes reflection oﬀ a ionised slab and is used instead Page 18 of 22 to describe the disc reflection component. The illuminating photon index is the same as the primary continuum and the Fe abundance is fixed to the solar value (except for MCG–6-30-15, see below). The model is convolved with the kernel of the KYRLINE model (Dovčiak et al. 2004), which allows including all relativistic eﬀects and measuring the relevant parameters. It is important to stress once again that the goal here is not to provide the best possible fitting statistics but rather to compare the results of the phenomenological model used in the paper (which does not account for emission lines and associated reflection continua in a self-consistent manner) with a more physical spectral model. In some cases, more spectral components are included as explained in the subsequent section. The results are reported in Table E.1, where the most important spectral parameters associated with the reflection components are considered. In Table E.2 the best-fitting relativistic parameters for both the phenomenological and the more physically motivated models are reported. Such a comparison implies that the more complex and self-consistent model for the reflection components does not significantly aﬀect the results. Since the cases considered here correspond to the highest signal-to-noise data within the whole sample, the test supports the analysis carried out on the whole available sample within the context of the less sophisticated and more phenomenological model. E.1. Notes on individual sources Slight modifications to the baseline model described in Appendix E are given here for some individual sources. In the remaining cases, the baseline model was applied without being modified. IC 4329A: absorption is best modelled with a neutral layer with moderate column density (4 × 1021 cm−2 ). A Gaussian absorption line with EW −15 eV was also included to model an absorption feature at 7.65 keV phenomenologically. MCG-5-23-16: absorption is best modelled with a neutral layer with column density NH 1.5 × 1022 cm−2 . The presence of a further ionised layer is possible but not statistically required. MCG-6-30-15: the Fe abundance of the two reflectors was left free to vary in this case resulting in an overabundance of ∼× 3 with respect to the solar value. The model also comprises a Gaussian absorption line at 6.7 keV with EW −15 eV. NGC 4051: a Gaussian absorption line with EW −30 eV was also included to model an absorption feature at 7.04 keV phenomenologically. NGC 3516: a Gaussian absorption line with EW −28 eV was also included to model an absorption feature at 6.7 keV phenomenologically. NGC 3783: a Gaussian absorption line with EW −20 eV was also included to model an absorption feature at 6.7 keV phenomenologically. Mrk 509: a Gaussian absorption line with EW −10 eV was also included to model an absorption feature at 7.3 keV phenomenologically. I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN Table A.1. Flux-limited sample: 33 sources from the RXTE Slew Survey with a count rate in the 3–8 keV energy range greater than 1 cts/sec and fulfilling the FERO source selection criteria. Source Type NGC 4593 MRK 704 ESO511-G030 NGC 7213 AKN 564 H1846-786 MRK 110 ESO198-G024 FAIRALL9 UGC 3973 NGC 4051 NGC 526A MCG-2-58-22 NGC 7469 MRK 766 MRK 590 IRAS05078+1626 NGC 3227 MR2251-178 MRK 279 ARK 120 NGC 7314 H0557-385 MCG+8-11-11 MCG-6-30-15 MRK 509 NGC 3516 NGC 5548 NGC 3783 MCG-5-23-16 IC 4329A UGC10683 ESO141-G055 BLSY BLSY NCSY BLSY NLSY NCSY NLSY BLSY BLQ BLSY NLSY NCSY BLSY BLSY NLSY BLSY NCSY BLSY BLQ BLSY BLSY NCSY NCSY BLSY BLSY BLQ BLSY BLSY BLSY NCSY BLSY SY SY RXTE slew survey (cts/s)3−8 keV 1.05 ± 0.16 1.06 ± 0.22 1.10 ± 0.09 1.12 ± 0.16 1.13 ± 0.02 1.15 ± 0.09 1.17 ± 0.10 1.21 ± 0.19 1.25 ± 0.02 1.32 ± 0.12 1.49 ± 0.02 1.61 ± 0.14 1.61 ± 0.08 1.69 ± 0.01 1.77 ± 0.12 1.95 ± 0.10 2.08 ± 0.19 2.10 ± 0.02 2.10 ± 0.11 2.13 ± 0.06 2.14 ± 0.03 2.16 ± 0.04 2.33 ± 0.34 2.36 ± 0.27 3.08 ± 0.02 3.12 ± 0.10 3.21 ± 0.01 3.58 ± 0.02 4.90 ± 0.04 6.46 ± 0.12 7.29 ± 0.07 1.27 ± 0.18 1.37 ± 0.12 XMM-Newton (cts/s)2−10 keV 4.613 ± 0.009 0.984 ± 0.009 2.284 ± 0.005 2.448 ± 0.009 2.246 ± 0.005 0.700 ± 0.011 3.362 ± 0.010 1.150 ± 0.003 1.313 ± 0.007 1.304 ± 0.019 2.783 ± 0.007 2.177 ± 0.007 3.459 ± 0.021 3.343 ± 0.005 1.962 ± 0.002 0.766 ± 0.003 2.691 ± 0.008 3.635 ± 0.006 2.387 ± 0.007 3.110 ± 0.006 4.553 ± 0.008 4.509 ± 0.012 4.254 ± 0.021 4.838 ± 0.014 4.719 ± 0.004 4.432 ± 0.005 1.841 ± 0.004 4.191 ± 0.007 5.539 ± 0.006 8.025 ± 0.008 10.713 ± 0.011 – – XMM-Newton exposure time (ks) 53.2 14.9 76.2 29.6 84.1 6.3 32.8 85.3 25.9 3.6 73.3 41.5 7.2 142.6 398.3 72.0 40.1 97.4 44.5 104.8 76.3 30.1 10.5 26.5 295.1 27.8 153.0 74.8 170.4 110.3 85.1 – – Notes. These sources, with the exception of the last two sources, have all been the target of XMM-Newton observations. The XMM-Newton EPICpn count rate in the 2–10 keV energy range and exposure time is also given. For two of them, UGC 10683 and ESO 0141-G055, no XMM-Newton data were available as of April 2008. This reduces the number of XSS-selected bright sources in the FERO sample to 31. Page 19 of 22 A&A 524, A50 (2010) Table B.1. List of sources within the FERO sample where multiple observations are available but only one has been used (the one with longest exposure time). Source 1H0707-495 ESO113-G010 ESO198-G24 IZW1 MR2251-178 MRK 841 NGC 2622 NGC 4051 NGC 4593 PG 1211+143 UGC 3973 PG 1116+215 IRASF12397+3333 List of XMM-Newton observation IDs combined 0148010301 0110890201 0301890101 0103861601 0305370101 0067190101 0112910101 0300470101 0110890301 0012940101 0112910301 0205340201 0112910201 0070740101 0070740301 0205340401 0302260201 0302260401 0109141401 0157560101 0059830101 0109970101 0112610101 0208020101 0400070201 0103860801 0103862101 0201940101 0201940201 0111290401 0202180201 0202180301 Date Exposure (ks) 2002-10-13 2000-10-21 2005-11-10 2001-05-03 2006-02-04 2001-01-24 2000-12-01 2005-07-18 2002-06-22 2002-05-18 2000-11-29 2005-01-16 2001-01-13 2001-01-13 2001-01-14 2005-07-17 2005-04-09 2005-10-08 2001-05-16 2002-11-22 2002-06-23 2000-07-02 2001-06-15 2004-06-21 2006-09-30 2000-10-09 2001-04-26 2004-12-17 2004-12-19 2001-12-02 2005-06-20 2005-06-23 70.0 36.4 90.6 4.0 85.3 26.4 6.8 58.0 18.4 44.5 3.5 30.1 5.9 7.6 9.3 17.3 6.6 4.5 73.3 46.2 53.2 8.8 49.5 43.6 14.5 1.7 3.6 69.3 5.0 5.5 68.7 9.1 Notes. Observation IDs in bold correspond to those observations that have been considered, while observation IDs not in bold have been discarded. Page 20 of 22 I. de la Calle Pérez et al.: Statistics of relativistic Fe Kα lines in Type 1 AGN Table C.1. Flux-limited sample: relevant best-fit-model parameters corresponding to the neutral reflection run and 6.4 keV relativistic Fe Kα line. Source AKN564 NGC526A MCG-6-30-15 MRK766 NGC5548 MCG+8-11-11 MRK704 NGC3516 MCG-5-23-16 NGC3227 H0557-385 NGC4593 NGC7469 NGC3783 ESO511-G030 IRAS05078+1626 MRK279 MRK509 NGC7314 NGC7213 IC4329A MR2251-178 H1846-786 FAIRALL9 UGC3973 MCG-2-58-22 NGC4051 MRK590 ARK120 MRK110 ESO198-G24 Power Law Γ +0.05 2.82−0.25 +0.10 1.52−0.02 +0.02 2.05−0.01 +0.06 2.10−0.05 +0.02 1.64−0.01 +0.05 1.78−0.04 +0.04 1.72−0.14 +0.06 1.61−0.06 +0.04 1.65−0.02 +0.01 1.52−0.02 +0.2 1.86−0.05 +0.03 1.84−0.04 >1.97 +0.01 1.70−0.01 +0.02 1.93−0.03 +0.03 1.72−0.06 +0.02 1.83−0.03 >1.96 +0.11 2.09−0.08 +0.09 1.76−0.02 +0.02 1.79−0.01 +0.03 1.32−0.01 +0.21 1.87−0.10 +0.05 1.90−0.11 +0.27 1.78−0.16 +0.10 1.69−0.04 +0.07 2.02−0.01 +0.08 1.61−0.02 +0.02 2.12−0.03 +0.02 1.88−0.06 +0.07 1.79−0.04 Warm Absorber NH ξ (1022 cm−2 ) (erg cm−1 s−1 ) +0.17 0.76−0.52 – +0.42 1.24−0.05 <0.47 +0.04 +1.97 0.80−0.14 6.30−2.98 +0.19 +8.71 0.40−0.12 10.37−4.92 <0.42 – <6.24 – +3.82 9.67−3.86 105.42+74.83 −30.54 +0.63 4.48−0.52 77.03+11.98 −16.48 +0.13 1.20−0.02 <0.02 +0.07 0.09−0.03 <25.38 +0.57 1.22−0.32 5.67+14.75 −5.24 <4.07 – <0.53 – +4.09 +0.08 1.64−0.06 54.21−4.70 <1.38 – +3.00 4.94−4.05 – <0.51 – <0.2 – +0.32 +5.88 1.35−0.29 4.86−3.81 <7.67 – +0.04 0.33−0.02 <0.47 <0.61 – <8.95 – <2.67 – <37.79 – <2.93 – +3.39 2.21−0.64 – <3.39 – <0.52 – <1.21 – <2.57 – Compton Reflection Norm (10−3 keV−1 cm−2 s−1 ) +0.62 +1.36 2.22−1.25 18.38−3.40 +0.49 <0.77 4.46−0.21 +0.29 +0.23 0.51−0.17 17.38−0.49 +0.77 0.78−0.42 >6.87 +2.82 +0.46 0.20−0.12 8.57−0.04 +0.54 +0.62 1.37−0.48 11.78−0.56 +0.63 <3.33 3.37−0.84 +0.22 +0.14 1.99−0.32 4.02−0.27 +0.19 +0.79 1.15−0.18 19.37−0.48 +0.09 +0.12 0.15−0.12 6.70−0.19 +4.12 <3.28 13.63−2.52 +0.32 +0.44 1.24−0.31 11.88−0.55 +0.25 +0.20 1.79−0.19 10.07−0.07 +0.04 +0.08 0.51−0.43 13.44−0.17 +0.62 +0.26 2.10−0.10 6.31−0.06 +0.28 +0.12 1.70−0.76 5.96−0.38 +0.10 +0.07 1.16−0.35 7.79−0.07 +0.07 +0.10 2.49−0.36 12.88−0.18 +1.00 +2.45 1.16−0.64 18.22−2.02 +0.38 <0.77 5.80−0.30 +0.07 +0.18 0.93−0.21 26.85−0.37 +0.24 +0.10 0.54−0.48 3.06−0.04 +0.45 >0 1.98−0.19 +0.65 +0.22 1.70−1.05 3.56−0.28 +1.64 >0 3.06−0.59 +0.97 <3.32 7.65−0.49 +1.09 <1.49 9.48−0.23 +0.92 +0.08 0.59−0.39 1.46−0.04 +0.35 +0.31 2.14−0.34 15.95−0.34 +0.25 +0.12 1.47−0.61 8.58−0.39 +0.83 +0.16 1.79−0.51 2.7−0.09 R EW Narrow Fe Kα Emission Lines FeI 6.4 keV FeXXV 6.7 keV FeXXVI 6.96 keV (eV) (eV) (eV) <18 13+10 <4 −11 42+13 <26 <22 −17 44+4 <5 <7 −4 32+4 <21 20+9 −6 −11 67+7 <4 <7 −12 +10 97−11 <10 <17 <83 <42 <45 140+9 <7 <5 −13 32+6 <5 <5 −6 59+9 <5 8+6 −5 −18 <31 <14 <26 39+26 <10 14+8 −7 −16 73+5 <3 <9 −6 90+3 <1 23+5 −8 −3 11+0 <14 <12 −0 38+21 11+10 <11 −23 −10 55+16 <3 7+7 −14 −6 34+4 <4 <3 −4 +10 <56 14−10 27+11 −11 +13 82+14 22 17+13 −15 −13 −15 +6 +4 34−9 <4 8−3 23+11 <5 <2 −8 +71 78+60 <35 104 −72 −58 +27 107−23 <21 <29 +57 156−68 <87 <38 <51 <20 21+27 −19 63+12 17+10 <17 −13 −11 113+16 <31 41+20 −18 −15 +6 65+7 15 9+7 −7 −7 −6 33+10 <6 <3 −10 60+12 <8 <8 −15 Notes. Errors are given at the 90% confidence level. Upper limits to the relevant parameters are also given at the 90% confidence level. Sources with a significant broad Fe Kα line detection are marked in bold (as listed in Table 2). Table D.1. Flux-limited sample: list of 20 sources with an upper limit to the relativistic Fe Kα line EW. Source MRK 704 FAIRALL9 AKN 564 MCG-2-58-22 H1846-786 MCG+8-11-11 NGC 526A MRK 110 NGC 7213 UGC 3973 H0557-385 MR2251-178 NGC 7314 NGC 4593 NGC 7469 IRAS05078+1626 MRK 279 NGC 5548 ESO198-G24 MRK 590 Cts2−10 keV (105 cts) 0.146 ± 0.012 0.341 ± 0.018 1.889 ± 0.043 0.250 ± 0.016 0.044 ± 0.007 1.284 ± 0.036 0.903 ± 0.030 1.104 ± 0.033 0.724 ± 0.027 0.389 ± 0.020 0.446 ± 0.021 1.062 ± 0.032 1.358 ± 0.037 2.455 ± 0.049 4.769 ± 0.069 1.079 ± 0.033 3.259 ± 0.057 3.133 ± 0.056 0.981 ± 0.031 0.552 ± 0.023 EW upper limit (eV) 362.1 320.4 292.3 208.5 173.7 158.4 122.1 115.6 107.4 100.2 99.3 99.2 87.8 84.2 84.0 61.4 38.2 37.6 35.8 33.4 Notes. The EW upper limit is provided at the 90% confidence level. Page 21 of 22 A&A 524, A50 (2010) Table E.1. Summary of the results of the self-consistent reflection model. Source IC 4329A MCG-5-23-16 ESO511-G030 MCG-6-30-15 NGC 4051 NGC 3516 NGC 3783 NGC 3227 MRK 509 MRK 766 ARK 120 Distant reflection Disc reflection 0.5 ± 0.2 0.3 ± 0.1 0.7 ± 0.2 0.2 ± 0.1 0.9 ± 0.3 1.5 ± 0.3 0.8 ± 0.2 0.6 ± 0.2 0.3 ± 0.1 0.3 ± 0.1 1.0 ± 0.3 0.3 ± 0.2 0.5 ± 0.3 0.8 ± 0.4 2.4 ± 0.8 1.0 ± 0.5 1.8 ± 0.5 0.8 ± 0.4 0.5 ± 0.3 1.0 ± 0.6 0.8 ± 0.4 0.7 ± 0.4 Disc ionisation (erg cm s−1 ) 150+80 −65 ≤70 70 ± 50 62+40 −50 140+90 −80 ≤70 ≤90 70+60 −60 80+40 −50 700+120 −350 80+80 −30 θ (◦ ) 34+11 −12 31+7 −12 37+21 20 39 ± 5 18+9 −13 34+7 −12 27+19 −20 26 ± 9 ≥45 28 ± 8 45 ± 10 a β χ2 /d.o.f. ≥0 ≥0 ≥0 ≥0.8 ≥0.3 ≥0.3 ≥0.2 ≥0 ≥0.4 ≥0.3 ≥0.1 +1.3 2.0−0.9 +1.5 1.8−0.8 +0.8 2.6−1.7 +0.8 3.4−0.5 +0.6 2.5−0.5 2.7±0.4 +0.5 3.1−0.4 +1.1 2.3−0.7 +0.8 2.8−0.5 +0.4 2.6−0.5 +0.9 2.7−1.2 197.2/163 247.3/164 185.0/162 192.1/164 172.0/162 212.7/161 221.4/163 226.5/162 244.2/160 262.5/162 188.7/160 Notes. All sources for which a detection of a relativistic line component is claimed in the paper have been described with two self-consistent reflection models, one for distant reflection (the PEXMON model), the other for disc reflection (the REFLION model). The relevant parameters and fitting statistics are reported in this table. Table E.2. Comparison of the phenomenological and self-consistent model. Source IC4329A MCG-5-23-16 ESO511-G030 MCG-6-30-15 NGC4051 NGC3516 NGC3783 NGC3227 MRK509 MRK766 ARK120 Phenomenological model θ a β (◦ ) 28+6 ≥0 ≤1.3 −11 21+8 ≥0 ≤1.6 −3 18 ± 7 ≥0 ≤1.1 +0.01 40+1 0.86−0.02 4.1±0.2 −3 +0.3 22 ± 6 ≥0.46 2.9−0.4 +0.2 27+2 ≥0.48 2.8 −3 −0.3 +0.1 ≤8 ≥0.16 2.7−0.2 +0.6 23 ± 4 ≥0 1.9−0.5 +0.03 53 ± 1 0.78−0.04 ≥3.8 +0.2 20+3 ≥0.47 2.7−0.1 −2 +0.6 ≥59 ≥0 2.2−0.3 Self-consistent model θ a β (◦ ) +0.8 34+11 ≥0 1.9−1.0 −12 +1.5 31+7 ≥0 1.8 −12 −0.8 +21 +0.8 37−20 ≥0 2.6−1.7 +0.8 39 ± 5 ≥0.8 3.4−0.5 +9 +0.6 18−13 ≥0.3 2.5−0.5 34+7 ≥0.3 2.7±0.4 −12 +0.5 27+19 ≥0.2 3.1−0.4 −20 +1.1 26 ± 9 ≥0 2.3−0.7 +0.8 ≥45 ≥0.4 2.8−0.5 +0.4 28 ± 8 ≥0.3 2.6−0.5 +0.9 48 ± 12 ≥0.1 2.7−1.2 Notes. This table shows a comparison of the best-fitting relativistic parameters (only disc parameters are shown) obtained by applying the phenomenological model discussed throughout the paper (whose full results are given in Table 2) and the more self-consistent one discussed in Appendix E. Here, only sources for which a detection of a relativistic line component is claimed in the paper are shown. Page 22 of 22
https://openalex.org/W2976775529	https://www.biorxiv.org/content/biorxiv/early/2019/09/30/787820.full.pdf	English	null	A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	19,044	. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint dependent homodimer polymerization 2 Andrea Sánchez-Vallet1,2,‡, Hui Tian1,‡, Luis Rodriguez-Moreno1,‡,†, Dirk-Jan Valkenburg1, 4 Raspudin Saleem-Batcha3,§, Stephan Wawra4, Anja Kombrink1, Leonie Verhage1, Ronnie de 5 Jonge1,ƒ, H. Peter van Esse1,ø, Alga Zuccaro4, Daniel Croll5, Jeroen R. Mesters3,+,* and Bart 6 P.H.J. Thomma1, 4,+,* 7 P.H.J. Thomma1, 4,+,* 7 8 1Laboratory of Phytopathology, Wageningen University& Research, Droevendaalsesteeg 1, 9 6708 PB Wageningen, The Netherlands. 10 2Plant Pathology Group, Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland. 11 3Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of 12 Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. 13 4University of Cologne, Botanical Institute, Cluster of Excellence on Plant Sciences 14 (CEPLAS), 50674 Cologne, Germany. 15 5Institute of Biology, University of Neuchâtel, Rue Emile-Argand 11, CH-2000 Neuchâtel, 16 Switzerland. 17 18 ‡ These authors contributed equally 19 + These authors contributed equally 20 §Present address: Center for Biological Systems Analysis, University of Freiburg, 21 Habsburgerstrasse 49, 79104 Freiburg, Germany 22 †Present address: Departamento de Biología Celular, Genética y Fisiología, Universidad de 23 Málaga, Málaga, Spain. 24 8 1Laboratory of Phytopathology, Wageningen University& Research, Droevendaalsesteeg 1, 9 6708 PB Wageningen, The Netherlands. 10 2Plant Pathology Group, Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland. 11 3Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of 12 Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. 13 2Plant Pathology Group, Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland. 11 3Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of 12 Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. 13 †Present address: Departamento de Biología Celular, Genética y Fisiología, Universidad de 23 Málaga, Málaga, Spain. 24 1 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. dependent homodimer polymerization 2 ; https://doi.org/10.1101/787820 doi: bioRxiv preprint øPresent address: The Sainsbury Laboratory, University of East Anglia, Norwich Research 25 Park, NR4 7UH, UK 26 ƒPresent address: Plant-Microbe Interactions, Department of Biology, Science4Life, Utrecht 27 University, Padualaan 8, Utrecht, 3584 CH, The Netherlands 28 To whom correspondence should be addressed. E-mail: bart.thomma@wur.nl and 29 jeroen.mesters@uni-luebeck.de 30 øPresent address: The Sainsbury Laboratory, University of East Anglia, Norwich Research 25 øPresent address: The Sainsbury Laboratory, University of East Anglia, Norwich Research 25 P k NR4 7UH UK 26 øPresent address: The Sainsbury Laboratory, University of East Anglia, Norwich Research 25 ƒPresent address: Plant-Microbe Interactions, Department of Biology, Science4Life, Utrecht 27 University, Padualaan 8, Utrecht, 3584 CH, The Netherlands 28 ƒPresent address: Plant-Microbe Interactions, Department of Biology, Science4Life, Utrecht 27 University, Padualaan 8, Utrecht, 3584 CH, The Netherlands 28 To whom correspondence should be addressed. E-mail: bart.thomma@wur.nl and 29 *To whom correspondence should be addressed. E-mail: bart.thomma@wur.nl and 29 jeroen.mesters@uni-luebeck.de 30 2 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint ABSTRACT 31 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint ABSTRACT 31 Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the 32 release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, 33 many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host 34 through scavenging of chitin oligomers. We previously showed that intrachain LysM 35 dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding 36 groove that competitively sequesters chitin oligomers from host immune receptors. 37 Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase 38 hydrolysis during host colonization. However, the molecular basis for the protection of fungal 39 cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the 40 single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria 41 tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin- 42 dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains 43 the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin- 44 independent Mg1LysM homodimers, a property that confers protection to fungal cell walls 45 against host chitinases. 46 Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the 32 release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, 33 many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host 34 through scavenging of chitin oligomers. We previously showed that intrachain LysM 35 dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding 36 groove that competitively sequesters chitin oligomers from host immune receptors. 37 Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase 38 hydrolysis during host colonization. However, the molecular basis for the protection of fungal 39 cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the 40 single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria 41 tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin- 42 dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains 43 the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin- 44 independent Mg1LysM homodimers, a property that confers protection to fungal cell walls 45 against host chitinases. 46 3 3 . INTRODUCTION 47 Fungi constitute an evolutionarily and ecologically diverse group of microorganisms that are 48 characterized by the presence of chitin, an N-acetyl-D-glucosamine (GlcNAc) homopolymer, 49 in their cell walls. In addition to providing strength, shape, rigidity and protection against 50 environmental hazards, chitin is also a well-known inducer of plant immune responses 51 (Boller, 1995; Sánchez-Vallet et al, 2015; Shibuya et al, 1993). A major mechanism of plant 52 defense against fungal invasion includes the secretion of microbial cell wall-degrading 53 enzymes that include chitin-degrading enzymes, known as chitinases, to hinder fungal 54 pathogen ingress (Schlumbaum et al, 1986; van Loon et al, 2006). Plant chitinases are diverse 55 in nature, and grouped into six different classes that belong to glycosyl hydrolase families 18 56 and 19 (Adrangi & Faramarzi, 2013; Hamid et al, 2013). Although many chitinases are 57 specifically produced upon pathogen invasion, others are constitutively expressed (van Loon 58 et al, 2006). 59 Chitin hydrolysis on the one hand inhibits fungal growth, and on the other hand 60 releases chitooligosacharides (Kasprzewska, 2003; Liu et al, 2014) that are recognized by cell 61 surface receptors of host cells to mount an appropriate immune response (Rovenich et al, 62 2016; Sánchez-Vallet et al, 2015). In plants, chitin is recognized in the extracellular space 63 through membrane-exposed Lysin motif (LysM)-containing receptor molecules (Kaku et al, 64 2006; Liu et al, 2012; Miya et al, 2007; Shibuya et al, 1993). In turn, many successful fungal 65 pathogens have evolved effector molecules that either protect their cell walls against plant 66 chitinases or prevent or perturb the elicitation of chitin-triggered host immunity (De Jonge et 67 al, 2010; Marshall et al, 2011; Ökmen et al, 2018; Rovenich et al, 2016; Sánchez-Vallet et al, 68 2015). 69 Since decades, the interaction between the foliar fungal pathogen Cladosporium 70 fulvum and its only host tomato has been studied to unravel the role of pathogen virulence and 71 4 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . INTRODUCTION 47 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint host defense mechanisms, including mechanisms that revolve around chitin (de Wit, 2016). 72 After leaf penetration, C. fulvum secretes an arsenal of apoplastic effector proteins, including 73 the chitin-binding effector proteins Avr4, which protects fungal hyphae against hydrolysis by 74 plant chitinases (van den Burg et al, 2006; Van Esse et al, 2007), and Ecp6, which perturbs 75 the activation of chitin-triggered host immunity (De Jonge et al, 2010; Sánchez-Vallet et al, 76 2013). Whereas Avr4 binds chitin through an invertebrate chitin-binding domain (Kohler et 77 al, 2016, van den Burg et al, 2004), Ecp6 utilizes LysM domains for chitin binding (De Jonge 78 et al, 2010; Sánchez-Vallet et al, 2013). Previous biochemical analysis revealed that Avr4 79 monomers require a stretch of at least three exposed GlcNAc residues for binding, and 80 positive allosteric interactions among Avr4 molecules occur during chitin binding to facilitate 81 the shielding of cell wall chitin against host chitinases (van den Burg et al, 2004). Based on 82 X-ray crystallography it was recently shown that two Avr4 molecules interact through their 83 chitohexaose ligand to form a three-dimensional molecular sandwich that encapsulates two 84 chitohexaose molecules within the dimeric assembly (Hurlburt et al, 2018). A crystal structure 85 of Ecp6 revealed chitin-induced dimerization of two of the three LysM domains, resulting in 86 the formation of an ultrahigh affinity (pM) chitin-binding groove, conferring the capacity to 87 outcompete plant receptors for chitin binding (Sánchez-Vallet et al, 2013). Interestingly, 88 whereas Avr4 homologs are found in other, C. fulvum-related, Dothideomycete plant 89 pathogens (Stergiopoulos et al, 2010), LysM effectors are widespread in the fungal kingdom 90 (Bolton et al, 2008; De Jonge & Thomma, 2009; Kombrink & Thomma, 2013). In several 91 plant pathogenic fungi, including the Dothideomycete Zymoseptoria tritici and the 92 Sodariomycetes Magnaporthe oryzae, Colletotrichum higginsianum and Verticillium dahliae, 93 LysM effectors have been shown to contribute to virulence through chitin binding (Kombrink 94 et al, 2017; Marshall et al, 2011; Mentlak et al, 2012; Takahara et al, 2016). 95 host defense mechanisms, including mechanisms that revolve around chitin (de Wit, 2016). INTRODUCTION 47 72 After leaf penetration, C. fulvum secretes an arsenal of apoplastic effector proteins, including 73 the chitin-binding effector proteins Avr4, which protects fungal hyphae against hydrolysis by 74 plant chitinases (van den Burg et al, 2006; Van Esse et al, 2007), and Ecp6, which perturbs 75 the activation of chitin-triggered host immunity (De Jonge et al, 2010; Sánchez-Vallet et al, 76 2013). Whereas Avr4 binds chitin through an invertebrate chitin-binding domain (Kohler et 77 al, 2016, van den Burg et al, 2004), Ecp6 utilizes LysM domains for chitin binding (De Jonge 78 et al, 2010; Sánchez-Vallet et al, 2013). Previous biochemical analysis revealed that Avr4 79 monomers require a stretch of at least three exposed GlcNAc residues for binding, and 80 positive allosteric interactions among Avr4 molecules occur during chitin binding to facilitate 81 the shielding of cell wall chitin against host chitinases (van den Burg et al, 2004). Based on 82 X-ray crystallography it was recently shown that two Avr4 molecules interact through their 83 chitohexaose ligand to form a three-dimensional molecular sandwich that encapsulates two 84 chitohexaose molecules within the dimeric assembly (Hurlburt et al, 2018). A crystal structure 85 of Ecp6 revealed chitin-induced dimerization of two of the three LysM domains, resulting in 86 the formation of an ultrahigh affinity (pM) chitin-binding groove, conferring the capacity to 87 outcompete plant receptors for chitin binding (Sánchez-Vallet et al, 2013). Interestingly, 88 whereas Avr4 homologs are found in other, C. fulvum-related, Dothideomycete plant 89 pathogens (Stergiopoulos et al, 2010), LysM effectors are widespread in the fungal kingdom 90 (Bolton et al, 2008; De Jonge & Thomma, 2009; Kombrink & Thomma, 2013). In several 91 plant pathogenic fungi, including the Dothideomycete Zymoseptoria tritici and the 92 Sodariomycetes Magnaporthe oryzae, Colletotrichum higginsianum and Verticillium dahliae, 93 LysM effectors have been shown to contribute to virulence through chitin binding (Kombrink 94 et al, 2017; Marshall et al, 2011; Mentlak et al, 2012; Takahara et al, 2016). 95 host defense mechanisms, including mechanisms that revolve around chitin (de Wit, 2016). 72 After leaf penetration, C. fulvum secretes an arsenal of apoplastic effector proteins, including 73 the chitin-binding effector proteins Avr4, which protects fungal hyphae against hydrolysis by 74 plant chitinases (van den Burg et al, 2006; Van Esse et al, 2007), and Ecp6, which perturbs 75 the activation of chitin-triggered host immunity (De Jonge et al, 2010; Sánchez-Vallet et al, 76 2013). INTRODUCTION 47 Whereas Avr4 binds chitin through an invertebrate chitin-binding domain (Kohler et 77 al, 2016, van den Burg et al, 2004), Ecp6 utilizes LysM domains for chitin binding (De Jonge 78 et al, 2010; Sánchez-Vallet et al, 2013). Previous biochemical analysis revealed that Avr4 79 monomers require a stretch of at least three exposed GlcNAc residues for binding, and 80 positive allosteric interactions among Avr4 molecules occur during chitin binding to facilitate 81 the shielding of cell wall chitin against host chitinases (van den Burg et al, 2004). Based on 82 X-ray crystallography it was recently shown that two Avr4 molecules interact through their 83 chitohexaose ligand to form a three-dimensional molecular sandwich that encapsulates two 84 chitohexaose molecules within the dimeric assembly (Hurlburt et al, 2018). A crystal structure 85 of Ecp6 revealed chitin-induced dimerization of two of the three LysM domains, resulting in 86 the formation of an ultrahigh affinity (pM) chitin-binding groove, conferring the capacity to 87 outcompete plant receptors for chitin binding (Sánchez-Vallet et al, 2013). Interestingly, 88 whereas Avr4 homologs are found in other, C. fulvum-related, Dothideomycete plant 89 pathogens (Stergiopoulos et al, 2010), LysM effectors are widespread in the fungal kingdom 90 (Bolton et al, 2008; De Jonge & Thomma, 2009; Kombrink & Thomma, 2013). In several 91 plant pathogenic fungi, including the Dothideomycete Zymoseptoria tritici and the 92 Sodariomycetes Magnaporthe oryzae, Colletotrichum higginsianum and Verticillium dahliae, 93 LysM effectors have been shown to contribute to virulence through chitin binding (Kombrink 94 5 5 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint The LysM effectors Mg1LysM and Mg3LysM, with one and three LysM domains, 96 respectively, have been characterized from the Septoria tritici blotch pathogen of wheat, Z. 97 tritici (Marshall et al, 2011). Functional characterization has revealed that Mg3LysM can 98 suppress chitin-induced immunity in a similar fashion as C. fulvum Ecp6. INTRODUCTION 47 Surprisingly, in 99 contrast to Ecp6 and similar to Avr4, Mg3LysM was additionally shown to have the ability to 100 protect fungal hyphae against chitinase hydrolysis. As expected, based on the presence of a 101 single LysM domain only, a role in suppression of chitin-triggered immunity could not be 102 demonstrated for Mg1LysM (Marshall et al, 2011). Intriguingly, however, Mg1LysM was 103 characterized as a functional homolog of Avr4 that protects hyphae against hydrolysis by host 104 chitinases (Marshall et al, 2011). In order to understand how a LysM effector that is 105 composed from little more than only a single LysM domain is able to confer protection of cell 106 wall chitin from hydrolysis by plant enzymes, we aimed to obtain a crystal structure of the Z. 107 tritici effector Mg1LysM in this study. Surprisingly, we discovered that Mg1LysM has the 108 ability to simultaneously undergo ligand-mediated dimerization as well as ligand-independent 109 homodimerization, allowing the formation of a contiguous oligomeric structure that anchors 110 to the fungal cell wall through chitin to confer its protection ability. 111 6 6 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint RESULTS 112 Crystal structure of Mg1LysM reveals ligand-dependent and -independent 113 intermolecular dimerization 114 In order to understand LysM effector functionality, and particularly how Mg1LysM is able to 115 protect chitin against chitinase hydrolysis, the crystal structure of Mg1LysM was determined. 116 To this end, Mg1LysM was heterologously produced in the yeast Pichia pastoris and purified 117 based on the presence of a His-FLAG affinity tag. The large Mg1LysM crystals that were 118 finally obtained by micro-seeding techniques (Bergfors, 2003) belonged to the space group P 119 61 2 2. Some crystals were soaked with the Ta6Br14 cluster and initial phases were determined 120 by the single-wavelength anomalous dispersion technique (SAD; Table 1). INTRODUCTION 47 The initial phases 121 were further improved with the help of an I3C soaked crystal. A native dataset was finally 122 refined to a resolution of 2.41 Å with an Rwork and Rfree of 17.96% and 22.03%, 123 respectively (Table 1). The structure model comprises in total four copies of the complete 124 mature protein sequence except for the first amino acid after the signal peptide for chains A to 125 C and the first two amino acids after the signal peptide for chain D, and one carbohydrate 126 molecule per asymmetric unit (a.u.). 127 A d h i f h d i f 1 i 128 Crystal structure of Mg1LysM reveals ligand-dependent and -independent 113 intermolecular dimerization 114 In order to understand LysM effector functionality, and particularly how Mg1LysM is able to 115 protect chitin against chitinase hydrolysis, the crystal structure of Mg1LysM was determined. 116 To this end, Mg1LysM was heterologously produced in the yeast Pichia pastoris and purified 117 based on the presence of a His-FLAG affinity tag. The large Mg1LysM crystals that were 118 finally obtained by micro-seeding techniques (Bergfors, 2003) belonged to the space group P 119 61 2 2. Some crystals were soaked with the Ta6Br14 cluster and initial phases were determined 120 by the single-wavelength anomalous dispersion technique (SAD; Table 1). The initial phases 121 were further improved with the help of an I3C soaked crystal. A native dataset was finally 122 refined to a resolution of 2.41 Å with an Rwork and Rfree of 17.96% and 22.03%, 123 respectively (Table 1). The structure model comprises in total four copies of the complete 124 mature protein sequence except for the first amino acid after the signal peptide for chains A to 125 C and the first two amino acids after the signal peptide for chain D, and one carbohydrate 126 molecule per asymmetric unit (a.u.). 127 As expected, the tertiary structure of the LysM domain of an Mg1LysM monomer is 128 similar to that of previously described LysM domains (Bateman & Bycroft, 2000; Bielnicki et 129 al, 2006; Bozsoki et al, 2017; Koharudin et al, 2011; Liu et al, 2012; Sánchez-Vallet et al, 130 2013) with a conserved βααβ-fold in which the antiparallel β-sheet lies adjacent to two α- 131 helices (Fig1 and S1 Fig). The compact LysM structure is stabilized by two disulfide bridges 132 between Cys44 and Cys78, and between Cys13 and Cys70 (Fig 1 and S1 Fig). In addition to the 133 single LysM domain, Mg1LysM comprises a relatively long N-terminal tail that contains a 134 short β-strand (Fig 1). 135 7 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Crystal structure of Mg1LysM reveals ligand-dependent and -independent 113 intermolecular dimerization 114 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint pastoris. Potentially, this finding indicates that Mg1LysM displays an increased affinity for 161 chitin (low micromolar range) when compared with other, single-acting, LysM domains. The 162 chitin binding site is formed by the loops between the first ß-strand and the first α-helix and 163 between the second α-helix and the second ß-strand of Mg1LysM, encompassing the residues 164 26GDTLT30 and 56NRI58 that are conserved in many other LysM domains including those of 165 Ecp6 (Sánchez-Vallet et al, 2013) (S1 Fig). Remarkably, besides the ligand-independent 166 Mg1LysM homodimerization described above, the crystal structure revealed that chitin 167 induces a dimerization of homodimers and, consequently, that a chitin-binding groove is 168 formed by two LysM domains from two independent protomers (Fig 1, Fig 2). In addition to 169 the amino acids that are in direct contact with the chitin trimer, the ligand-induced 170 dimerization is strengthened by several hydrogen bonds that occur between residues from the 171 two protomers involved. One salt bridge between residues K31 and D54 of the different 172 protomers stabilizes the binding of the single chitin molecule and adds further strength to the 173 dimerization, resulting in a tight binding pocket in which the chitin trimer is strongly bound 174 (Fig 2). Arguably, we would expect an increased chitin-binding affinity of Mg1LysM when 175 compared with a single-acting LysM domain, which can explain in turn why the chitin 176 remained adhered to the Mg1LysM protein during the protein purification procedure. 177 161 In order to determine the Mg1LysM chitin-binding affinity, isothermal titration 178 calorimetry (ITC) analysis was used. Since the crystal structure revealed that a portion of the 179 Mg1LysM binding sites were occupied by chitin in the P. Crystal structure of Mg1LysM reveals ligand-dependent and -independent 113 intermolecular dimerization 114 It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint The four Mg1LysM monomers within the a.u. form two pairs of homodimers that are 136 each very tightly packed. The large homodimerization interface that occurs between two 137 monomers was calculated to be 1113 Å2 using PISA (Protein Interfaces, Surfaces and 138 Assemblies; http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) (Krissinel & Henrick, 2007) and 139 is stabilized by a total of 25 hydrogen bonds between residues of each of the monomers. In 140 addition, the crystal structure revealed that the N-terminal 12 amino acid tails of the 141 homodimer run anti-parallel and form a small but stable β-sheet (5ITI7 of each chain) that is 142 stabilized by clustering of all four isoleucine side-chain residues and by threonine-threonine 143 sidechain hydrogen bonding, potentially strengthening the homodimer (Fig 1). The latter 144 hypothesis is further supported by the formation of two additional salt bridges formed 145 between Arg2 of one subunit and Asp12 of the other one (Fig 1B and 1C). The root mean 146 square deviations (rmsd) between the Cα atoms of the two homodimers of the a.u. is 0.267 Å 147 as calculated with Lsqkab of the CCP4 suite (Winn, et al, 2011). 148 Surprisingly, when determining the crystal structure for the C. fulvum LysM effector 149 Ecp6 in the absence of exogenously added chitin we found a chitin tetramer in a large 150 interdomain groove between two of the three intrachain LysM domains that appeared to 151 constitute an ultra-high affinity binding site, while no chitin binding was observed to the 152 remaining, third LysM domain of Ecp6 (Sánchez-Vallet et al, 2013). Unexpectedly, the 153 calculated 2\|F0\| – \|Fc\| electron density map of the Mg1LysM crystal structure assembly 154 similarly revealed well-defined electron density for a single chitin trimer bound to one 155 monomer of the a.u. only (S2 Fig). Inspection of the crystal packing interactions unveiled the 156 presence of a chitin binding pocket formed between two Mg1LysM protomers of 157 neighbouring homodimers (Fig 1). Since protein purification and crystallization was 158 performed without exogenous addition of chitin in this case as well, we concluded that the 159 chitin once more was derived from the cell wall of the heterologous protein production host P. 160 8 8 . Crystal structure of Mg1LysM reveals ligand-dependent and -independent 113 intermolecular dimerization 114 Interestingly, none of these non-synonymous SNPs localized 196 within the signal peptide, the homodimerization surface, the chitin-binding groove or 197 concerned the residues involved in disulfide or salt bridge formation (Fig 4), pointing towards 198 the relevance of these sites for the functionality of Mg1LysM. 199 200 Chitin-induced Mg1LysM polymerization is crucial for protection of hyphae against the 201 hydrolytic activity of plant chitinases 202 We first attempted to evaluate the contribution of the ligand-independent Mg1LysM 203 homodimerization to hyphal protection against chitinases. To this end, we pursued to produce 204 an Mg1LysM mutant that lacked the 12-amino acid tail that is involved in ligand-independent 205 homodimerization. Unfortunately, production of this mutant in the heterologous host P. 206 pastoris was not successful, most probably because of protein instability. 207 Subsequently, we evaluated the role of chitin-induced Mg1LysM homodimerization in 208 the protection of fungal hyphae against chitinases by generating three mutant proteins. The 209 T28 residue that makes direct contact with the chitin substrate in the binding site that was 210 Crystal structure of Mg1LysM reveals ligand-dependent and -independent 113 intermolecular dimerization 114 pastoris-produced Mg1LysM 180 preparation, we pursued production of Mg1LysM in the bacterium Escherichia coli as a 181 heterologous system that is devoid of chitin, in order to obtain chitin-free protein. Subsequent 182 ITC analysis based on chitohexaose (GlcNAc)6 titration revealed that this protein preparation 183 bound chitin with a binding affinity of 4.36 µM (Fig 3). Furthermore, a stoichiometry of 1:2 184 was observed (n = 0.504) based on a single-binding-site model, analogous to the observation 185 9 9 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint that two Mg1LysM protomers originating from two Mg1LysM homodimers bind a single 186 chitin trimer as disclosed in the crystal structure model. Obviously, this ratio also implies a 187 polymerisation reaction in solution upon addition of the ligand chitohexaose. 188 that two Mg1LysM protomers originating from two Mg1LysM homodimers bind a single 186 chitin trimer as disclosed in the crystal structure model. Obviously, this ratio also implies a 187 polymerisation reaction in solution upon addition of the ligand chitohexaose. 188 that two Mg1LysM protomers originating from two Mg1LysM homodimers bind a single 186 chitin trimer as disclosed in the crystal structure model. Obviously, this ratio also implies a 187 polymerisation reaction in solution upon addition of the ligand chitohexaose. 188 189 Mg1LysM sequence conservation in a world-wide collection of Z. tritici isolates 190 In order to evaluate Mg1LysM conservation in Z. tritici, the occurrence of sequence 191 polymorphisms was evaluated in a collection of 149 isolates from four different populations 192 collected in Switzerland, Australia, Israel and the USA (Hartmann et al, 2017). This analysis 193 revealed that the Mg1LysM protein sequence is highly conserved. Only 5 non-synonymous 194 mutations were identified in the full length Mg1LysM protein, three of which were previously 195 identified (Marshall et al, 2011). Mg1LysM sequence conservation in a world-wide collection of Z. tritici isolates 190 In order to evaluate Mg1LysM conservation in Z. tritici, the occurrence of sequence 191 polymorphisms was evaluated in a collection of 149 isolates from four different populations 192 collected in Switzerland, Australia, Israel and the USA (Hartmann et al, 2017). This analysis 193 revealed that the Mg1LysM protein sequence is highly conserved. Only 5 non-synonymous 194 mutations were identified in the full length Mg1LysM protein, three of which were previously 195 identified (Marshall et al, 2011). Interestingly, none of these non-synonymous SNPs localized 196 within the signal peptide, the homodimerization surface, the chitin-binding groove or 197 concerned the residues involved in disulfide or salt bridge formation (Fig 4), pointing towards 198 the relevance of these sites for the functionality of Mg1LysM. 199 Chitin-induced Mg1LysM polymerization is crucial for protection of hyphae against the 201 We first attempted to evaluate the contribution of the ligand-independent Mg1LysM 203 homodimerization to hyphal protection against chitinases. To this end, we pursued to produce 204 an Mg1LysM mutant that lacked the 12-amino acid tail that is involved in ligand-independent 205 homodimerization. Unfortunately, production of this mutant in the heterologous host P. 206 pastoris was not successful, most probably because of protein instability. 207 Subsequently, we evaluated the role of chitin-induced Mg1LysM homodimerization in 208 the protection of fungal hyphae against chitinases by generating three mutant proteins. The 209 T28 residue that makes direct contact with the chitin substrate in the binding site that was 210 Subsequently, we evaluated the role of chitin-induced Mg1LysM homodimerization in 208 the protection of fungal hyphae against chitinases by generating three mutant proteins. The 209 T28 residue that makes direct contact with the chitin substrate in the binding site that was 210 10 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint previously shown to be essential for chitin binding in the C. fulvum LysM effector Ecp6 was 211 substituted by arginine. In addition, the two residues involved in the formation of the 212 intermolecular salt bridge near the chitin binding site (K31 and D54) were substituted by 213 alanines, respectively. In order to obtain chitin-free proteins, production in E. coli was 214 pursued. 215 Based on previous findings for Ecp6 (Sánchez-Vallet et al, 2013), we predicted that 216 the T28R mutant was incapable of binding chitin, but the chitin binding capacity of the 217 mutants impaired in the intermolecular salt bridge formation remained to be elucidated 218 (Sánchez-Vallet et al, 2013). Indeed, ITC analysis with the mutant T28R revealed a 219 significantly reduced binding affinity of 88.5 μM, which is twenty times weaker when 220 compared with wild-type Mg1LysM protein produced in E. coli (4.36 μM; Fig 3). Chitin-induced Mg1LysM polymerization is crucial for protection of hyphae against the 201 However, 221 also the binding affinity of the mutants K31A and D54A decreased, to 338.9 µM and 46.3 µM, 222 respectively (Fig 3). Furthermore, besides a lower chitin-binding capacity, the stoichiometry 223 calculated for the mutants K31A and D54A changed from 1:2 as observed for the wild-type 224 Mg1LysM protein to 1:1. This finding implies that a single monomer of K31A or D54A binds a 225 single chitohexaose in solution, whereas a single chitohexaose is bound by two wild-type 226 Mg1LysM protomers, supporting the hypothesis that the chitin-induced dimerization is 227 impaired in K31A and D54A by disruption of the intermolecular salt bridge (Fig 3). 228 Subsequently, we tested the ability of the Mg1LysM mutants to protect fungal hyphae 229 against the hydrolytic activity of plant chitinases. To this end, spores of Fusarium oxysporum 230 and Trichoderma viride were germinated, incubated with a plant extract containing hydrolytic 231 enzymes including chitinases, and supplemented with wild-type or mutant Mg1LysM. As 232 expected, wild-type Mg1LysM protein produced in E. coli prevented the hydrolysis of 233 Fusarium oxysporum f. sp. lycopersici (Fig 5) and Trichoderma viride (S3 Fig) hyphae. 234 Furthermore, mutant T28R that is mutated in the substrate-binding loop did not protect F. 235 11 11 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint oxysporum and T. viride hyphae against these hydrolytic enzymes (Fig 5 and S3 Fig), 236 confirming that chitin-binding of Mg1LysM is required to confer protection of cell walls 237 against hydrolysis by plant enzymes. Considering the even lower chitin-binding activity, it is 238 not surprising that also mutant K31A did not protect cell walls against enzymatic hydrolysis. 239 However, also mutant D54A no longer protected cell walls, suggesting that a ten-fold 240 reduction of chitin-binding affinity is sufficient to disrupt the protective activity of Mg1LysM. Chitin-induced Mg1LysM polymerization is crucial for protection of hyphae against the 201 241 Unfortunately, based on these findings it is impossible to determine the contribution of the 242 dimerization to the protection activity of Mg1LysM. 243 We previously determined that LysM effector Ecp6 has two sites that bind chitin with 244 1:1 stoichiometry, one with ultra-high affinity (kd = 280 pM) and one in the range with which 245 Mg1LysM binds chitin (kd = 1.70 µM) (Bateman & Bycroft, 2000; Bielnicki et al, 2006; 246 Bozsoki et al, 2017; Koharudin et al, 2011; Liu et al, 2012; Sánchez-Vallet et al, 2013), but 247 both of which bind chitin with higher affinity than Mg1LysM. Intriguingly, Ecp6 fails to 248 protect hyphae against hydrolysis by chitinases (De Jonge et al, 2010; Sánchez-Vallet et al, 249 2013). Nevertheless, localization experiments making use of constitutive expression of C- 250 terminally GFP-tagged Ecp6 in Verticillium dahliae, and of BODIPY-labelled Ecp6 protein 251 exogenously applied to Botrytis cinerea, two fungal species that expose chitin on their cell 252 walls during growth in vitro, revealed that Ecp6 can bind to fungal cell walls (S4 Fig) in a 253 similar fashion as Cladosporium fulvum effector protein Avr4 that protects fungal cell walls 254 against hydrolysis by chitin binding through an invertebrate chitin-binding domain (van den 255 Burg et al, 2006; Van Esse et al, 2007). These findings suggest that binding of a LysM 256 effector to cell wall chitin with high affinity is not sufficient to mediate protection against 257 hydrolytic enzymes. Moreover, from these observations we infer that chitin-induced 258 dimerization of Mg1LysM may be crucial for hyphal protection against plant enzymatic 259 hydrolysis. 260 12 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. Chitin-induced Mg1LysM polymerization is crucial for protection of hyphae against the 201 ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Considering that Mg1LysM homodimers possess two chitin-binding sites on opposite 261 sides of the protein complex (Fig 1), combined with the observed chitin-induced dimerization 262 that may be responsible for the protective activity, we hypothesized that Mg1LysM will form 263 highly oligomeric super-complexes in which ligand-independent Mg1LysM homodimers 264 dimerize on both ends in a chitin-dependent manner (Fig 6A). Moreover, we hypothesized 265 that LysM effectors that do not protect hyphae against chitinase hydrolysis would not display 266 such oligomerisation. To test these hypotheses, we first assessed whether we could alter the 267 particle size distribution of Mg1LysM in solutions by adding chitohexaose. Using dynamic 268 light scattering (DLS) we observed that, upon chitin addition at a molar ratio of 1:2 the radius 269 distribution of Mg1LysM particles shifted from around 10 nm in the absence of chitin to 100 270 nm in the presence of chitin. Moreover, further increase of the chitin concentration to a 1:5 271 ratio induced a strong signal appearing at 100 µM, demonstrating clear ligand-induced 272 polymerisation of Mg1LysM protein to large protein complexes. Next, we assessed the effect 273 of chitohexaose on the distribution of Ecp6 particles in solution. Interestingly, although the 274 addition of chitohexaose smoothened the Ecp6 particle size distribution, suggesting the 275 stabilization of Ecp6 molecules, chitin addition did not lead to an increased particle size. 276 Thus, in contrast to Mg1LysM, Ecp6 does not display chitin-induced polymerization. 277 261 13 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint DISCUSSION 278 DISCUSSION 278 Studies on many plant pathogenic fungi have shown that the perception of microbial cell wall- 279 derived glycans by plant hosts plays a central role in microbe–host interactions (Rovenich et 280 al, 2016). Among these glycans, fungal cell wall chitin has emerged as one of the most potent 281 fungal elicitors of host immune responses (Rovenich et al, 2016; Sánchez-Vallet et al, 2015). 282 The widespread glycan perception capacity in plants has spurred the evolution of various 283 fungal strategies to evade glycan perception (Rovenich et al, 2016; Sánchez-Vallet et al, 284 2015). Many fungal pathogens secrete LysM effectors to perturb the induction of chitin- 285 triggered immunity. Structural analysis of the C. fulvum LysM effector Ecp6 has revealed that 286 this activity could be attributed to the presence of an ultra-high chitin binding affinity site in 287 the LysM effector protein that is established by intramolecular LysM domain dimerization 288 (Sánchez-Vallet et al, 2013). However, some LysM effectors rather, or additionally, are able 289 to prevent the hydrolysis of fungal cell wall chitin by plant chitinases. Moreover, functional 290 characterization of Mg1LysM, a LysM effector that is merely composed of a single LysM 291 domain and a few additional amino acids, suggested that the ability to protect cell walls is 292 conferred simply by the chitin-binding ability of the LysM domain (Marshall et al, 2011). Yet, 293 the observation that various other LysM effectors, including C. fulvum Ecp6, M. oryzae Slp1 294 and C. higginsianum ELP1 and ELP2, are not able to protect hyphae challenged this 295 hypothesis (De Jonge et al, 2010; Mentlak et al, 2012; Takahara et al, 2016). Thus, the 296 mechanism by which some LysM effectors are able to protect fungal cell walls remained to be 297 characterized. The crystal structure model that was generated in this study has revealed that 298 Mg1LysM is able to undergo chitin-mediated dimerization such that a chitin molecule is 299 deeply buried in the protein dimer. DISCUSSION 278 Nevertheless, the structure of the dimer allows a chitin 300 chain to protrude into the solvent on either side of the binding groove, such that it is 301 conceivable that the dimer can be formed on long-chain chitin polymers of any length, 302 DISCUSSION 278 Studies on many plant pathogenic fungi have shown that the perception of microbial cell wall- 279 derived glycans by plant hosts plays a central role in microbe–host interactions (Rovenich et 280 al, 2016). Among these glycans, fungal cell wall chitin has emerged as one of the most potent 281 fungal elicitors of host immune responses (Rovenich et al, 2016; Sánchez-Vallet et al, 2015). 282 The widespread glycan perception capacity in plants has spurred the evolution of various 283 fungal strategies to evade glycan perception (Rovenich et al, 2016; Sánchez-Vallet et al, 284 2015). Many fungal pathogens secrete LysM effectors to perturb the induction of chitin- 285 triggered immunity. Structural analysis of the C. fulvum LysM effector Ecp6 has revealed that 286 this activity could be attributed to the presence of an ultra-high chitin binding affinity site in 287 the LysM effector protein that is established by intramolecular LysM domain dimerization 288 (Sánchez-Vallet et al, 2013). However, some LysM effectors rather, or additionally, are able 289 to prevent the hydrolysis of fungal cell wall chitin by plant chitinases. Moreover, functional 290 characterization of Mg1LysM, a LysM effector that is merely composed of a single LysM 291 domain and a few additional amino acids, suggested that the ability to protect cell walls is 292 conferred simply by the chitin-binding ability of the LysM domain (Marshall et al, 2011). Yet, 293 the observation that various other LysM effectors, including C. fulvum Ecp6, M. oryzae Slp1 294 and C. higginsianum ELP1 and ELP2, are not able to protect hyphae challenged this 295 hypothesis (De Jonge et al, 2010; Mentlak et al, 2012; Takahara et al, 2016). Thus, the 296 mechanism by which some LysM effectors are able to protect fungal cell walls remained to be 297 characterized. The crystal structure model that was generated in this study has revealed that 298 Mg1LysM is able to undergo chitin-mediated dimerization such that a chitin molecule is 299 deeply buried in the protein dimer. DISCUSSION 278 Combined with the observed chitin-induced 316 dimerization, we postulate that this provides Mg1LysM with the ability to form highly 317 oligomeric super-complexes in the fungal cell wall, in which ligand-independent Mg1LysM 318 homodimers dimerize on both ends in a chitin-dependent manner, leading to the formation of 319 a contiguous structure throughout the cell wall (Fig 6A). Possibly, it is such contiguous 320 structure that provides steric hindrance that renders fungal cell wall chitin inaccessible to 321 chitinase enzymes. Thus, both ligand-independent homodimerization as well as ligand- 322 induced dimerization of Mg1LysM appear to be required for its cell wall protective function. 323 Accordingly, residues shaping these regions are fully conserved in all Mg1LysM isoforms 324 h h b id ifi d d 325 including polymeric cell wall chitin. In addition to several noncovalent bounds between the 303 two Mg1LysM protomers and between the protein and the chitin, a salt bridge between the 304 two Mg1LysM protomers strengthens the chitin-binding affinity and thus supports the chitin- 305 induced dimerization by stabilizing the chitin binding groove. Arguably, it is this particular 306 trait that confers the ability to protect hyphae against plant chitinases, as disruption of the ion 307 bond in the K31A mutant abolished hyphal protection and chitin binding by itself is not 308 sufficient to confer cell wall protection. The crystal structure further revealed that Mg1LysM 309 undergoes ligand-independent homodimerization whereby a large dimerization interface of 310 two Mg1LysM monomers is stabilized by several noncovalent bounds and further 311 strengthened by two salt bridges between the interlaced N-terminal regions of the protein. 312 Despite various efforts, we have not been able to produce monomeric Mg1LysM protein, 313 suggesting that ligand-independent homodimerization is required for proper folding of the 314 protein. Consequently, Mg1LysM homodimers are released and possess two chitin-binding 315 sites on opposite sides of the protein (Fig 1). Combined with the observed chitin-induced 316 dimerization, we postulate that this provides Mg1LysM with the ability to form highly 317 oligomeric super-complexes in the fungal cell wall, in which ligand-independent Mg1LysM 318 homodimers dimerize on both ends in a chitin-dependent manner, leading to the formation of 319 a contiguous structure throughout the cell wall (Fig 6A). Possibly, it is such contiguous 320 structure that provides steric hindrance that renders fungal cell wall chitin inaccessible to 321 chitinase enzymes. DISCUSSION 278 Nevertheless, the structure of the dimer allows a chitin 300 chain to protrude into the solvent on either side of the binding groove, such that it is 301 conceivable that the dimer can be formed on long-chain chitin polymers of any length, 302 Studies on many plant pathogenic fungi have shown that the perception of microbial cell wall- 279 14 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint including polymeric cell wall chitin. In addition to several noncovalent bounds between the 303 two Mg1LysM protomers and between the protein and the chitin, a salt bridge between the 304 two Mg1LysM protomers strengthens the chitin-binding affinity and thus supports the chitin- 305 induced dimerization by stabilizing the chitin binding groove. Arguably, it is this particular 306 trait that confers the ability to protect hyphae against plant chitinases, as disruption of the ion 307 bond in the K31A mutant abolished hyphal protection and chitin binding by itself is not 308 sufficient to confer cell wall protection. The crystal structure further revealed that Mg1LysM 309 undergoes ligand-independent homodimerization whereby a large dimerization interface of 310 two Mg1LysM monomers is stabilized by several noncovalent bounds and further 311 strengthened by two salt bridges between the interlaced N-terminal regions of the protein. 312 Despite various efforts, we have not been able to produce monomeric Mg1LysM protein, 313 suggesting that ligand-independent homodimerization is required for proper folding of the 314 protein. Consequently, Mg1LysM homodimers are released and possess two chitin-binding 315 sites on opposite sides of the protein (Fig 1). Protein production and purification 327 Mg1LysM and mutants were produced in Pichia pastoris strain GS115 and in Escherichia 328 coli. P. pastoris production was performed as previously described (Marshall et al, 2011). 329 Purification was performed by gel filtration chromatography (Superdex 75: GE Healthcare, 330 Chicago, IL, US) in 20 mM HEPES, pH 7.0, and 50 mM NaCl. P. pastoris produced protein 331 (6-10 mg/mL) was used for protein crystallization. E. coli protein production was performed 332 using the pET-SUMO (Thermo Fisher, Waltham, MA, USA) expression system in E. coli 333 ORIGAMI (DE3, Merck, Darmstadt, Germany) cells according to the manufacturer´s 334 instructions. Transformants were selected and grown in Luria broth (LB) medium until an 335 optical density of 0.8 at 600 nm was reached. Protein production was induced with the 336 addition of 0.05 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 28°C. Cells were 337 harvested by centrifugation ~20 h after induction, the cell pellets were dissolved and lysed 338 using lysozyme from chicken egg (Sigma-Aldrich, St. Louis, MO, US) and the 6xHis-SUMO 339 tagged proteins were purified from the soluble protein fraction after centrifugation using an 340 Ni2+-NTA Superflow column (Qiagen, Venlo, Netherlands). Next, purified proteins were 341 incubated with the SUMO protease ULP1 from Saccharomyces cerevisiae (Thermo Fisher, 342 Waltham, MA, USA), dialysed over night against 200 mM NaCl at 4°C, and again passed 343 through a Ni2+-NTA Superflow column. Native proteins were finally dialysed against 50 mM 344 NaH2PO4, 300 mM NaCl at pH 8.0, concentrated to 0.6 mg/mL over Amicon ultracentrifugal 345 filter units (Sigma-Aldrich, St. Louis, MO, USA) and used for subsequent assays. 346 347 DISCUSSION 278 Thus, both ligand-independent homodimerization as well as ligand- 322 induced dimerization of Mg1LysM appear to be required for its cell wall protective function. 323 Accordingly, residues shaping these regions are fully conserved in all Mg1LysM isoforms 324 that have been identified to date. 325 15 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Crystallization conditions and structure determination 348 First crystal hits with 1,4-dioxane as the reservoir solution were obtained overnight in a small 349 initial vapor-diffusion crystallization screening campaign using the Phoenix robot (Art 350 16 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Robbins Instrument LLC, Sunnyvale, CA, USA) with 96-well Intelli Plates (Dunn 351 Labortechnik GmbH, Asbach, Germany) and several different commercial screens (Hampton 352 Research, Aliso Viejo, CA, USA; Molecular Dimensions, Newmarket, Suffolk, UK) 353 (Newman et al, 2005). Conditions were further optimized and useful crystals were finally 354 obtained by micro-seeding techniques using 0.1 M sodium citrate pH 5.6, 5%-20% PEG4000 355 and 5% isopropanol as the reservoir solution (Bergfors, 2003). 0.2 M sodium acetate pH 4.6 356 with 20% ethylene glycol was used as the crystal cryo-buffer. Several crystals were soaked 357 with either I3C (Jena Bioscience GmbH, Jena, Germany), 2 mM in cryo-buffer, quick soak, or 358 Ta6Br14 (Jena Bioscience GmbH, Jena, Germany), 1 mM in cryo-buffer, 1 hr soak, brief wash 359 and prolonged back soak. X-ray diffraction data were collected on BL14.1 at the BESSY II 360 electron storage ring operated by the Helmholz-Zentrum Berlin (Gerlach et al, 2016). Using 361 the Phenix AutoSol wizard (Adams et al, 2010), initial phases were obtained from the 362 Ta6Br12 2- derivatized crystals by single-wavelength anomalous dispersion techniques (SAD) 363 that were improved by phase information from the I3C derivatized crystals by single 364 isomorphous replacement with anomalous scattering (SIRAS). 365 The structure was refined using REFMAC5 (Murshudov et al, 2011) and phenix 366 (Adams et al, 2010) and manually built using Coot (Emsley et al, 2010). All figures showing 367 structural representations were prepared with the program PyMOL (The PyMOL Molecular 368 Graphics System, Version 2.0 Schrödinger, LLC, DeLano Scientific, Palo Alto, CA, USA]. 369 The quality of the final model was validated with MolProbity (Chen et al, 2010). Crystallization conditions and structure determination 348 coli-produced wild-type Mg1LysM (20 µM) and the mutants T28R (15 µM), K31A (30 386 µM) and D54A (30 µM) were titrated with a single injection of 2 µL, followed by 26 387 injections of 10 µL of (GlcNAc)6 (Isosep AB, Tullinge, Sweden) at 400 µM. Before the 388 experiment, all proteins were dialyzed against 20 mM of sodium chloride, pH 7.0. 389 Chitohexaose (Megazyme, Wicklow, Ireland) was freshly dissolved in the dialysis buffer. 390 Data were analyzed using Origin (OriginLab, Northampton, MA, USA) and fitted to a one- 391 binding-site model. 392 393 Dynamic light scattering (DLS) measurements 394 Mg1LysM and Ecp6 were dialyzed overnight against water, and subsequently incubated with 395 0.01% Triton X-100 for 4 hours to improve protein solubility. Next, chitohexaose 396 (Megazyme, Wicklow, Ireland) was added in a molar ratio of 1:0, 1:2 and 1:5 (protein:chitin) 397 Trichoderma viride were incubated overnight at room temperature in 40 μL of half-strength 376 potato dextrose broth (PDB; Becton Dickinson, Franklin Lakes, NJ, USA) in a 96-well 377 microtiter plate. Subsequently, wild-type or mutant Mg1LysM protein was added at a final 378 concentration of 20 μM. After a 2 h incubation period, 10 μL of tomato extract containing 379 hydrolytic enzymes was added (van den Burg et al, 2004). Fungal growth was assessed 380 microscopically after 4 h of incubation at room temperature. 381 Isothermal titration calorimetry 383 Isothermal titration calorimetry (ITC) experiments were performed at 20°C following 384 standard procedures using a Microcal VP-ITC calorimeter (GE Healthcare, Chicago, IL, US). 385 The E. coli-produced wild-type Mg1LysM (20 µM) and the mutants T28R (15 µM), K31A (30 386 µM) and D54A (30 µM) were titrated with a single injection of 2 µL, followed by 26 387 injections of 10 µL of (GlcNAc)6 (Isosep AB, Tullinge, Sweden) at 400 µM. Before the 388 experiment, all proteins were dialyzed against 20 mM of sodium chloride, pH 7.0. 389 Chitohexaose (Megazyme, Wicklow, Ireland) was freshly dissolved in the dialysis buffer. 390 Data were analyzed using Origin (OriginLab, Northampton, MA, USA) and fitted to a one- 391 binding-site model. 392 Crystallization conditions and structure determination 348 Refinement 370 and phasing statistics are summarized in Table 1. 371 372 Chitinase-protection assay 373 In-vitro chitinase protection assays were performed as described previously (van den Burg et 374 al, 2004). Essentially, ~103 conidiospores of Fusarium oxysporum f. sp. lycopersici or 375 The structure was refined using REFMAC5 (Murshudov et al, 2011) and phenix 366 (Adams et al, 2010) and manually built using Coot (Emsley et al, 2010). All figures showing 367 structural representations were prepared with the program PyMOL (The PyMOL Molecular 368 Graphics System, Version 2.0 Schrödinger, LLC, DeLano Scientific, Palo Alto, CA, USA]. 369 The quality of the final model was validated with MolProbity (Chen et al, 2010). Refinement 370 and phasing statistics are summarized in Table 1. 371 In-vitro chitinase protection assays were performed as described previously (van den Burg et 374 al, 2004). Essentially, ~103 conidiospores of Fusarium oxysporum f. sp. lycopersici or 375 17 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Trichoderma viride were incubated overnight at room temperature in 40 μL of half-strength 376 potato dextrose broth (PDB; Becton Dickinson, Franklin Lakes, NJ, USA) in a 96-well 377 microtiter plate. Subsequently, wild-type or mutant Mg1LysM protein was added at a final 378 concentration of 20 μM. After a 2 h incubation period, 10 μL of tomato extract containing 379 hydrolytic enzymes was added (van den Burg et al, 2004). Fungal growth was assessed 380 microscopically after 4 h of incubation at room temperature. 381 382 Isothermal titration calorimetry 383 Isothermal titration calorimetry (ITC) experiments were performed at 20°C following 384 standard procedures using a Microcal VP-ITC calorimeter (GE Healthcare, Chicago, IL, US). 385 The E. Localisation of Ecp6 401 Conidiospores of a V. dahliae transformant were grown in a few micro liters of PDB on a 402 glass slide with coverslip. To prevent the samples from drying out, the slides were mounted 403 on top of moistened tissue in an empty pipette box with water on the bottom. After 404 approximately 6 hr of growth at room temperature, the slides were used for localization 405 studies. Labelling of Ecp6 and Avr4 with BODIPY TMR-X amine reactive probe (Invitrogen, 406 Carlsbad, CA, USA) was performed as described previously (van den Burg et al, 2006; Van 407 Esse et al, 2007). Conidiospores of Botrytis cinerea were harvested and germinated overnight 408 in PDB at room temperature. BODIPY-labeled proteins were applied at a concentration of 4 409 μM and incubated for 2-3 hrs at room temperature in the dark. All localisation studies were 410 performed using a Nikon eclipse 90i UV microscope and NIS-Elements AR 2.3 software 411 (Nikon Instruments Inc., Melville, USA). 412 p p pp 9 μM and incubated for 2-3 hrs at room temperature in the dark. All localisation studies were 410 performed using a Nikon eclipse 90i UV microscope and NIS-Elements AR 2.3 software 411 (Nikon Instruments Inc., Melville, USA). 412 413 Assembly and alignment of Mg1LysM sequences 414 Illumina whole-genome sequencing data from a global collection of Z. tritici isolates was 415 used to extract Mg1LysM sequences (Hartmann & Croll, 2017). We used the SPAdes 416 assembler version 3.6.2 (Bankevich et al, 2012) to generate de-novo genome assemblies. The 417 Dynamic light scattering (DLS) measurements 394 We defined the k-mer range as 21, 35, 419 49, 63 and 77. We used the “—careful” option to reduce mismatches and indel errors in the 420 assembly. Polished assemblies were then used to retrieve the contigs containing Mg1LysM 421 orthologs based on blastn (Camacho et al, 2009). High-confidence sequence matches were 422 extracted with samtools (Li et al, 2009) from each draft assembly and aligned using MAFFT 423 version 7.305b (Katoh & Standley, 2013) using the --auto option and 1,000 iterative 424 Dynamic light scattering (DLS) measurements 394 Mg1LysM and Ecp6 were dialyzed overnight against water, and subsequently incubated with 395 0.01% Triton X-100 for 4 hours to improve protein solubility. Next, chitohexaose 396 (Megazyme, Wicklow, Ireland) was added in a molar ratio of 1:0, 1:2 and 1:5 (protein:chitin) 397 and incubated overnight. Particle size distribution was measured by a SpectroSize 300 (Xtal 398 Concepts, Hamburg, Germany). 399 400 18 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Localisation of Ecp6 401 Conidiospores of a V. dahliae transformant were grown in a few micro liters of PDB on a 402 glass slide with coverslip. To prevent the samples from drying out, the slides were mounted 403 on top of moistened tissue in an empty pipette box with water on the bottom. After 404 approximately 6 hr of growth at room temperature, the slides were used for localization 405 studies. Labelling of Ecp6 and Avr4 with BODIPY TMR-X amine reactive probe (Invitrogen, 406 Carlsbad, CA, USA) was performed as described previously (van den Burg et al, 2006; Van 407 Esse et al, 2007). Conidiospores of Botrytis cinerea were harvested and germinated overnight 408 in PDB at room temperature. BODIPY-labeled proteins were applied at a concentration of 4 409 μM and incubated for 2-3 hrs at room temperature in the dark. All localisation studies were 410 performed using a Nikon eclipse 90i UV microscope and NIS-Elements AR 2.3 software 411 (Nikon Instruments Inc., Melville, USA). 412 413 Assembly and alignment of Mg1LysM sequences 414 Illumina whole-genome sequencing data from a global collection of Z. tritici isolates was 415 used to extract Mg1LysM sequences (Hartmann & Croll, 2017). We used the SPAdes 416 assembler version 3.6.2 (Bankevich et al, 2012) to generate de-novo genome assemblies. The 417 SPAdes pipeline includes the BayesHammer read error correction module to build contigs in a 418 stepwise procedure based on increasing k-mer lengths. Assembly and alignment of Mg1LysM sequences 414 Illumina whole-genome sequencing data from a global collection of Z. tritici isolates was 415 used to extract Mg1LysM sequences (Hartmann & Croll, 2017). We used the SPAdes 416 assembler version 3.6.2 (Bankevich et al, 2012) to generate de-novo genome assemblies. The 417 SPAdes pipeline includes the BayesHammer read error correction module to build contigs in a 418 stepwise procedure based on increasing k-mer lengths. We defined the k-mer range as 21, 35, 419 49, 63 and 77. We used the “—careful” option to reduce mismatches and indel errors in the 420 assembly. Polished assemblies were then used to retrieve the contigs containing Mg1LysM 421 orthologs based on blastn (Camacho et al, 2009). High-confidence sequence matches were 422 extracted with samtools (Li et al, 2009) from each draft assembly and aligned using MAFFT 423 version 7.305b (Katoh & Standley, 2013) using the --auto option and 1,000 iterative 424 19 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint refinement cycles. Alignments were processed using JalView (Waterhouse et al, 2009) and 425 CLC Genomic Workbench 9 (Qiagen, Venlo, Netherlands). 426 427 Accession codes 428 All whole-genome sequencing data is accessible on the Nucleotide Short Read Archive 429 (accession numbers PRJNA327615 and PRJNA178194). The atomic coordinates and 430 experimental structure factors were deposited with the Protein Data Bank under accession 431 code 6Q40. 432 433 ACKNOWLEDGEMENTS 434 Work in the laboratory of B.P.H.J.T. is supported by the Research Council Earth and Life 435 Sciences (ALW) of the Netherlands Organization of Scientific Research (NWO). Assembly and alignment of Mg1LysM sequences 414 436 437 AUTHOR CONTRIBUTIONS 438 ASV, JRM, BPHJT conceived the study; ASV, HT, LRM, RdJ, HPvE designed experiments 439 ASV, HT, LRM, DJV, RSB, SW, AK, LV, DC performed experiments; RdJ, HPvE, AZ 440 analyzed data; ASV, HT, LRM, JRM, BPHJT wrote the manuscript; JRM, BPHJT supervised 441 the project; all authors discussed the results and contributed to the final manuscript. 442 443 CONFLICT OF INTEREST 444 The authors declare no conflict of interest exists. 445 refinement cycles. Alignments were processed using JalView (Waterhouse et al, 2009) and 425 CLC Genomic Workbench 9 (Qiagen, Venlo, Netherlands). 426 ACKNOWLEDGEMENTS 434 Work in the laboratory of B.P.H.J.T. is supported by the Research Council Earth and Life 435 Sciences (ALW) of the Netherlands Organization of Scientific Research (NWO). 436 Sciences (ALW) of the Netherlands Organization of Scientific Research (NWO). 436 AUTHOR CONTRIBUTIONS 438 It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint REFERENCES 446 Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, 447 Kapral GJ, Grosse-Kunstleve RW (2010) PHENIX: a comprehensive Python-based 448 system for macromolecular structure solution. Acta Crystallographica Section D: 449 Biological Crystallography 66: 213-221 450 Adrangi S, Faramarzi MA (2013) From bacteria to human: a journey into the world of 451 chitinases. Biotechnology Advances 31: 1786-1795 452 Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, 453 Nikolenko SI, Pham S, Prjibelski AD (2012) SPAdes: a new genome assembly 454 algorithm and its applications to single-cell sequencing. Journal of Computational 455 Biology 19: 455-477 456 Bateman A, Bycroft M (2000) The structure of a LysM domain from E. coli membrane-bound 457 lytic murein transglycosylase D (MltD) 1. Journal of Molecular Biology 299: 1113- 458 1119 459 Bergfors T (2003) Seeds to crystals. Journal of Structural Biology 142: 66-76 460 Bielnicki J, Devedjiev Y, Derewenda U, Dauter Z, Joachimiak A, Derewenda ZS (2006) B. 461 subtilis ykuD protein at 2.0 Å resolution: insights into the structure and function of a 462 novel, ubiquitous family of bacterial enzymes. Proteins: Structure, Function, and 463 Bioinformatics 62: 144-151 464 Boller T (1995) Chemoperception of microbial signals in plant cells. 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Molecular Microbiology 69: 119-136 470 AUTHOR CONTRIBUTIONS 438 AUTHOR CONTRIBUTIONS 438 ASV, JRM, BPHJT conceived the study; ASV, HT, LRM, RdJ, HPvE designed experiments 439 ASV, HT, LRM, DJV, RSB, SW, AK, LV, DC performed experiments; RdJ, HPvE, AZ 440 analyzed data; ASV, HT, LRM, JRM, BPHJT wrote the manuscript; JRM, BPHJT supervised 441 the project; all authors discussed the results and contributed to the final manuscript. 442 443 CONFLICT OF INTEREST 444 The authors declare no conflict of interest exists. 445 ASV, JRM, BPHJT conceived the study; ASV, HT, LRM, RdJ, HPvE designed experiments 439 ASV, HT, LRM, DJV, RSB, SW, AK, LV, DC performed experiments; RdJ, HPvE, AZ 440 analyzed data; ASV, HT, LRM, JRM, BPHJT wrote the manuscript; JRM, BPHJT supervised 441 the project; all authors discussed the results and contributed to the final manuscript. 442 ASV, JRM, BPHJT conceived the study; ASV, HT, LRM, RdJ, HPvE designed experiments 439 ASV, HT, LRM, DJV, RSB, SW, AK, LV, DC performed experiments; RdJ, HPvE, AZ 440 analyzed data; ASV, HT, LRM, JRM, BPHJT wrote the manuscript; JRM, BPHJT supervised 441 the project; all authors discussed the results and contributed to the final manuscript. 442 ASV, JRM, BPHJT conceived the study; ASV, HT, LRM, RdJ, HPvE designed experiments 439 ASV, HT, LRM, DJV, RSB, SW, AK, LV, DC performed experiments; RdJ, HPvE, AZ 440 analyzed data; ASV, HT, LRM, JRM, BPHJT wrote the manuscript; JRM, BPHJT supervised 441 the project; all authors discussed the results and contributed to the final manuscript. 442 The authors declare no conflict of interest exists. 445 The authors declare no conflict of interest exists. 445 20 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. REFERENCES 446 Proceedings of the National 473 Academy of Sciences of the USA 114: E8118-E8127 474 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) 475 BLAST+: architecture and applications. BMC Bioinformatics 10: 421 476 Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, 477 Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for 478 macromolecular crystallography. Acta Crystallographica Section D: Biological 479 Crystallography 66: 12-21 480 De Jonge R, Thomma BP (2009) Fungal LysM effectors: extinguishers of host immunity? 481 Trends in Microbiology 17: 151-157 482 De Jonge R, Van Esse HP, Kombrink A, Shinya T, Desaki Y, Bours R, Van Der Krol S, 483 Shibuya N, Joosten MH, Thomma BP (2010) Conserved fungal LysM effector Ecp6 484 prevents chitin-triggered immunity in plants. Science 329: 953-955 485 de Wit PJ (2016) Cladosporium fulvum effectors: weapons in the arms race with tomato. 486 Annual Review of Phytopathology 54: 1-23 487 Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta 488 Crystallographica Section D: Biological Crystallography 66: 486-501 489 Gerlach M, Mueller U, Weiss MS (2016) The MX beamlines BL14. 1-3 at BESSY II. Journal 490 of Large-Scale Research Facilities 2: 47 491 Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S (2013) 492 Chitinases: an update. Journal of Pharmacy & Bioallied Sciences 5: 21-29 493 Bozsoki Z, Cheng J, Feng F, Gysel K, Vinther M, Andersen KR, Oldroyd G, Blaise M, 471 Radutoiu S, Stougaard J (2017) Receptor-mediated chitin perception in legume roots is 472 functionally separable from Nod factor perception. Proceedings of the National 473 Academy of Sciences of the USA 114: E8118-E8127 474 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) 475 BLAST+: architecture and applications. BMC Bioinformatics 10: 421 476 Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, 477 Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for 478 macromolecular crystallography. Acta Crystallographica Section D: Biological 479 Crystallography 66: 12-21 480 De Jonge R, Thomma BP (2009) Fungal LysM effectors: extinguishers of host immunity? REFERENCES 446 Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, 447 Kapral GJ, Grosse-Kunstleve RW (2010) PHENIX: a comprehensive Python-based 448 system for macromolecular structure solution. Acta Crystallographica Section D: 449 Biological Crystallography 66: 213-221 450 Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, 447 Adrangi S, Faramarzi MA (2013) From bacteria to human: a journey into the world of 451 chitinases. Biotechnology Advances 31: 1786-1795 452 Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, 453 Nikolenko SI, Pham S, Prjibelski AD (2012) SPAdes: a new genome assembly 454 algorithm and its applications to single-cell sequencing. Journal of Computational 455 Biology 19: 455-477 456 Bateman A, Bycroft M (2000) The structure of a LysM domain from E. coli membrane-bound 457 lytic murein transglycosylase D (MltD) 1. Journal of Molecular Biology 299: 1113- 458 1119 459 Bergfors T (2003) Seeds to crystals. Journal of Structural Biology 142: 66-76 460 Bielnicki J Devedjiev Y Derewenda U Dauter Z Joachimiak A Derewenda ZS (2006) B 461 21 21 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Bozsoki Z, Cheng J, Feng F, Gysel K, Vinther M, Andersen KR, Oldroyd G, Blaise M, 471 Radutoiu S, Stougaard J (2017) Receptor-mediated chitin perception in legume roots is 472 functionally separable from Nod factor perception. REFERENCES 446 481 Trends in Microbiology 17: 151-157 482 De Jonge R, Van Esse HP, Kombrink A, Shinya T, Desaki Y, Bours R, Van Der Krol S, 483 Shibuya N, Joosten MH, Thomma BP (2010) Conserved fungal LysM effector Ecp6 484 prevents chitin-triggered immunity in plants. Science 329: 953-955 485 de Wit PJ (2016) Cladosporium fulvum effectors: weapons in the arms race with tomato. 486 Annual Review of Phytopathology 54: 1-23 487 Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. 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It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Hartmann FE, Croll D (2017) Distinct trajectories of massive recent gene gains and losses in 494 populations of a microbial eukaryotic pathogen. Molecular Biology and Evolution 34: 495 2808-2822 496 Hartmann FE, Sánchez-Vallet A, McDonald BA, Croll D (2017) A fungal wheat pathogen 497 evolved host specialization by extensive chromosomal rearrangements. The ISME 498 Journal 11: 1189-1204 499 Hurlburt NK, Chen L-H, Stergiopoulos I, Fisher AJ (2018) Structure of the Cladosporium 500 fulvum Avr4 effector in complex with (GlcNAc) 6 reveals the ligand-binding 501 mechanism and uncouples its intrinsic function from recognition by the Cf-4 502 resistance protein. 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CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. REFERENCES 446 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, 544 Kaku H, Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin 545 elicitor signaling in Arabidopsis. Proceedings of the National Academy of Sciences of 546 the USA 104: 19613-19618 547 Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, 548 Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal 549 structures. Acta Crystallographica Section D: Biological Crystallography 67: 355-367 550 Newman J, Egan D, Walter TS, Meged R, Berry I, Ben Jelloul M, Sussman JL, Stuart DI, 551 Perrakis A (2005) Towards rationalization of crystallization screening for small-to 552 medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta 553 Crystallographica Section D: Biological Crystallography 61: 1426-1431 554 Ökmen B, Kemmerich B, Hilbig D, Wemhöner R, Aschenbroich J, Perrar A, Huesgen PF, 555 Schipper K, Doehlemann G (2018) Dual function of a secreted fungalysin 556 metalloprotease in Ustilago maydis. New Phytologist 220: 249-261 557 Rovenich H, Zuccaro A, Thomma BP (2016) Convergent evolution of filamentous microbes 558 towards evasion of glycan‐triggered immunity. New Phytologist 212: 896-901 559 Sánchez-Vallet A, Mesters JR, Thomma BP (2015) The battle for chitin recognition in plant- 560 microbe interactions. FEMS Microbiology Reviews 39: 171-183 561 Sánchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg D-J, Thomma BP, 562 Mesters JR (2013) Fungal effector Ecp6 outcompetes host immune receptor for chitin 563 binding through intrachain LysM dimerization. eLife 2: e00790 564 Schlumbaum A, Mauch F, Vögeli U, Boller T (1986) Plant chitinases are potent inhibitors of 565 Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, 544 Kaku H, Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin 545 elicitor signaling in Arabidopsis. Proceedings of the National Academy of Sciences of 546 the USA 104: 19613-19618 547 Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, 548 Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal 549 structures. REFERENCES 446 ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Kombrink A, Rovenich H, Shi‐Kunne X, Rojas‐Padilla E, van den Berg GC, Domazakis E, 519 De Jonge R, Valkenburg DJ, Sánchez‐Vallet A, Seidl MF (2017) Verticillium dahliae 520 LysM effectors differentially contribute to virulence on plant hosts. Molecular Plant 521 Pathology 18: 596-608 522 Kombrink A, Thomma BP (2013) LysM effectors: secreted proteins supporting fungal life. 523 PLoS Pathogens 9: e1003769 524 Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. 525 Journal of Molecular Biology 372: 774-797 526 Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R 527 (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25: 2078- 528 2079 529 Liu T, Chen L, Ma Q, Shen X, Yang Q (2014) Structural insights into chitinolytic enzymes 530 and inhibition mechanisms of selective inhibitors. Current Pharmaceutical Design 20: 531 754-770 532 Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Fan F, Wang J, Jin C, Chang J (2012) Chitin- 533 induced dimerization activates a plant immune receptor. Science 336: 1160-1164 534 Marshall R, Kombrink A, Motteram J, Loza-Reyes E, Lucas J, Hammond-Kosack K, 535 Thomma B, Rudd J (2011) Analysis of two in planta expressed LysM effector 536 homologues from the fungus Mycosphaerella graminicola reveals novel functional 537 properties and varying contributions to virulence on wheat. Plant Physiology 156: 538 756-769 539 Mentlak TA, Kombrink A, Shinya T, Ryder LS, Otomo I, Saitoh H, Terauchi R, Nishizawa Y, 540 Shibuya N, Thomma BP (2012) Effector-mediated suppression of chitin-triggered 541 immunity by Magnaporthe oryzae is necessary for rice blast disease. The Plant Cell 542 24 322 335 543 24 24 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . REFERENCES 446 Acta Crystallographica Section D: Biological Crystallography 67: 355-367 550 Newman J, Egan D, Walter TS, Meged R, Berry I, Ben Jelloul M, Sussman JL, Stuart DI, 551 Perrakis A (2005) Towards rationalization of crystallization screening for small-to 552 medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta 553 Crystallographica Section D: Biological Crystallography 61: 1426-1431 554 Ökmen B, Kemmerich B, Hilbig D, Wemhöner R, Aschenbroich J, Perrar A, Huesgen PF, 555 Schipper K, Doehlemann G (2018) Dual function of a secreted fungalysin 556 metalloprotease in Ustilago maydis. New Phytologist 220: 249-261 557 Sánchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg D-J, Thomma BP, 562 Mesters JR (2013) Fungal effector Ecp6 outcompetes host immune receptor for chitin 563 binding through intrachain LysM dimerization. eLife 2: e00790 564 Schlumbaum A, Mauch F, Vögeli U, Boller T (1986) Plant chitinases are potent inhibitors of 565 fungal growth. Nature 324: 365-367 566 25 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Shibuya N, Kaku H, Kuchitsu K, Maliarik MJ (1993) Identification of a novel high‐affinity 567 binding site for N‐acetylchitooligosaccharide elicitor in the membrane fraction from 568 suspension‐cultured rice cells. FEBS Letters 329: 75-78 569 Stergiopoulos I, van den Burg HA, Ökmen B, Beenen HG, van Liere S, Kema GH, de Wit PJ 570 (2010) Tomato Cf resistance proteins mediate recognition of cognate homologous 571 effectors from fungi pathogenic on dicots and monocots. REFERENCES 446 Proceedings of the National 572 Academy of Sciences of the USA 107: 7610-7615 573 Takahara H, Hacquard S, Kombrink A, Hughes HB, Halder V, Robin GP, Hiruma K, 574 Neumann U, Shinya T, Kombrink E (2016) Colletotrichum higginsianum extracellular 575 LysM proteins play dual roles in appressorial function and suppression of chitin‐ 576 triggered plant immunity. New Phytologist 211: 1323-1337 577 van den Burg HA, Harrison SJ, Joosten MH, Vervoort J, de Wit PJ (2006) Cladosporium 578 fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases 579 accumulating during infection. Molecular Plant-Microbe Interactions 19: 1420-1430 580 van den Burg HA, Spronk CA, Boeren S, Kennedy MA, Vissers JP, Vuister GW, de Wit PJ, 581 Vervoort J (2004) Binding of the AVR4 elicitor of Cladosporium fulvum to chitotriose 582 units is facilitated by positive allosteric protein-protein interactions. Journal of 583 Biological Chemistry 279: 16786-16796 584 Van Esse HP, Bolton MD, Stergiopoulos I, de Wit PJ, Thomma BP (2007) The chitin-binding 585 Cladosporium fulvum effector protein Avr4 is a virulence factor. Molecular Plant- 586 Microbe Interactions 20: 1092-1101 587 van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins 588 Shibuya N, Kaku H, Kuchitsu K, Maliarik MJ (1993) Identification of a novel high‐affinity 567 binding site for N‐acetylchitooligosaccharide elicitor in the membrane fraction from 568 suspension‐cultured rice cells. FEBS Letters 329: 75-78 569 Stergiopoulos I, van den Burg HA, Ökmen B, Beenen HG, van Liere S, Kema GH, de Wit PJ 570 (2010) Tomato Cf resistance proteins mediate recognition of cognate homologous 571 effectors from fungi pathogenic on dicots and monocots. Proceedings of the National 572 Academy of Sciences of the USA 107: 7610-7615 573 Academy of Sciences of the USA 107: 7610-7615 573 Takahara H, Hacquard S, Kombrink A, Hughes HB, Halder V, Robin GP, Hiruma K, 574 Neumann U, Shinya T, Kombrink E (2016) Colletotrichum higginsianum extracellular 575 LysM proteins play dual roles in appressorial function and suppression of chitin‐ 576 triggered plant immunity. New Phytologist 211: 1323-1337 577 van den Burg HA, Harrison SJ, Joosten MH, Vervoort J, de Wit PJ (2006) Cladosporium 578 fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases 579 accumulating during infection. REFERENCES 446 It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint FIGURE LEGENDS 597 FIGURE LEGENDS 597 Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 structure model in which a dimer of two Mg1LysM homodimers is shown, with each of the 599 Mg1LysM molecules in a different colour (orange, yellow, red and green). While the two 600 monomers that form a ligand-independent homodimer on the right are represented as a surface 601 model, the two monomers that form a ligand-independent homodimer on the left are 602 represented by ribbons with the (putative) chitin binding sites indicated in blue and the 603 disulfide bridges as yellow sticks. The chitin trimer that mediates the dimerization of two 604 ligand-independent Mg1LysM homodimers is depicted by grey sticks. The two salt bridges 605 between R2 and D12 in the dimer interface on the left are indicated with grey discontinuous 606 lines. 607 608 Fig. 2. The chitin binding groove formed by two Mg1LysM protomers. (A) A chitin 609 trimer (GlcNAc)3, displayed as grey sticks, was identified in a binding pocket formed by two 610 Mg1LysM protomers (indicated in yellow and red, respectively). (B) Representation of the 611 binding pocket from the top. (C) Detail of the chitin binding site. The amino acids involved in 612 direct chitin trimer binding (26GDTLT30 and 56NRI58) are represented with blue sticks. In 613 addition, K31 and D54 (represented in green) of the two different Mg1LysM protomers form a 614 salt bridge that tightly closes the binding pocket. Grey discontinuous lines represent the salt 615 bridge and the hydrogen bonds between the protomers. 616 617 Fig. 3. Two Mg1LysM protomers bind a single chitin hexamer with high affinity. 618 Isothermal titration calorimetry of (GlcNAc)6 binding by wild-type Mg1LysM produced in E. 619 coli, and the mutants T28R, K31A and D54A. The dissociation constant (Kd) and the 620 stoichiometry (N) of the interactions are indicated. 621 REFERENCES 446 Molecular Plant-Microbe Interactions 19: 1420-1430 580 van den Burg HA, Spronk CA, Boeren S, Kennedy MA, Vissers JP, Vuister GW, de Wit PJ, 581 Vervoort J (2004) Binding of the AVR4 elicitor of Cladosporium fulvum to chitotriose 582 Takahara H, Hacquard S, Kombrink A, Hughes HB, Halder V, Robin GP, Hiruma K, 574 Neumann U, Shinya T, Kombrink E (2016) Colletotrichum higginsianum extracellular 575 LysM proteins play dual roles in appressorial function and suppression of chitin‐ 576 triggered plant immunity. New Phytologist 211: 1323-1337 577 Van Esse HP, Bolton MD, Stergiopoulos I, de Wit PJ, Thomma BP (2007) The chitin-binding 585 Cladosporium fulvum effector protein Avr4 is a virulence factor. Molecular Plant- 586 Microbe Interactions 20: 1092-1101 587 van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins 588 in infected plants. Annual Review of Phytopathology 44: 135-162 589 26 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version 2— 590 a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 591 1189-1191 592 Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel 593 EB, Leslie AG, McCoy A (2011) Overview of the CCP4 suite and current 594 developments. Acta Crystallographica Section D 67: 235-242 595 596 27 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 structure model in which a dimer of two Mg1LysM homodimers is shown, with each of the 599 Mg1LysM molecules in a different colour (orange, yellow, red and green). While the two 600 monomers that form a ligand-independent homodimer on the right are represented as a surface 601 model, the two monomers that form a ligand-independent homodimer on the left are 602 represented by ribbons with the (putative) chitin binding sites indicated in blue and the 603 disulfide bridges as yellow sticks. The chitin trimer that mediates the dimerization of two 604 ligand-independent Mg1LysM homodimers is depicted by grey sticks. The two salt bridges 605 between R2 and D12 in the dimer interface on the left are indicated with grey discontinuous 606 lines. 607 Fig. 2. The chitin binding groove formed by two Mg1LysM protomers. (A) A chitin 609 trimer (GlcNAc)3, displayed as grey sticks, was identified in a binding pocket formed by two 610 Mg1LysM protomers (indicated in yellow and red, respectively). (B) Representation of the 611 binding pocket from the top. (C) Detail of the chitin binding site. The amino acids involved in 612 direct chitin trimer binding (26GDTLT30 and 56NRI58) are represented with blue sticks. In 613 addition, K31 and D54 (represented in green) of the two different Mg1LysM protomers form a 614 salt bridge that tightly closes the binding pocket. Grey discontinuous lines represent the salt 615 bridge and the hydrogen bonds between the protomers. 616 Fig. 3. Two Mg1LysM protomers bind a single chitin hexamer with high affinity. 618 Isothermal titration calorimetry of (GlcNAc)6 binding by wild-type Mg1LysM produced in E. 619 coli, and the mutants T28R, K31A and D54A. The dissociation constant (Kd) and the 620 stoichiometry (N) of the interactions are indicated. 621 28 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 ; https://doi.org/10.1101/787820 doi: bioRxiv preprint 622 Fig. 4. Mg1LysM sequence polymorphisms in Z. tritici. (A) 5 non-synonymous SNPs were 623 identified in 149 Z. tritici strains from four different populations. Arrows indicate the position 624 of the residues involved in the formation of salt bridges, while green underlining indicates the 625 signal peptide and red underlining the chitin-binding loops. Red and green underlines indicate 626 the signal peptide and the chitin binding sites, respectively. (B) While the mutations (shown 627 in blue sticks) do not co-localize but occur dispersed over the Mg1LysM protein, none of 628 them is in the chitin-binding site or in the (homo-)dimerization surface. 629 630 Fig. 5. Mg1LysM mutants are impaired in protection against chitinases. Microscopic 631 pictures of Fusarium oxysporum f. sp. lycopersici grown in vitro in the absence or presence of 632 wild-type or mutant Mg1LysM, 4 hours after addition of tomato hydrolytic enzymes (HE) that 633 include chitinases, or water as control. 634 635 Fig. 6. Chitin-induced polymerization of Mg1LysM homodimers. (A) Model inferred from 636 the crystal structure of Mg1LysM in which a continuous structure of Mg1LysM homodimers 637 and chitin is formed. Alternating chitin molecules (in grey sticks) and Mg1LysM homodimers 638 (in red and green), each of them with two chitin-binding sites, are shown. (B) Dynamic light 639 scattering (DLS) heat maps of Mg1LysM and of C. fulvum Ecp6 treated with chitohexaose in 640 molar ratios of 1:0, 1:2 and 1:5 (protein: chitohexaose), respectively. The particle size 641 distribution is indicated as a color scale ranging from blue (lowest amount) to red (highest 642 amount) for a particle size range of 1 nm to 100 um. 643 644 622 622 Fig. 4. Mg1LysM sequence polymorphisms in Z. tritici. (A) 5 non-synonymous SNPs were 623 identified in 149 Z. tritici strains from four different populations. Arrows indicate the position 624 of the residues involved in the formation of salt bridges, while green underlining indicates the 625 signal peptide and red underlining the chitin-binding loops. Red and green underlines indicate 626 the signal peptide and the chitin binding sites, respectively. (B) While the mutations (shown 627 in blue sticks) do not co-localize but occur dispersed over the Mg1LysM protein, none of 628 them is in the chitin-binding site or in the (homo-)dimerization surface. 629 630 Fig. 4. Mg1LysM sequence polymorphisms in Z. tritici. The values in the parentheses refer to the highest resolution shell. # │ │ y y q y §I/σI is the mean reflection intensity divided by the estimated error. 649 m = (∑│Ihkl – <Ihkl>│) / (∑ Ihkl), where the average intensity <Ihkl> is taken over a metry equivalent measurements and Ihkl is the measured intensity for any given reflection Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 (A) 5 non-synonymous SNPs were 623 identified in 149 Z. tritici strains from four different populations. Arrows indicate the position 624 of the residues involved in the formation of salt bridges, while green underlining indicates the 625 signal peptide and red underlining the chitin-binding loops. Red and green underlines indicate 626 the signal peptide and the chitin binding sites, respectively. (B) While the mutations (shown 627 in blue sticks) do not co-localize but occur dispersed over the Mg1LysM protein, none of 628 them is in the chitin-binding site or in the (homo-)dimerization surface. 629 Fig. 4. Mg1LysM sequence polymorphisms in Z. tritici. (A) 5 non-synonymous SNPs were 623 identified in 149 Z. tritici strains from four different populations. Arrows indicate the position 624 of the residues involved in the formation of salt bridges, while green underlining indicates the 625 signal peptide and red underlining the chitin-binding loops. Red and green underlines indicate 626 the signal peptide and the chitin binding sites, respectively. (B) While the mutations (shown 627 in blue sticks) do not co-localize but occur dispersed over the Mg1LysM protein, none of 628 them is in the chitin-binding site or in the (homo-)dimerization surface. 629 Fig. 5. Mg1LysM mutants are impaired in protection against chitinases. Microscopic 631 pictures of Fusarium oxysporum f. sp. lycopersici grown in vitro in the absence or presence of 632 wild-type or mutant Mg1LysM, 4 hours after addition of tomato hydrolytic enzymes (HE) that 633 include chitinases, or water as control. 634 635 Fig. 6. Chitin-induced polymerization of Mg1LysM homodimers. (A) Model inferred from 636 the crystal structure of Mg1LysM in which a continuous structure of Mg1LysM homodimers 637 and chitin is formed. Alternating chitin molecules (in grey sticks) and Mg1LysM homodimers 638 (in red and green), each of them with two chitin-binding sites, are shown. (B) Dynamic light 639 scattering (DLS) heat maps of Mg1LysM and of C. fulvum Ecp6 treated with chitohexaose in 640 molar ratios of 1:0, 1:2 and 1:5 (protein: chitohexaose), respectively. The particle size 641 distribution is indicated as a color scale ranging from blue (lowest amount) to red (highest 642 amount) for a particle size range of 1 nm to 100 um. 643 644 29 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. #Rsym = (∑│Ihkl – <Ihkl>│) / (∑ Ihkl), where the average intensity <Ihkl> is 647 symmetry equivalent measurements and Ihkl is the measured intensity for any g 648 Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint Table 1. Data collection and refinement statistics. 645 Native Derivative-1 (SAD) (Ta6Br12)2+ Derivative-2 (SIRAS) (I3C) Data collection statistics Beamline BL14.1 - BESSY Wavelength (Å) 0.91841 1.24845 1.88313 Space group P 61 2 2 Cell dimensions a, b, c (Å) 119.4, 119.4, 157.7 119.5, 119.5, 157.6 119.3, 119.3, 157.3 Resolution (Å) 49.12–2.41 (2.54–2.41) 26.97–2.96 (3.12–2.96) 39.37–5.00 (5.27–5.00) Rsym # (%) 8.3 (50.4) 7.8 (20.1) 12.9 (20.7) I / σI § 18.0 (4.5) 28.2 (11.5) 17.6 (17.3) Completeness (%) 92.1 (100) 91.5 (100) 99.8 (100) Redundancy 10.7 (10.7) 17.6 (16.3) 19.1 (19.9) Anomalous completeness (%) - 91.4 (100) 100 (100) Anomalous multiplicity - 9.5 (8.6) 11.1 (11.0) Phasing statistics Figure of Merit (FOM) - 0.57 0.36 Map Skew - 1.40 0.03 Correlation of local RMS density - 0.92 0.60 Correlation Coefficient (CC) - 0.56 0.27 Refinement statistics Resolution (Å) 2.41 - - No. of reflections (total / free) 24,112 / 1,219 - - Rwork / Rfree † (%) 17.96 / 22.03 - - No. of atoms / average B- factor Overall 2,595 / 55.2 - - Protein 2,419 / 55.6 - - Ligand 43 / 53.7 - - R.m.s. deviations bond lengths (Å) 0.010 - - R.m.s. deviations bond angles (°) 1.195 - - Ramachandran plot (% favored / % outliers) 95.8 / 0.0 - - MolProbity (clash- & overall score) 6.42 / 1.65 - - The values in the parentheses refer to the highest resolution shell. #Rsym = (∑│Ihkl – <Ihkl>│) / (∑ Ihkl), where the average intensity <Ihkl> is taken over all symmetry equivalent measurements and Ihkl is the measured intensity for any given reflection. §I/σI is the mean reflection intensity divided by the estimated error. 30 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . †Rwork = ││Fo│ - │Fc││ / │Fo│, where Fo and Fc are the observed and calculated structure 650 factor amplitudes, respectively. Rfree is equivalent to Rwork but calculated for 5% of the 651 reflections chosen at random and omitted from the refinement process. 652 Fig. 1. Overall crystal structure of the Zymoseptoria tritici effector Mg1LysM. Crystal 598 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint †Rwork = ││Fo│ - │Fc││ / │Fo│, where Fo and Fc are the observed and calculated structure 650 factor amplitudes, respectively. Rfree is equivalent to Rwork but calculated for 5% of the 651 reflections chosen at random and omitted from the refinement process. 652 31 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint SUPPORTING INFORMATION 1 2 S1 Fig. Protein alignment of Ecp6 and Mg1LysM. (A) Protein sequence alignment of Ecp6 3 and Mg1LysM. The two chitin binding loops of Mg1LysM are indicated with a blue line and 4 the signal peptide with a green line. Red and blue asterisks indicate the position of the residues 5 in ol ed in the formation of salt bridges in the binding groo e and in the dimeri ation s rface 6 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whi this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint SUPPORTING INFORMATION 1 SUPPORTING INFORMATION 1 2 S1 Fig. Protein alignment of Ecp6 and Mg1LysM. (A) Protein sequence alignment of Ecp6 3 and Mg1LysM. The two chitin binding loops of Mg1LysM are indicated with a blue line and 4 the signal peptide with a green line. Red and blue asterisks indicate the position of the residues 5 involved in the formation of salt bridges in the binding groove and in the dimerization surface, 6 respectively. (B) Structural alignment of the LysM1 domain from Ecp6 (in blue) and the LysM 7 domain from Mg1LysM (in red). The chitin trimer is shown in grey sticks. The chitin binding 8 loops are shown in dark blue and green for LysM1 and for Mg1LysM, respectively. 9 2 2 S1 Fig. Protein alignment of Ecp6 and Mg1LysM. (A) Protein sequence alignment of Ecp6 3 and Mg1LysM. The two chitin binding loops of Mg1LysM are indicated with a blue line and 4 the signal peptide with a green line. Red and blue asterisks indicate the position of the residues 5 involved in the formation of salt bridges in the binding groove and in the dimerization surface, 6 respectively. (B) Structural alignment of the LysM1 domain from Ecp6 (in blue) and the LysM 7 domain from Mg1LysM (in red). The chitin trimer is shown in grey sticks. The chitin binding 8 loops are shown in dark blue and green for LysM1 and for Mg1LysM, respectively. 9 S1 Fig. Protein alignment of Ecp6 and Mg1LysM. (A) Protein sequence alignment of Ecp6 3 and Mg1LysM. The two chitin binding loops of Mg1LysM are indicated with a blue line and 4 the signal peptide with a green line. Red and blue asterisks indicate the position of the residues 5 involved in the formation of salt bridges in the binding groove and in the dimerization surface, 6 respectively. (B) Structural alignment of the LysM1 domain from Ecp6 (in blue) and the LysM 7 domain from Mg1LysM (in red). The chitin trimer is shown in grey sticks. The chitin binding 8 loops are shown in dark blue and green for LysM1 and for Mg1LysM, respectively. 9 1 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . SUPPORTING INFORMATION 1 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint 10 S2 Fig. 2\|F0\| – \|Fc\| map. 2\|F0\| – \|Fc\| electron density map around the chitin trimer (carbon atoms 11 coloured yellow) is contoured at 1 sigma above the mean. Amino acids of the chitin binding 12 motif (26GDTLT30 and 56NRI58) are represented as sticks (carbon atoms coloured light-grey). 13 10 10 10 S2 Fig. 2\|F0\| – \|Fc\| map. 2\|F0\| – \|Fc\| electron density map around the chitin trimer (carbon atoms 11 coloured yellow) is contoured at 1 sigma above the mean. Amino acids of the chitin binding 12 motif (26GDTLT30 and 56NRI58) are represented as sticks (carbon atoms coloured light-grey). 13 S2 Fig. 2\|F0\| – \|Fc\| map. 2\|F0\| – \|Fc\| electron density map around the chitin trimer (carbon atoms 11 coloured yellow) is contoured at 1 sigma above the mean. Amino acids of the chitin binding 12 motif (26GDTLT30 and 56NRI58) are represented as sticks (carbon atoms coloured light-grey). 13 2 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint 14 S3 Fig. Mg1LysM mutants are impaired in protection of Trichoderma viride against 15 chitinases. Microscopic pictures of Trichoderma viride grown in vitro in the absence or 16 presence of wild-type or mutant Mg1LysM, 4 hours after addition of tomato hydrolytic enzymes 17 (HE) that include chitinases, or water as control. 18 14 S3 Fig. Mg1LysM mutants are impaired in protection of Trichoderma viride against 15 chitinases. SUPPORTING INFORMATION 1 Microscopic pictures of Trichoderma viride grown in vitro in the absence or 16 presence of wild-type or mutant Mg1LysM, 4 hours after addition of tomato hydrolytic enzymes 17 (HE) that include chitinases, or water as control. 18 14 Mg1LysM mutants are impaired in protection of Trichoderma viride against S3 Fig. Mg1LysM mutants are impaired in protection of Trichoderma 15 S3 Fig. Mg1LysM mutants are impaired in protection of Trichoderma viride against 15 chitinases. Microscopic pictures of Trichoderma viride grown in vitro in the absence or 16 presence of wild-type or mutant Mg1LysM, 4 hours after addition of tomato hydrolytic enzymes 17 (HE) that include chitinases, or water as control. 18 S3 Fig. Mg1LysM mutants are impaired in protection of Trichoderma viride against 15 chitinases. Microscopic pictures of Trichoderma viride grown in vitro in the absence or 16 presence of wild-type or mutant Mg1LysM, 4 hours after addition of tomato hydrolytic enzymes 17 (HE) that include chitinases, or water as control. 18 3 3 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 30, 2019. ; https://doi.org/10.1101/787820 doi: bioRxiv preprint 19 S4 Fig. The Cladosporium fulvum LysM effector Ecp6 protein localizes to fungal cell walls. 20 (A) Brightfield image (left), fluorescence image (middle) and the overlay image (right) of a 21 hypha from an Ecp6-GFP transformant of Verticillium dahliae. The chitin-binding C. fulvum 22 LysM effector Ecp6 (B) and chitin-binding effector Avr4 (C) that carries an invertebrate chitin- 23 binding domain were labeled with the amine-reactive fluorescent dye BODIPY and incubated 24 with Botrytis cinerea spores for 2-3 hours and observed with fluorescence microscopy. 25 A B C A C 9 B B B 19 S4 Fig. The Cladosporium fulvum LysM effector Ecp6 protein localizes to fungal cell walls. 20 (A) Brightfield image (left), fluorescence image (middle) and the overlay image (right) of a 21 hypha from an Ecp6-GFP transformant of Verticillium dahliae. The chitin-binding C. SUPPORTING INFORMATION 1 fulvum 22 LysM effector Ecp6 (B) and chitin-binding effector Avr4 (C) that carries an invertebrate chitin- 23 binding domain were labeled with the amine-reactive fluorescent dye BODIPY and incubated 24 with Botrytis cinerea spores for 2-3 hours and observed with fluorescence microscopy. 25 S4 Fig. The Cladosporium fulvum LysM effector Ecp6 protein localizes to fung 20 The Cladosporium fulvum LysM effector Ecp6 protein localizes to fungal cell walls. S4 Fig. The Cladosporium fulvum LysM effector Ecp6 protein localizes to fungal cell walls. 20 (A) Brightfield image (left), fluorescence image (middle) and the overlay image (right) of a 21 hypha from an Ecp6-GFP transformant of Verticillium dahliae. The chitin-binding C. fulvum 22 LysM effector Ecp6 (B) and chitin-binding effector Avr4 (C) that carries an invertebrate chitin- 23 binding domain were labeled with the amine-reactive fluorescent dye BODIPY and incubated 24 with Botrytis cinerea spores for 2-3 hours and observed with fluorescence microscopy. 25 4 4
https://openalex.org/W3122330867	https://scholarship.law.duke.edu/cgi/viewcontent.cgi?article=1552&context=faculty_scholarship	English	null	Foucault in Cyberspace: Surveillance, Sovereignty, and Hardwired Censors	Routledge eBooks	2,017	public-domain	18,170	* Copyright 1997 James Boyle, Professor of Law, Washington College of Law, American University. My remarks at the Symposium dealt more specifically with the law and policy of proposed changes to copyright on the Interet. However, I have already outlined some of those views in print, and at some length. S&eJAMES BOYLE, SHAMANS, SOFTWARE AND SPLEENS: LAW AND THE CONSTRUCTION OFTHE INFORMATION SOCIETY, 136-39, 192-200 (1996); James Boyle, Intellectual Property Poliy On-Line: A Young Person's Guide, 10 HARV.J.L. &TECH. 47 (1996); James Boyle, Sold Out, N.Y. TIMES, Mar. 31, 1996, at EIS; James Boyle, Q Is Congress Turning the Inteme into an Inforntion Toll Road?, INSIGHT ON THE NEWS, Jan. 15, 1996, at 24; James Boyle, A Politics ofIntellectual Property: Environmentalismfor the Ve4 DUKE LJ. (forthcoming). The editors of the Universiy ofCininati Law Rie= were kind enough to allow me to address myself in this Article to a slightly different issue, though one of profound importance to the Symposium as a whole-the extent to which state regulation of the Internet is possible at all, and the costs and benefits of technical solutions. In the course of that discussion, I use a number of examples drawn from the recent proposals on Internet copyright. 1. Michel Foucault, Two Lectures, in MICHEL FOUCAULT, POWER/KNOWLEDGE: SELECTED INTERVIEWS AND OTHER WRITINGS, 1972-1977, 78,103-05 (Colin Gordon ed. & Colin Gordon et al. trans., 1980). FOUCAULT IN CYBERSPACE: SURVEILLANCE, SOVEREIGNTY, AND HARDWIRED CENSORS James Boyle* 2. Communications Decency Act of 1996, 47 U.S.C.A. § 223 (West Supp. 1997). 3. There are a variety of versions of the claim but the content is pretty consistent. See, e.g., John P. Barlow, Parsing the Buck on Porn (visited June 24, 1996) <http://www.cff.org/pub/Publications/john-Perry--Barlow/HTML/pom-and-responsibility.html> ('The Internet, in the words of... John Gilmore, 'deals with censorship as though it were a malfunction and routes around it."'); Judith Lewis, Whyl ohnny Can't Surf, LAWKLY., Feb. 21, 1997, at 43. ("Mt's not easy to push standards of 'decency' on a network that, as.. .John Gilmore allegedly put it (though even he can't remember where), treats censorship as damage and mutes around it."). I. INTRODUCTION [T]he problems to which the theory of.sovereignty was addressed were in effect confined to the general mechanisms of power, to the way in which its forms of existence at the higher level of society influenced its exercise at the lowest levels .... In effect, the mode in which power was exercised could be defined in its essentials in terms of the relationship sovereign-subject. But . . . we have the . . . emergence, or rather the invention, of a new mechanism of power possessed of highly specific procedural techniques ... which is also, I believe, absolutely incompatible with the relations of sovereignty.... It is a type of power which is constantly exercised by means of surveillance rather than in a discontinuous manner by means of a system of levies or obligations distributed over time. It presupposes a tightly knit grid of material coercions rather than the physical existence of a sovereign .... This non-sovereign power, which lies outside the form of sovereignty, is disciplinary power.' This is an Article about law in cyberspace. It focuses on three interdependent phenomena: a set of political and legal assumptions that I call the jurisprudence of digital libertarianism, a separate but related set of beliefs about the state's supposed inability to regulate the Internet, and a preference for technological solutions to hard legal issues on-line. 178 [Vol.66 UNIVERSITY OF CIN)VA TI L4 WREVIEW 66 I make the familiar criticism that digital libertarianism is inadequate because of its blindness to the effects of private power, and the less familiar claim that digital libertarianism is also surprisingly blind to the state's own power in cyberspace. In fact, I argue that the conceptual structure and jurisprudential assumptions of digital libertarianism lead its practitioners to ignore the ways in which the state can often use privatized enforcement and state-backed technologies to evade some of the supposed practical (and constitutional) restraints on the exercise of legal power over the Internet. Finally, I argue that technological solutions which provide the keys to the first two phenomena are neither as neutral nor as benign as they are currently perceived to be. Some of my illustrations will come from the Clinton administration's proposals for Internet copyright regulation, others from the Communications Decency Act (CDA)2 and the cryptography debate. II. THE INTERNET TRINITY For a long time, the Internet's enthusiasts have believed that it would be largely immune from state regulation. The theory was not so much that nation states would not want to regulate the Internet, it was that they would be unable to do so, forestalled by the technology of the medium, the geographical distribution of its users, and the nature of its content. This tripartite immunity came to be a kind of "Internet Holy Trinity"; faith in it was a condition of acceptance into the community. Indeed, the ideas I am about to discuss are so well known on the Internet that they have actually acquired the highest status that a culture can confer: they have become cliches. I. INTRODUCTION In the process, I make opportunistic and unsystematic use of the late Michel Foucault's work to criticize some of the jurisprudential orthodoxy of the Internet. 4. See generally Todd Flaming, An Introduction to the Internet, 83 ILL. Bj., 311, (1995); JOSHUA EDDINGS, HOWTHE INTERNET WORKS 13 (1994); Bruce Sterling, Short Histoiy ofthe Internet (last modified Feb. 1993) <gopher://gopher.metronet.com:70/00/inet/A..Short..History-oLthejntemet>. For background information on Internet legal issues, see generally Lawrence Lessig, The Zones Of Cyberspace, 48 STAN. L REV. 1403 (1996); Lawrence Lessig, The Path Of Cyberlaw, 104 YALE Lj. 1743 (1995); David R. Johnson & David Post, Law and Borders-the Rise Of Law In Cyberspace, 48 STAN. L. REV. 1367 (1996). 5. See, e.g., John P. Barlow, Leaving the Physical World (visited June 24, 1997) <http://www.eff.org/pub/Publicadons/john-Perty-Barlow/HTML/leaving-the-physica-world.html> (discussing the inapplicability of physical-world standards in cyberspace). A. "The Net interprets censorship as damage and routes around it." This quote fromJohn Gilmore,' one of the founders of the Electronic Frontier Foundation, has the twin advantages of being pithy and CORPORA TE LA W SYMPOSIUM 1997] technologically accurate. The Internet was originally designed to survive a nuclear war; its distributed architecture and its technique of packet switching were designed to get messages delivered despite blockages, holes, and malfunctions." Imagine the poor censor faced with such a system. There is no central exchange to seize and hold; messages actively "seek out" alternative routes so that even if one path is blocked another may open up. Here was the civil libertarian's dream: a technology with a comparatively low cost of entry to speakers and listeners alike, technologically resistant to censorship, yet politically and economically important enough that it cannot easily be ignored. The Internet offers obvious advantages to the countries, research communities, cultures, and companies that use it, but it is extremely hard to control the amount and type of information available; access is like a tap that only has two settings-"off' and "full." For govern- ments, this has been seen as one of the biggest problems posed by the Internet. To the Internet's devotees, most of whom embrace some variety of libertarianism, the Internet's structural resistance to censor- ship, or any externally imposed filtration, is "not a bug but a feature." B. "In Cyberspace, the First Amendment is a local ordinance. "5 To the technological obstacles the Internet raises against externally imposed content filtration, one must add the geographic obstacles raised by its global extent. Because a document can be retrieved as easily from a server five-thousand miles away or a server five miles away, geographical proximity and content availability are independent of each other. If the king's writ reaches only as far as the king's sword, then much of the content on the Internet might be presumed to be free from the regulation of any particular sovereign. The libertarian culture that dominates the Internet at present posits that state intervention into private action is only necessary to prevent the infliction of harm. Seeing the Internet as a speech-dominated realm of human activity in which harm would be comparatively hard to inflict, libertarians have been even more resistant to state regulation of the 180 UNIVERSITY OF CLCLNNATI I WREVIEW [Vol.66 digital environment than of the disdainfully named, "meatspace." "Sticks and stones can break my bones but bytes can never hurt me," or so goes their assumption. 'Thus; the postulate that a global Internet cannot be regulated by national governments has been seen as an unequivocally positive thing. John Perry Barlow's description of the First Amendment as a local ordinance offers the sobering reminder that it is not merely "bad" state traditions, interventions, and regulations that are enfeebled by cyberspace. There is a difference between speech being constitutionally protected and practically immune from regulation; a free speech tradition that pins all of its hopes on physical immunity from regulation is likely to be particularly vulnerable if that immunity proves illusory. C. "Information Wants to be Free." Esther Dyson, et al., A Magna Cartaforthe KnowledgeAge, 11 NEW PERSPECTIVES QUARTERLY 26, 29 (1994) (emphasis added) If this analysis is correct, copyright and patent protection ofImowledge (or at Last many forns of it) may no longer be necessary. In fact, the marketplace may already be creating vehicles to compensate creators of customized knowledge outside the cumbersome copyright/patent process, as suggested byJohn Perry Barlow. Esther Dyson, et al., A Magna Cartaforthe KnowledgeAge, 11 NEW PERSPECTIVES QUARTERLY 26, 29 (1994) (emphasis added). Esther Dyson, et al., A Magna Cartaforthe KnowledgeAge, 11 NEW PERSPECTIVES QUARTERLY 26, 29 (1994) (emphasis added). 7. Huber, in fact, took a direct shot at the notion that "information wants to be free." See Peter Huber, Tangked 1rres: The Intellctual Confusion and Hypocrisy of the Wired Crowd, SLATE, Oct. 18, 1996 <http://www.slate.com/Features/TangledWires/TangledWires.asp>. In this article, Huber labeled the intellectual property rights skeptics as hypocrites whose real attitude reflects a desire for liberal redistribution of everyone else's stuff. See id His views are frankly dismissive and puzzlingly so. He criticized a group of people with widely varying political views, linked mainly by an opposition to the expansion of intellectual property rights. Some have argued in favor of maintaining the existing intellectual property rules in cyberspace, others have claimed that reliance on rules rather than technological innovation would actually inhibit the operation of capitalism on-line. Yet Huber's description of this "Wired Crowd," many of whom make Ayn Rand sound like Vladimir Ilyich, is that their position is that of a hypocritical New Dealer-"My property is mine; yours is for sharing." Id. Wired, we are supposed to believe, is the Economic and Philosophical Manuscripts in cyberspace. (Would that it were true! In fact, Wird's ideal of scathing social commentary is to claim that someone's computer is out of date.) Huber then seeks to restore normative appeal to intellectual property by arguing that it "isjust a commercial form of privacy law. Indeed for some, it's the only kind of privacy they still own." Id. This powerful argument suffers a little from the example that follows. "Madonna can no longer stop you from gazing at her breasts. Copyright at least makes you pay for the pleasure." Id. C. "Information Wants to be Free." To a person interested in political theory, one of the most striking things about the Internet is the instability of its political cartography. We divide our world up into contiguous and opposing terr- itories-public and private, property and sovereignty, regulation and laissez faire-solving problems by deciphering their placement on this map. In the everyday world, these divisions seem comparatively solid and lumpish, even if clever academic critics may harp on their theoretical indeterminacy. On the Internet, things are different. Concepts and political forces seem to be up for grabs. Nothing illustrates this point better than the debate over intellectual property on- line. In the digital environment, is intellectual property just property, the precondition to an unregulated market, just another example of the rights that libertarians believe the state was specifically created to protect? Or is intellectual property actually public regulation, artificial rather than natural, an invented monopoly imposed by a sovereign state, a distorting and liberty-reducing intervention in an otherwise free domain? Although it would be hard to find anyone who believes entirely in either of these two stereotypes, recognizable versions of both do exist in the debate over intellectual property and-more interestingly-can be found across the political spectrum. George Gilder of the conservative Manhattan Institute, a fervent booster of capitalism and laissez faire economics, has shown considerable skepticism about intellectual CORPORA TE LA W SYMPOSIUM 1997] 181 property.' Peter Huber, from the same conservative think tank, has pronounced it the very acme of liberty, privacy, and natural right.7 The Clinton administration attempted to extend intellectual property rights property.' Peter Huber, from the same conservative think tank, has pronounced it the very acme of liberty, privacy, and natural right.7 The p y y p y g Clinton administration attempted to extend intellectual property right 6. One of the strongest statements of his position comes in the manifesto he co-authored with a number of other prominent members of the digerati: Unlike the mass knowledge of the Second Wave-public good knowledge that was useful to everyone because most people's information needs were standardized-Third Wave customized knowledge is by nature a private good. If this analysis is correct, copyright and patent protection ofImowledge (or at Last many forns of it) may no longer be necessary. In fact, the marketplace may already be creating vehicles to compensate creators of customized knowledge outside the cumbersome copyright/patent process, as suggested byJohn Perry Barlow. 6. One of the strongest statements of his position comes in the manifesto he co-authored with a number of other prominent members of the digerati: Unlike the mass knowledge of the Second Wave-public good knowledge that was useful to everyone because most people's information needs were standardized-Third Wave customized knowledge is by nature a private good. If this analysis is correct, copyright and patent protection ofImowledge (or at Last many forns of it) may no longer be necessary. In fact, the marketplace may already be creating vehicles to compensate creators of customized knowledge outside the cumbersome copyright/patent process, as suggested byJohn Perry Barlow. Esther Dyson, et al., A Magna Cartaforthe KnowledgeAge, 11 NEW PERSPECTIVES QUARTERLY 26, 29 (1994) (emphasis added). 6. One of the strongest statements of his position comes in the manifesto he co-authored with a number of other prominent members of the digerati: 8. See eraffy INFORMATION INFRASTRUCTURE TASK FORCE, INTE ICTUAL PROPERTY AND THE NATIONAL INFORMATION INFRASTRUCTURE: THE REPORT OF THE WORKING GROUP ON INTELLECTUAL PROPERTY RIGHTS (Sept. 1995). 9. SeeJames Boyle, Intellctual Property Poticy On-Line: A Young Person's Guide, 10 HARv.J.L. & TECH. 47, 52 (1996). 10. John P. Barlow, Selling Wine Wirhout Bottles: The Economy of Mind on the Global Net, WIRED 2.03 1993, at 86 (visited June 24, 1997) <http://www.eff.org/pub/Publications/John_-Perry.-Bar- low/HTML/idea.economy-article.html> (crediting Stewart Brand with the statement). 11. Id. (quoting THOMASJEFFERSON, 13 THE WRITINGS OF THOMASJEFFERSON 333-34 (Albert E. Bergh ed., Monticello ed., Thomas Jefferson Memorial Ass'n 1904) (letter from Jefferson to Isaac C. "Information Wants to be Free." Our sympathies are with her (and with him if this is the best illustration that comes to mind.) Stopping the world from gazing at her breasts has never seemed to be particularly high on Madonna's list of priorities-at least as a matter of "privacy." True, Madonna might prefer a legal regime that would allow her to wring the maximum commercial advantage in every market for images of her and references to her-by making people like Huber pay if they wished to use her as an example, restricting the fair use privilege, or limiting news reporting and biography to authorized images. Yet it is not clear why this desire, in itself, makes the notion of such a regime normatively compelling as a matter of social policy. There is also a danger in labeling critics of extensive intellectual property rights "anti-privacy." If there is a privacy interest consisting solely in the extraction of the maximum rent for one's intellectual property, then was the Justice Department's investigation of Microsoft's allegedly anticompetitive practices an attempt to cut down on Bill Gates's privacy interest in Windows '95? Or are we referring simply to spin-off effects in a particular case? Are federal automobile emissions standards "anti-privacy" if they make it harder for me to leave the paparazzi in the dust? Surely one must distinguish occasional opportunistic uses of a right from the underlying purposes of that right? We have all used things for other than their intended purpose. Intellectual property can sometimes be used to preserve privacy and I have used a stout and WASP-y pair of wingtips to hammer in a nail; this does not mean that the manufacturers of Birkenstock sandals are "anti-carpentry." There are indeed profound and interesting linkages and tensions between property and privacy, and this point has been made for some time. Compare Samuel D. Warren & Louis D. Brandeis, The Right to Privacy, 4 HARV. L. REV. 193 (1890), with Michael Madow, Private Ownership ofPub/i Imag" Popu/ar Culture and Pubiciy Rights, 81 CAL. L. REV. 125 (1993). C. "Information Wants to be Free." Inventions then cannot, in nature, be a subject of property." CORPORATE L4 W 997] CORPORA TE L4 W SYMPOSIUM 1997] The quotation expresses perfectly the mixture of Enlightenment values and upbeat, public-goods theory that typifies Internet analysis of information flows. Information is costless to copy, should be spread widely, and cannot be confined. Beyond the Jeffersonian credo lies a kind of Darwinian anthropomorphism. Information really does want to be free. John Perry Barlow credited Brand's phrase with "recognizing both the natural desire of secrets to be told and the fact that they might be capable of possessing something like a 'desire' in the first place."12 Barlow continued: English biologist and philosopher Richard Dawkins proposed the idea of "memes," self-replicating, patterns of information which propagate themselves across the ecologies of mind, saying they were like life forms. I believe they are life forms in every respect but a basis in the carbon atom. They self-reproduce, they interact with their surroundings and adapt to them, they mutate, they persist. Like any other life form they evolve to fill the possibility spaces of their local environments, which are, in this case the surrounding belief systems and cultures of their hosts, namely, us. I d d th i bi l i t lik D ki k .Indeed, the sociobiologists like Dawkins make a plausible case that carbon-based life forms are information as well, that, as the chicken is an egg's way of making another egg, the entire biological spectacle is just the DNA molecule's means of copying out more information strings exactly like itself.'3 Viewed through this lens, the Internet is the ultimate natural environment for information; trying to regulate the Internet is like trying to prohibit evolution. Taken together, the three quotations assert that the technology of the medium, the geographical distribution of its users, and the nature of its content all make the Internet specially resistant to state regulation. The state is too big, too slow, and too geographically and technically limited to regulate a global citizenry's fleeting interactions over a mercurial medium. Though I do not subscribe to the full-throated versions of any of these slogans, I have sympathy with each of them. C. "Information Wants to be Free." Yet, as these articles both show, intellectual property most definitely is not 'just a commercial form ofprivacy law " UNIVERSITY OF CMCLNATILAWREVIEW [Vol.66 [Vol.66 182 on-line,8 and has been roundly criticized by both civil liberties groups and right wing intellectuals.9 This is not just a disagreement as to tactics among people who might be said to share the same ideology: it is a fundamental set of disputes over the very social construction and normative significance of a particular phenomenon-as if the Libertarian party couldn't agree on whether its motto was to be "Taxation is theft" or "Property is theft." In this contested terrain, Stewart Brand's phrase, "information wants to be free," marks out the territory of those who are skeptical of both the need for, and the utility of restraints on the flow of information and who frequently extend that skepticism to intellectual property rights. p As a phrase, "information wants to be free,"'" has sufficiently penetrated the culture that it is now actually parodied in advertisements. Yet its ubiquitous nature may work to conceal the claims that it makes. John Perry Barlow began his famous essay, "Selling Wine Without Bottles: The Economy of Mind on the Global Net," with this quote from Thomas Jefferson: If nature has made any one thing less susceptible than all others of exclusive property, it is the action of the thinking power called an idea, which an individual may exclusively possess as long as he keeps it to himself; but the moment it is divulged, it forces itself into the possession of everyone, and the receiver cannot dispossess himself of it. Its peculiar character, too, is that no one possesses the less, because every other possesses the whole of it. He who receives an idea from me, receives instruction himself without lessening mine; as he who lights his taper at mine, receives light without darkening me. That ideas should freely spread from one to another over the globe, for the moral and mutual instruction of man, and improvement of his condition, seems to have been peculiarly and benevolently designed by nature, when she made them, like fire, expansible over all space, without lessening their density at any point, and like the air in which we breathe, move, and have our physical being, incapable of confinement or exclusive appropriation. C. "Information Wants to be Free." It does excite me that the Internet is highly resistant to externally imposed content filtration, although I tend to worry about structural private filters as well as command-based public filters, and I also recognize that speech and information can and will produce harm as well as good. I do think that the global nature of the Internet is, by and large, a positive thing, though McPherson, Aug. 13, 1813)). 12. Id 13. Id [Vol.66 66 184 UNIVERSITY OF CININNA TI LA WREVIEW we need to pay more attention to things like the cost of the technology required to play the game, or the effects on workers of a networked economy in which companies can relocate around the world and find a new on-line workforce in an afternoon. 4 Finally, I am optimistic about the historical conjunction of technologies based on nearly costless copying and a political tradition that treats information in a more egalitarian way than other resources.15 It is possible, of course, to conjure up a world in which rampant info-kleptocracy undermines scientific and artistic development. I have argued elsewhere that the main danger is not that information will be unduly free, but that intellectual property rights will become so extensive that they will actually stifle innovation, free speech, and educational potential. In any event, I want to set aside my agreement or disagreement with the values behind the Internet catechism, and focus instead on the factual and legal assumptions on which it relies. My argument is that info-libertarians should not be so quick to write off the state. In fact, I argue that the work of the distinctively nondigital philosopher, Michel Foucault, provides some suggestive insights into the ways in which power can be exercised on the Internet and the reasons why much contemporary analysis is so dismissive of the power of law and the state. 14. Global, lightspeed mobility of labor is not something that Adam Smith had contemplated; is it a quantitative or a qualitative distinction? 15. SeeJAMES BOYLE, SHAMANS, SOFTWARE AND SPLEENS: LAW AND THE CONSTRUCTION OF THE INFORMATION SOCIETY at 182-83 (1996) ("To someone like me, who believes a lot of our social ills come from the restriction of egalitarian norms, [the] fact [that our current ideas about information have strong egalitarian underpinnings] has an optimistic ring."); see also Eugene Volokh, Cheap Speech and IWhat It Wdl Do, 104 YALE LJ. 1805, 1847 (1995). [I]he [Supreme] Court has based its jurisprudence on an idealized view of the world, a view that doesn't quite correspond to the world in which we live .... [T]his idealized world ... is much closer to the electronic media world of the future than it is to the print and broadcast media world of the present. If my predictions are right, the new technologies will make it much easier for all ideas, whether backed by the rich or the poor, to participate in the marketplace .... [D]uring the print age, the Supreme Court created a First Amendment for the electronic age. The fictions the Court found necessary to embrace are turning, at least in part, into fact. Id.; cf. C. Edwin Baker, New Media Technologies, the First Amendment and Public Policy, 1 Communications Review 315 (1996) (arguing that Volokh's view reduces the First Amendment to the marketplace of ideas theory that, in fact, represents only a part of First Amendment doctrine and theory). 16. JOHN AUSTIN, THE PROVINCE OFJURISPRUDENCE DETERMINED (Isaiah Berlin, et al. eds., 16. JOHN AUSTIN, THE PROVINCE OFJURISPRUDENCE DETERMINED (Isaiah Berlin, et al. eds., 14. Global, lightspeed mobility of labor is not something that Adam Smith had contemplated; is it a quantitative or a qualitative distinction? Id.; cf. C. Edwin Baker, New Media Technologies, the First Amendment and Public Policy, 1 Communications Review 315 (1996) (arguing that Volokh's view reduces the First Amendment to the marketplace of ideas theory that in fact represents only apart ofFirst Amendment doctrine and theory) It Wdl Do, 104 YALE LJ. 1805, 1847 (1995). [I]he [Supreme] Court has based its jurisprudence on an idealized view of the world, a view that doesn't quite correspond to the world in which we live .... [T]his idealized world ... is much closer to the electronic media world of the future than it is to the print and broadcast media world of the present. If my predictions are right, the new technologies will make it much easier for all ideas, whether backed by the rich or the poor, to participate in the marketplace .... [D]uring the print age, the Supreme Court created a First Amendment for the electronic age. The fictions the Court found necessary to embrace are turning, at least in part, into fact. 15. SeeJAMES BOYLE, SHAMANS, SOFTWARE AND SPLEENS: LAW AND THE CONSTRUCTION OF THE INFORMATION SOCIETY at 182-83 (1996) ("To someone like me, who believes a lot of our social ills come from the restriction of egalitarian norms, [the] fact [that our current ideas about information have strong egalitarian underpinnings] has an optimistic ring."); see also Eugene Volokh, Cheap Speech and IWhat I Wdl D 104 YALE LJ 1805 1847 (1995) theory that, in fact, represents only a part of First Amendment doctrine and theory). Id.; cf. C. Edwin Baker, New Media Technologies, the First Amendment and Public Policy, 1 Communications Review 315 (1996) (arguing that Volokh's view reduces the First Amendment to the marketplace of ideas theory that, in fact, represents only a part of First Amendment doctrine and theory). theory that, in fact, represents only a part of First Amendment doctrine and theory). 16. JOHN AUSTIN, THE PROVINCE OFJURISPRUDENCE DETERMINED (Isaiah Berlin, et al. eds., 19. Michel Foucault, What Is an Author?, in TEXTUAL STRATEGIES: PERSPECTIVES IN POST- STRUCTURALIST CRITICISM 141, 141-160 (osue V. Harari ed., 1979); MICHEL FOUCAULT, DISCIPLINE AND PUNISH: THE BIRTH OFTHE PRISON (Alan Sheridan cd. & trans., 1979) [hercinafter FOUCAULT]. III. FOUCAULT & THEJURISPRUDENCE OF DIGITAL LIBERTARIANISM When "Netizens" think of law, they tend to conjure up a positivist, even Austinian, image: 6 law is a command backed by threats, issued by 16. JOHN AUSTIN, THE PROVINCE OFJURISPRUDENCE DETERMINED (Isaiah Berlin, et al. eds., CORPORA TE LA W SYMPOSIUM 1997] a sovereign who acknowledges no superior, directed to a geographically defined population which renders that sovereign habitual obedience. 7 Thus, Netizens think of the state's law as a blunt instrument, incapable of imposing its will on the global subjects of the Internet and their evanescent and geographically unsituated transactions. Indeed, if there was ever a model of law designed to fail at regulating the Internet, it is the Austinian model. Fortunately or unfortunately for the Internet, however, the Austinian model is both crude and inaccurate. That is where the work of the late Michel Foucault comes in. Michel Foucault was one of the most interesting postwar French philosophers and social theorists. His work was wide-ranging, sometimes obscure,'8 indeed deliberately so, and his historical generalizations would have been insufferable if they were not so often provocatively useful. 9 Above all, Foucault possessed a knack for posing problems in a new way-reorienting the inquiry in a way that was 17. The overwhelmingly libertarian cast.to Internet politics in the United States might provide an explanation for this view. Libertarians tend to concentrate on state power rather than private power, and focus on the obvious restraints on freedom imposed by criminal law's impact against the citizen, rather than the subtler restraints imposed by the rules constituting and structuring market and other relationships. Both ideas fit the Austinian image. By making a criminal statute the paradigm of the exercise of state power, and the citizen's right against the government the paradigm of its fimikn, the libertarians code their normative ideas about political problems and solutions into the very image of law itself. You will recall my work here, such as it has been .... None of it does more than mark time. Repetitive and disconnected, it advances nowhere. Since indeed it never ceases to say the same thing, it perhaps says nothing. It is tangled up into an indecipherable, disorganised muddle. In a nutshell, it is inconclusive. You will recall my work here, such as it has been .... None of it does more than mark time. Repetitive and disconnected, it advances nowhere. 1954); see alsoJames Boyle, Thomas Hobbes and the Invented Tradition of Pos"tL Reflections on Language, Power, and Essentiaism, 135 U. PA. L. REV. 383 (1987). 1954); see alsoJames Boyle, Thomas Hobbes and the Invented Tradition of Pos"tL Reflections on Language, Po and Essentiaism, 135 U. PA. L. REV. 383 (1987). 17. The overwhelmingly libertarian cast.to Internet politics in the United States might provid explanation for this view. Libertarians tend to concentrate on state power rather than private power, focus on the obvious restraints on freedom imposed by criminal law's impact against the citizen, rather the subtler restraints imposed by the rules constituting and structuring market and other relationships. B ideas fit the Austinian image. By making a criminal statute the paradigm of the exercise of state power, the citizen's right against the government the paradigm of its fimikn, the libertarians code their norma ideas about political problems and solutions into the very image of law itself. 18. Foucault wrote: You will recall my work here, such as it has been .... None of it does more than mark time. Repetitive and disconnected, it advances nowhere. Since indeed it never ceases to say the same thing, it perhaps says nothing. It is tangled up into an indecipherable, disorganised muddle. In a nutshell, it is inconclusive. Still, I could claim that after all these were only trails to be followed, it mattered little where they led; indeed, it was important that they did not have a predetermined starting point and destination. They were merely lines laid down for you to pursue or to divert elsewhere, or re-design as the case might be. They are, in the final analysisjust fragments, and it is up to you or me to see what we can make of them. For my part, it has struck me that I might have seemed a bit like a whale that leaps to the surface of the water disturbing it momentarily with a tiny jet of spray and lets it be believed, or pretends to believe, or wants to believe, or himself does in fact believe, that down in the depths where no one sees him any more, where he is no longer witnessed nor controlled by anyone, he follows a more profound, coherent and reasoned trajectory. Well, anyway, that was more or less how I at least conceived the situation; it could be that you perceived it differently. Foucault, supra note 1, at 78-79. 19. Michel Foucault, What Is an Author?, in TEXTUAL STRATEGIES: PERSPECTIVES IN P III. FOUCAULT & THEJURISPRUDENCE OF DIGITAL LIBERTARIANISM Since indeed it never ceases to say the same thing, it perhaps says nothing. It is tangled up into an indecipherable, disorganised muddle. In a nutshell, it is inconclusive. Still, I could claim that after all these were only trails to be followed, it mattered little where they led; indeed, it was important that they did not have a predetermined starting point and destination. They were merely lines laid down for you to pursue or to divert elsewhere, or re-design as the case might be. They are, in the final analysisjust fragments, and it is up to you or me to see what we can make of them. For my part, it has struck me that I might have seemed a bit like a whale that leaps to the surface of the water disturbing it momentarily with a tiny jet of spray and lets it be believed, or pretends to believe, or wants to believe, or himself does in fact believe, that down in the depths where no one sees him any more, where he is no longer witnessed nor controlled by anyone, he follows a more profound, coherent and reasoned trajectory. Well, anyway, that was more or less how I at least conceived the situation; it could be that you perceived it differently. cault, supra note 1, at 78-79. UNIVERSITY OF CINCINNA TI LA W REVIEW [Vol.66 186 [Vol.66 66 manifestly helpful for those who followed. Thinkers whose politics and methodology are very far from Foucault's own have testified to this facility.2" From the point of view of this Article, one of Foucault's most interesting contributions was to challenge a particular notion of power, power-as-sovereignty, and to juxtapose against it a vision of "surveillance" and "discipline."2 At the heart of this project was a belief that both our analyses of the operation of political power and our strategies for its restraint or limitation were inaccurate or misguided. In a series of essays and books Foucault argued that, rather than the public and formal triangle of sovereign, citizen, and right, we should focus on a series of subtler private, informal, and material forms of coercion organized around the concepts of surveillance and discipline. The paradigm for the idea of surveillance was the Panopticon, Bentham's plan for a prison constructed in the shape of a wheel around the hub of an observing warden. 23. In many ways, Foucault himself was most interested in a portion of this analysis that I shall pursue here only episodically. In a series of works on penology and the treatment of insanity, he argued that the emergence of the academic and intellectual "disciplines" as we know them now is reciprocally linked in important ways to this minute and quotidian regulation of behavior. At the same time, retrofitting some of his earlier work on the human sciences into this new theoretical mold, he suggested that our conception of"an individual" was not some naturally occurring fact of nature from which analyses could begin, but instead, in part, a result of the concatenation of discipline and surveillance. Elsewhere I have explored the connections between power and knowledge on the one hand, see James Boyle, 77Te Politics of Reason: Critical Legal 7eoy and Local Socal Thought, 133 U. PA. L. REv. 685 (1985), and the effects of the construction of subjectivity on the other, see James Boyle, Is Subjecviy Possible? The Poshnodem Subject in Legal 20. See, e.g., RICHARD A. POSNER, SEX AND REASON 23, 182 (1992) (describing Foucault's writings on sexuality as "remarkable" and "eloquent"). 21. FOUCAULT, supra note 19. 22. SeeJANEr SEMPLE, BENTHAM'S PRISON: A STUDY OFTHE PANOPTICON PENITENTIARY (1993). The two writers to have used Foucault's ideas most notably in the legal privacy and cyberspace context are J.M. Balkin, hkalt is a Postmodern Constitutionalisrm, 90 MICH. L. REV. 1966, 1987 (1992), and Larry Lessig, Reading the Cons itudion in Cyberspace, 45 EMORY L.J. 869,895 (1996) (citing FOUCAULT, THE BIRTH OF THE PRISON, supra note 19, at 139-40). 23. In many ways, Foucault himself was most interested in a portion of this analysis that I shall pursue here only episodically. In a series of works on penology and the treatment of insanity, he argued that the emergence of the academic and intellectual "disciplines" as we know them now is reciprocally linked in important ways to this minute and quotidian regulation of behavior. At the same time, retrofitting some of his earlier work on the human sciences into this new theoretical mold, he suggested that our conception of"an individual" was not some naturally occurring fact of nature from which analyses could begin, but instead, in part, a result of the concatenation of discipline and surveillance. Elsewhere I have explored the connections between power and knowledge on the one hand, see James Boyle, 77Te Politics of Reason: Critical Legal 7eoy and Local Socal Thought, 133 U. PA. L. REv. 685 (1985), and the effects of the construction of subjectivity on the other, see James Boyle, Is Subjecviy Possible? The Poshnodem Subject in Legal 22. SeeJANEr SEMPLE, BENTHAM'S PRISON: A STUDY OFTHE PANOPTICON PENITENTIARY (1993). The two writers to have used Foucault's ideas most notably in the legal privacy and cyberspace context are J.M. Balkin, hkalt is a Postmodern Constitutionalisrm, 90 MICH. L. REV. 1966, 1987 (1992), and Larry Lessig, Reading the Cons itudion in Cyberspace, 45 EMORY L.J. 869,895 (1996) (citing FOUCAULT, THE BIRTH OF THE PRISON, supra note 19, at 139-40). III. FOUCAULT & THEJURISPRUDENCE OF DIGITAL LIBERTARIANISM At any moment the warden might have the prisoner under observation through a nineteenth century version of the closed-circuit TV.22' Unsure when authority might in fact be watching, the prisoner would strive always to conform his behavior to its presumed desires. Bentham had hit upon a behavioralist equivalent of the superego, formed from uncertainty about when one was being observed by the powers that be. The echo of contemporary laments about the "privacy-free state" is striking. To this, Foucault added the notion of discipline-crudely put, the multitudinous private methods of regulation of individual behavior ranging from workplace time-and-motion efficiency directives to psychiatric evaluation." 20. See, e.g., RICHARD A. POSNER, SEX AND REASON 23, 182 (1992) (describing Foucault's writings on sexuality as "remarkable" and "eloquent"). 21. FOUCAULT, supra note 19. 22. SeeJANEr SEMPLE, BENTHAM'S PRISON: A STUDY OFTHE PANOPTICON PENITENTIARY (1993). The two writers to have used Foucault's ideas most notably in the legal privacy and cyberspace context are J.M. Balkin, hkalt is a Postmodern Constitutionalisrm, 90 MICH. L. REV. 1966, 1987 (1992), and Larry Lessig, Reading the Cons itudion in Cyberspace, 45 EMORY L.J. 869,895 (1996) (citing FOUCAULT, THE BIRTH OF THE PRISON, supra note 19, at 139-40). 23. In many ways, Foucault himself was most interested in a portion of this analysis that I shall pursue here only episodically. In a series of works on penology and the treatment of insanity, he argued that the emergence of the academic and intellectual "disciplines" as we know them now is reciprocally linked in important ways to this minute and quotidian regulation of behavior. At the same time, retrofitting some of his earlier work on the human sciences into this new theoretical mold, he suggested that our conception of"an individual" was not some naturally occurring fact of nature from which analyses could begin, but instead, in part, a result of the concatenation of discipline and surveillance. Elsewhere I have explored the connections between power and knowledge on the one hand, see James Boyle, 77Te Politics of Reason: Critical Legal 7eoy and Local Socal Thought, 133 U. PA. L. REv. 685 (1985), and the effects of the construction of subjectivity on the other, see James Boyle, Is Subjecviy Possible? The Poshnodem Subject in Legal 23. In many ways, Foucault himself was most interested in a portion of this analysis that I shall pursue here only episodically. 20. See, e.g., RICHARD A. POSNER, SEX AND REASON 23, 182 (1992) (describing Foucault's writings on sexuality as "remarkable" and "eloquent"). 21. FOUCAULT, supra note 19. 22. SeeJANEr SEMPLE, BENTHAM'S PRISON: A STUDY OFTHE PANOPTICON PENITENTIARY (1993). The two writers to have used Foucault's ideas most notably in the legal privacy and cyberspace context are J.M. Balkin, hkalt is a Postmodern Constitutionalisrm, 90 MICH. L. REV. 1966, 1987 (1992), and Larry Lessig, Reading the Cons itudion in Cyberspace, 45 EMORY L.J. 869,895 (1996) (citing FOUCAULT, THE BIRTH OF THE PRISON, supra note 19, at 139-40). Theory, 62 U. COLO. L. REV. 489 (1991). Although there are interesting things to be said about the construction of subjectivity in cyberspace, my goal here is more mundane. 24. Foucault, supra note 1, at 105. 25. Id at 104. 26. Id at 105. III. FOUCAULT & THEJURISPRUDENCE OF DIGITAL LIBERTARIANISM In a series of works on penology and the treatment of insanity, he argued that the emergence of the academic and intellectual "disciplines" as we know them now is reciprocally linked in important ways to this minute and quotidian regulation of behavior. At the same time, retrofitting some of his earlier work on the human sciences into this new theoretical mold, he suggested that our conception of"an individual" was not some naturally occurring fact of nature from which analyses could begin, but instead, in part, a result of the concatenation of discipline and surveillance. Elsewhere I have explored the connections between power and knowledge on the one hand, see James Boyle, 77Te Politics of Reason: Critical Legal 7eoy and Local Socal Thought, 133 U. PA. L. REv. 685 (1985), and the effects of the construction of subjectivity on the other, see James Boyle, Is Subjecviy Possible? The Poshnodem Subject in Legal CORPORA TE LIA W SYMPOSIUM 1997] Foucault pointed out the apparent conflict between a formal language of politics organized around relations between sovereign and citizen, expressed through rules backed by sanctions, and an actual experience of power being exercised through multitudinous non-state sources, often dependent on material or technological means of enforcement. Writing in a manner that managed to be simultaneously coy and sinister, Foucault suggested that there was something strange going on in the coexistence of these two systems: Impossible to describe in the terminology of the theory of sovereignty from which it differs so radically, this disciplinary power ought by rights to have led to the disappearance of the grand juridical edifice created by that theory. But in reality, the theory of sovereignty has continued to exist not only as an ideology of right, but also to provide the organising principle of the legal codes .... g Why has the theory of sovereignty persisted in this fashion ... ? For two reasons, I believe. On the one hand, it has been.., a permanent instrument of criticism of the monarchy and all the obstacles that can thwart the development of a disciplinary society. But at the same time, the theory of sovereignty, and the organisation of a legal code centered upon it, have allowed a system of right to be superimposed upon the mechanisms of discipline in such a way as to conceal its actual procedures .... 24 Foucault was not writing about the Internet. III. FOUCAULT & THEJURISPRUDENCE OF DIGITAL LIBERTARIANISM He was not even writing about the twentieth century. But his words provide a good starting place from which to examine the catechism of Internet inviolability. They are a good starting point precisely because, when viewed within the discourse of sovereign "commands backed by threats" aimed at a defined territory and population, the Internet does indeed look almost invulnerable. Things look rather different when viewed from the perspective of"a type of power which is constantly exercised by means of surveillance rather than in a discontinuous manner by means of a system of levies or obligations distributed over time [and which].... presupposes a tightly knit grid of material coercions rather than the physical existence of a sovereign." 25 What is more, there is a sense in which the "system of right [is] superimposed upon the mechanism of discipline in such a way as to conceal its actual procedures ... ,, 26 The jurisprudence of digital libertarianism is not simply inaccurate; it may Foucault was not writing about the Internet. He was not even writing about the twentieth century. But his words provide a good starting place from which to examine the catechism of Internet inviolability. They are a good starting point precisely because, when viewed within the discourse of sovereign "commands backed by threats" aimed at a defined territory and population, the Internet does indeed look almost invulnerable. Things look rather different when viewed from the perspective of"a type of power which is constantly exercised by means of surveillance rather than in a discontinuous manner by means of a system of levies or obligations distributed over time [and which].... i h l k i id f i l i h h presupposes a tightly knit grid of material coercions rather than the physical existence of a sovereign." 25 What is more, there is a sense in which the "system of right [is] superimposed upon the mechanism of discipline in such a way as to conceal its actual procedures ... ,, 26 The jurisprudence of digital libertarianism is not simply inaccurate; it may [Vol.66 UNIVERSITY OF CINCINNATIL4 WREVIEW [Vol.66 188 actually obscure our understanding of what is going on. Cyber Rights Now: Tyranny in dthInfastrucsa, WIRED 5:07 (July 1997) <http://www.wired. com/wired/5.07 /cyber_.rights.html>; see also Lawrence Lessig, The Zones of Cyberspace, 48 STAN. L. REV. 1403, 1408 (1996) ("In the well implemented system, there is no civil disobedience. Law as code is a start to the perfect technology ofjustice."). I discovered during the writing of this piece that Lessig's most recent paper comes even closer to my concerns here. See Lawrence Lessig, What Things Regulate Speech, (visited October 22, 1997), <http://www.si.umich.edu/-prie/tprc/abstracts97/lessig.pdf ">. Jonathan Weinberg's work provides a thoughtful description of the techniques and consequences of various Internet rating systems. SeeJonathan Weinberg, Rating the Net, 19 HASTINGS COMM. & ENT. LJ. 453 (1997). Julie Cohen has provided a very thought-provoking analysis of the significance of technologically hardwired regimes in the context of intellectual property law in Julie Cohen, Reverse Enginenzzg and the Rise of Electronic Viilantism: Intellectual Froperty Implications of "Lock-out" Programs, 68 S. CAL. L. REv. 1091 (1995), and of the jurisprudential assumptions of the supporters of hardwiring, inJulie Cohen, Lochner in Cyberspace, (visited October 22, 1997) <http://wvw.si.umich.edu/-prie/tprc/abstracts97/0 Il.txt>. 27. The best chronicler of the role of hard and softwired legal regimes is Lawrence Lessig. He wrote: I don't take issue with the values inherent in any one particular system of code. My criticism is directed against those who think about cyber regulation solely in terms of "law." Laws affect the pace of technological change, but the strictures of software can do even more to curtail freedom. In the long run, the shackles built by programmers may we constrain us most. 28. Compare 47 U.S.C.A. §223(a)(l)(A)(ii) ("obscene, lewd, lascivious, filthy, or indecent") with §223(a)(l)(B)(ii) ("obscene or indecent") and §223(d)(l)(B) ("in terms patently offensive as measured by contemporary community standards"). Communications Decency Act of 1996, 47 U.S.C.A. §223 (West Supp. 1997). None of these terms is defined, and it is not clear that they are intended to be distinct from each other. With some reservations, the courts that have scrutinized the Communications Decency Act (CDA) have treated both phrases as equivalent to "indecency" as defined in Pacifka. FCC v. Pacifica Foundation, 438 U.S. 726 (1978). The Supreme Court was less willing to waive away the statute's internal inconsistencies: R dl f h CDA i h f III. FOUCAULT & THEJURISPRUDENCE OF DIGITAL LIBERTARIANISM Thus, even the digerati may find the analysis that follows of interest, if only to see how far the Internet can be made to treat censorship as a feature not a bug, how far local ordinances may reach in cyberspace, and how information's desire for freedom may be curbed. The examples I will give are drawn from different areas of regulation of communications technology. Some of them deal explicitly with the Internet-the CDA, the proposed National Information Infrastructure (NII) Copyright Protection Act, and the regulation of cryptography. Others are directed towards technologies outside of the Internet, at least for the present-the V-chip, the Clippe'r Chip, digital telephony, and digital audio recorders. All of them share one thing--the state has worked actively to embed or hardwire the legal regime in the technology itself.27 In most of them, the exercise of power is much more a matter of the quotidian shaping and surveillance of activity than of imposing sanctions after the fact. Yet these examples also present revealing differences, illustrating a range of goals, tactics, and results. Sometimes technology has been mandated by legislation, sometimes facilitated through state-sanctioned, standard-setting bodies. Sometimes the legis- lation defines technological safe harbors to sanctions that would other- wise apply, and sometimes the state uses the power of the purse to create a de facto standard by refusing to purchase any equipment that does not conform to the desired technical' or legal standards. I will begin with the CDA, turn to the use of strict liability and digital fences in Internet copyright policy, and conclude with a sampler of hardwired regulation drawn from a number of areas of communications technology. 1997] CORPORA TE LA W SYMPOSIUM 30. Reno v. ACLU, 117 S. Ct. 2329. Regardless of whether the CDA is so vague that it violates the Fifth Amendment, the many ambiguities concerning the scope of its coverage render it problematic for purposes of the First Amendment. For instance, each of the two parts of the CDA uses a different linguistic form. The first uses the word "indecent," while the second speaks of material that "in context, depicts or describes, in terms patently offensive as measured by contemporary community standards, sexual or excretory activities or organs." Given the absence of a definition of either term, this difference in language will provoke uncertainty among speakers about how the two standards relate to each other and just what they mean. R ACLU 29. SeeACLU, 929 F. Supp. 824. In striking down the CDA, the district court held that, "0]ust as the strength of the Internet is chaos, so the strength of our liberty depends upon the chaos and cacophony of the unfettered speech the First Amendment protects. For these reasons, I without hesitation hold that the CDA is unconstitutional on its face." Id at 883 (Dalzel,J., concurring). Reno v. ACLU, 117 S. Ct. 2329, 2344 (1997) (citations omitted). Perhaps in desperation the government ended up by declaring that the CDA was intended to regulate only "commercial pornography," a phrase that appears nowhere within it. ACLU v. Reno, 929 F. Supp. 824, 854-55 (E.D. Pa. 1996). 28. Compare 47 U.S.C.A. §223(a)(l)(A)(ii) ("obscene, lewd, lascivious, filthy, or indecent") with §223(a)(l)(B)(ii) ("obscene or indecent") and §223(d)(l)(B) ("in terms patently offensive as measured by contemporary community standards"). Communications Decency Act of 1996, 47 U.S.C.A. §223 (West Supp. 1997). None of these terms is defined, and it is not clear that they are intended to be distinct from each other. With some reservations, the courts that have scrutinized the Communications Decency Act (CDA) have treated both phrases as equivalent to "indecency" as defined in Pacifka. FCC v. Pacifica Foundation, 438 U.S. 726 (1978). The Supreme Court was less willing to waive away the statute's internal inconsistencies: Regardless of whether the CDA is so vague that it violates the Fifth Amendment, the many ambiguities concerning the scope of its coverage render it problematic for purposes of the First Amendment. For instance, each of the two parts of the CDA uses a different linguistic form. The first uses the word "indecent," while the second speaks of material that "in context, depicts or describes, in terms patently offensive as measured by contemporary community standards, sexual or excretory activities or organs." Given the absence of a definition of either term, this difference in language will provoke uncertainty among speakers about how the two standards relate to each other and just what they mean. Reno v ACLU 117 S Ct 2329 2344 (1997) (citations omitted) Perhaps in desperation the government 30. Reno v. ACLU, 117 S. Ct. 2329. 28. Compare 47 U.S.C.A. §223(a)(l)(A)(ii) ("obscene, lewd, lascivious, filthy, or indecent") with §223(a)(l)(B)(ii) ("obscene or indecent") and §223(d)(l)(B) ("in terms patently offensive as measured by contemporary community standards"). Communications Decency Act of 1996, 47 U.S.C.A. §223 (West Supp. 1997). None of these terms is defined, and it is not clear that they are intended to be distinct from each other. With some reservations, the courts that have scrutinized the Communications Decency Act (CDA) have treated both phrases as equivalent to "indecency" as defined in Pacifka. FCC v. Pacifica Foundation, 438 U.S. 726 (1978). The Supreme Court was less willing to waive away the statute's internal inconsistencies: Regardless of whether the CDA is so vague that it violates the Fifth Amendment, the many ambiguities concerning the scope of its coverage render it problematic for purposes of the First Amendment. For instance, each of the two parts of the CDA uses a different linguistic form. The first uses the word "indecent," while the second speaks of material that "in context, depicts or describes, in terms patently offensive as measured by contemporary community standards, sexual or excretory activities or organs." Given the absence of a definition of either term, this difference in language will provoke uncertainty among speakers about how the two standards relate to each other and just what they mean. Reno v. ACLU, 117 S. Ct. 2329, 2344 (1997) (citations omitted). Perhaps in desperation the government ended up by declaring that the CDA was intended to regulate only "commercial pornography," a phrase that appears nowhere within it. ACLU v. Reno, 929 F. Supp. 824, 854-55 (E.D. Pa. 1996). 29. SeeACLU, 929 F. Supp. 824. In striking down the CDA, the district court held that, "0]ust as the strength of the Internet is chaos, so the strength of our liberty depends upon the chaos and cacophony of the unfettered speech the First Amendment protects. For these reasons, I without hesitation hold that the CDA is unconstitutional on its face." Id at 883 (Dalzel,J., concurring). 30. Reno v. ACLU, 117 S. Ct. 2329. IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES The CDA has been hailed as the nadir of congressional regulation of communications technology. Badly drafted, inconsistently worded,28 and palpably unconstitutional, it appeared to most of the Internet community to be a case of technological ignorance run rampant. Congress regulated what it did not understand, and did so in a way that would be practically futile because of the amount of content that came from beyond the jurisdiction of the United States. The reactions ranged from condescending amusement at the lack of Congress's technological knowledge, to proprietary anger that the state was overtly asserting its power over the electronic frontier. "Keep your laws off our Net," went the slogan. When the CDA was struck down by two different three-judge panels29 and then by a unanimous Supreme Court,3" the decisions were seen as an inevitable vindication of these libertarian views. The victory was only sweetened when the lower court opinions pointed out that some of the CDA's constitutional problems came from its practical inability to reach much of the content on the Internet. Federal judges had come a long way towards recognizing both the technological resistance of the Internet to censorship, and the fact that a global net could never be Reno v. ACLU, 117 S. Ct. 2329, 2344 (1997) (citations omitted). Perhaps in desperation the government ended up by declaring that the CDA was intended to regulate only "commercial pornography," a phrase that appears nowhere within it. ACLU v. Reno, 929 F. Supp. 824, 854-55 (E.D. Pa. 1996). 29. SeeACLU, 929 F. Supp. 824. In striking down the CDA, the district court held that, "0]ust as the strength of the Internet is chaos, so the strength of our liberty depends upon the chaos and cacophony of the unfettered speech the First Amendment protects. For these reasons, I without hesitation hold that the CDA is unconstitutional on its face." Id at 883 (Dalzel,J., concurring). UNIVERSITY OF CIA'CLIlNA T7 LA WREVIEW [Vol.66 190 effectively regulated by a single national jurisdiction." Thus, two of the three parts of the Internet Trinity had been acknowledged in the federal reporters. What is more, they had actually been plugged into the framework of conventional First Amendment analysis. 3 1. ACLU, 929 F. Supp. at 824 (discussing findings of fact). "There is no centralized storage location, control point, or communications channel for the Intemet, and it would not be technically feasible for a single entity to control all of the information conveyed on the Interet." Id. But cf ACLU Cyber- Uberties, Transcript of Supreme Court Oral Argument, Reno v. ACLU, Oral Argument of BruceJ. Ennis (visitedJune 24, 1997) <http://www.aclu.org/issues/cyber/trial/sctran.html> (statement of ChiefJustice Rehnquist) ("But if 70 percent [of indecent speech on the Internet] is shielded and 30 percent isn't, what kind of an argument is that against the constitutionality of the statute?"). 3 1. ACLU, 929 F. Supp. at 824 (discussing findings of fact). "There is no centralized storage location, control point, or communications channel for the Intemet, and it would not be technically feasible for a single entity to control all of the information conveyed on the Interet." Id. But cf ACLU Cyber- Uberties, Transcript of Supreme Court Oral Argument, Reno v. ACLU, Oral Argument of BruceJ. Ennis (visitedJune 24, 1997) <http://www.aclu.org/issues/cyber/trial/sctran.html> (statement of ChiefJustice Rehnquist) ("But if 70 percent [of indecent speech on the Internet] is shielded and 30 percent isn't, what kind of an argument is that against the constitutionality of the statute?"). 32. Charles Nesson & David Marglin, 77m Day Lim Intermt Met the Frst Amedme- Tme and the Communications LiecnyAct, 10 HARV.J.L. & TECH. 113, 115 (1996). 33. Buderv. Michigan, 352 U.S. 380,383 (1957), quoted in Sable Communications v. FCC, 492 U.S. 115, 127 (1989). 34. See 47 U.S.C.A. § 223(e)(5)(A) (West Supp. 1997). 35. 47 U.S.C.A. § 223(e)(5)(B). 32. Charles Nesson & David Marglin, 77m Day Lim Intermt Met the Frst Amedme- Tme and the Communications LiecnyAct, 10 HARV.J.L. & TECH. 113, 115 (1996). 33. Buderv. Michigan, 352 U.S. 380,383 (1957), quoted in Sable Communications v. FCC, 492 U.S. 115, 127 (1989). IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES Given the fact that the CDA was likely to be ineffective, could we possibly say that it passed strict First Amendment scrutiny?32 Was this not a case of substantially restricting the freedom of speech without effectively achieving the compelling state interest? Seen through the lens provided by the jurisprudence of digital libertarianism, these reactions were entirely warranted. A command backed by threats, uttered by a sovereign, and directed towards a geographically defined population had met and been annihilated by a right held by citizens against intrusion by state power, in part, because of the sovereign's inability to regulate those outside its borders. The CDA vanishes as if it had never been--an utter failure. Yet this analysis misses the developments surrounding the CDA, not the public criminal sanction, but rather the shaping and development of privately deployed, materially based, technological methods of surveillance and censorship. The CDA aimed to protect minors from indecent material. However, if it did so by substantially limiting the speech of adults, it would be held unconstitutional as overbroad; in the words of Justice Frankfurter, "burning down the house to roast the pig."33 The CDA's answer to this problem was to create safe harbors for indecent, but constitutionally protected speech aimed at adults, provided that speech was kept from the eyes of minors.34 The CDA offered a number of methods to achieve this goal, such as "requiring [the] use of a verified credit card, debit account, adult access code, or adult personal identification number."35 Given the technology and economics of the Internet, however, the most important safe harbor for nonprofit organizations was clearly going to be that provided by § 223(e)(5)(A), which offered immunity to those who Given the technology and economics of the Internet, however, the most important safe harbor for nonprofit organizations was clearly going to be that provided by § 223(e)(5)(A), which offered immunity to those who 1997] CORPORA TE LA W SYMPOSIUM had used "any method which is feasible under available technology."36 had used any method which is feasible under available technology. It is here that the irony begins. When the CDA was first proposed, a number of computer scientists and software engineers decided that they would do something more than merely rail against its unconstitutionality. 36. 47 U.S.C.A. §223(e)(5)(A). The statute provides: (5) It is a defense to a prosecution under subsection (a)(1)(B) or (d) of this section, or under subsection (a)(2) of this section with respect to the use of a facility for an activity under subsection (a)(1)(B) of this section that a person- (A) has taken, in good faith, reasonable, effective, and appropriate actions under the circumstances to restrict or prevent access by minors to a communication specified in such subsections, which may involve any appropriate measures to restrict minors from such communications, including any method which is feasible under available technology .... 47 U.S.C.A. § 223(e)(5)(A). 37. See Paul Resnick &Jim Miller, The CDA's Silver Lining, WIRED, Aug. 1996, at 109. 38. See generally Platform for Internet Content &lection: What Does It Do? (visited June 24, 1997) <http://www.w3.org/PICS/951030/AV/StartHere.html>. 39. Self-rating israting provided by the person posting the information. Third party rating is rating provided by some other entity. World Wide Web Consortium, PICS Statement of Principle (visitedJune 24, 1997) <http://www.w3.org/PICS/principles.html>. 39. Self-rating israting provided by the person posting the information. Third party rating is rating provided by some other entity. World Wide Web Consortium, PICS Statement of Principle (visitedJune 24, 1997) <http://www.w3.org/PICS/principles.html>. 38. See generally Platform for Internet Content &lection: What Does It Do? (visited June 24, 1997) <http://www.w3.org/PICS/951030/AV/StartHere.html>. 47 U.S.C.A. § 223(e)(5)(A). 37. See Paul Resnick &Jim Miller, The CDA's Silver Lining, WIRED, Aug. 1996, at 109. 37. See Paul Resnick &Jim Miller, The CDA's Silver Lining, WIRED, Aug. 1996, at 109. IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES They were convinced that an answer to the perceived need for regulation could be met within the language of the Internet itself." I am not using the phrase, "language of the Internet" as part of some deconstructive or Saussurean trope-the idea was literally to provide a filtering system built into the same language that makes the World Wide Web possible: Hyper Text Markup Language (HTML). Conceiving of technical solutions as intrinsically more desirable than the exercise of state power by a sovereign, as facilitators of private choice rather than threats of public sanction, they offered an alternative designed to show that the CDA was, above all, unnecessary. This technological alternative to the CDA, called the Platform for Internet Content Selection (PICS), allows tags rating a web page to be embedded within "meta-file" information provided by the page about itself.8 PICS can be adapted to provide both first party and third party content labeling and rating.39 The system is touted as value neutral because it could be used to promote any value-system. Sites could be rated for violence, for sexism, for adherence to some particular religious belief, indeed for any set of criteria that was thought worthwhile. The third party filtering site could be the Christian Coalition, the National Organization for Women, or the Society for Protecting the Manifest Truths of Zoroastrianism. Of course in practice, we might believe that the PICS technology would be disproportionately used to favor a particular set of ideas and values and exclude others, just as we might believe that in practice, a Lochner regime of "free contract" would actually favor some groups and hurt others, despite the fact that each 192 UNIVERSITY OF CLNCLMNATI LA WREVIEW [Vol.66 66 is-on its face-value neutral. However, as Owen Fiss,Jack Balkin, and Richard Delgado have each pointed out (though in very different contexts), this kind of legal realist insistence on looking at actual effects, and scrutinizing actual, rather than formal power, is much rarer in the context of the First Amendment than it is in private law." p While PICS and a variety of other systems offered a technical solution at the "speaker" end of the connection, other software programs also offered technical solutions at the "listener" end. These programs would not offer speakers a safe harbor from the reach of the CDA. IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES Rather, they "empowered" computer users to protect their families from unwanted content by using software filters, thus raising in civil libertarians' hearts the hope that the whole Act was unnecessary. Programs such as SurfWatch, CyberPatrol, NetNanny, and CyberSitter blocked access to unsuitable material and did so without the need for constant parental intervention.4' Typically, these programs maintained a list of forbidden sites as well as a text-search filter, which would not load documents containing forbidden strings of words. The irony that I mentioned is that these technical solutions were used by both sides in the dispute over the CDA. Those challenging the CDA 40. See Owen M. Fiss, Free Speech and Social Structure, 71 IOWA L. REV. 1405, 1424-25 (1986). Today abolition of the fairness doctrine can be passed off as just one more instance of "deregulation." It seems to me, however, that there is much to regret in this stance of the Court and the [First Amendment] Tradition upon which it rests. The received Tradition presupposes a world that no longer exists and that is beyond our capacity to recall-a world in which the principal political forum is the street corner. IL; see also OWEN M. FISS, LIBERALISM DIVIDED (1996); J.M. Balkin, Some Realism About Pluralism. L R li A h h Fi A d 1990 DUKE 375 427 (1990) Realist Approaches to the FirstAmendment, 1990 DUKE LJ. 375, 427 (1990). In assessing what constitutes substantial overbreadth or vagueness, I do not think it inappropriate to employ common sense judgments about the way the world works. Although the distinction between public power and private power is significant, even more significant for me are what power relations (public or private) exist in the standard case in which the statute operates. Il See also Richard Delgado, First Anudment Fomai m Is Giving Way to First AmentLenm Legal Realism, 29 HARV. C.R.-C.L L REV. 169,170 (1994) ('The transition to the new [legal realist] paradigm is, however, far from complete."). But cf. Steven G. Gey, The Case Against Postmodern Censorship Theoty, 145 U. PA. L. REV. 193, 195-97 (1996). The theoretical advances celebrated by Delgado and other progressive critics of the First Amendment are not really advances at all. They are simply refurbished versions of arguments used since the beginning of modem First Amendment jurisprudence to justify government authority to control the speech (and thought) of citizens.... 41. See generally Kathryn Munro, Filtering Utilities, PC MAG., Apr. 8, 1997, at 235 (describing and reviewing various filtering software products). 40. See Owen M. Fiss, Free Speech and Social Structure, 71 IOWA L. REV. 1405, 1424-25 (1986). Today abolition of the fairness doctrine can be passed off as just one more instance of "deregulation." It seems to me, however, that there is much to regret in this stance of the Court and the [First Amendment] Tradition upon which it rests. The received Tradition presupposes a world that no longer exists and that is beyond our capacity to recall-a world in which the principal political forum is the street corner. IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES Moreover, despite the different objectives of the new censors, their reasons for supporting government control over speech are not significantly different from those of their reactionary predecessors.... The postmodem censorship theory offered by this new generation of politically progressive legal scholars is neither progressive nor, for that matter, even "postmodern." In the end, it isjust censorship. Id. 41. See generally Kathryn Munro, Filtering Utilities, PC MAG., Apr. 8, 1997, at 235 (describing and reviewing various filtering software products). CORPORA TE IA W SYMPOSIUM 1997] argued that the availability of privately implemented technological solutions meant that the CDA failed First Amendment scrutiny: clearly it was not the least restrictive means available to achieve the objective. "Listener-based" blocking software allowed parents to control what their children saw while "speaker-based," or third party rating systems such as PICS offered a private solution to the problem of rating the content available on the Internet. The government took the opposite position, arguing that the availability of systems such as PICS meant that the CDA was not overbroad. Adult speakers would not be burdened by the law because such systems provided adequate methods for adult speakers to segregate their indecent but protected speech from the eyes of minors. Thus, in their eyes, the PICS scheme, developed to destroy the CDA, actually saved it.42 The Supreme Court ultimately disagreed, though Justice O'Connor left open the possibility that future technical developments might change that conclusion.43 Before the decision was even handed down, President Clinton was already signaling his political preference for a technical solution to the question of regulating speech on-line, talking vaguely of a "V-chip for the Internet."" Bills have already been advanced in Congress that would require Internet service providers to provide filtering software to customers and aim at the development of an "E-chip. 45 43. Justice O'Connor wrote: Despite this progress, the transformation ofcyberspace is not complete. Although gateway technology has been available on the World Wide Web for some time now, it is not available to all Web speakers, and is just now becoming technologically feasible for chat rooms and USENET newsgroups. Gateway technology is not ubiquitous in cyberspace, and because without it "there is no means of age verification," cyberspace still remains largely unzoned--and unzoneable. User based zoning is also in its infancy. 42. For a fuller version of this argument, see James Boyle et al., Before th Supreme Un-Court ofthw United Stt& (visitedJune 24, 1997) <http://www.wcl.american.edu/pub/faculty/boyle/unreno.htm> (Un-Scalia, J., dissenting). 45. S, e.g., Communications Privacy and Consumer Empowerment Act, H.R. 1964, 105th Cong. (1997). IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES For it to be effective, (i) an agreed upon code (or "tag") would have to exist; (ii) screening software or browsers with screening capabilities would have to be able to recognize the "tag"; and (iii) those programs would have to be widely available-and widely used-by Internet users. At present, none of these conditions is true. Screening software "is not in wide use today" and "only a handful of browsers have screening capabilities." There is, moreover, no agreed- upon "tag" for those programs to recognize. Reno v. ACLU, 117 S. Ct. 2329, 2354 (1997) (O'ConnorJ, concurring in part and dissenting in part) (citations omitted). Reno v. ACLU, 117 S. Ct. 2329, 2354 (1997) (O'ConnorJ, concurring in part and dissenting in part) (citations omitted). 44. Remarks by President Clinton at Town Hall meeting in Bridgeport, W. Va. (May 22, 1997), in 33 WEEKLY COMPIIATION OF PRESIDENTIAL DOCUMENTS, 758 [hereinafter President Remarks]. "[I]t may be that what we have to do is to try to develop something like the equivalent of what we are developing for you for television, like the V-chip .... It's technically more difficult with the Internet .... But I think that is the answer. Something like the V-chip for televisions. And we are working on it." Id. 44. Remarks by President Clinton at Town Hall meeting in Bridgeport, W. Va. (May 22, 1997), in 33 WEEKLY COMPIIATION OF PRESIDENTIAL DOCUMENTS, 758 [hereinafter President Remarks]. "[I]t may be that what we have to do is to try to develop something like the equivalent of what we are developing for you for television, like the V-chip .... It's technically more difficult with the Internet .... But I think that is the answer. Something like the V-chip for televisions. And we are working on it." Id. 45. S, e.g., Communications Privacy and Consumer Empowerment Act, H.R. 1964, 105th Cong. (1997). 45. S, e.g., Communications Privacy and Consumer Empowerment Act, H.R. 1964, 105th Cong. (1997). 194 UNIVERSITY OF CLVCLNATI 4 WREVIEW [Vol.66 So where does on-line speech stand after the Supreme Court's decision in Rno v. ACLU? From the perspective of the digital libertarian, the Internet remains unregulated and the Internet Trinity, is undisturbed. From the perspective I have been developing here, things seem much more mixed. As the CDA was being constitutionally voided, the technological "solutions" were proceeding apace, some because of the CDA, some in spite of the CDA. IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES In contrast to the extensive attention given to CDA, much of this process was effectively insulated from scrutiny because of the assumptions about law and state that I have been exploring here. PICS is a wonderful tool for content selection and, in many ways, if one assumes a world very much like the idealized version of the marketplace of ideas, an unthreatening and beneficial one. Yet its technological goal-to facilitate third- and first-party rating and blocking of content-helps to weaken the Internet's supposed resistance to censorship at the same moment that it helps provide a filter for user- based selection. If national networks can be more easily run through a kind of PICS-filtered firewall, what happens to the notion that the Internet tap can only be turned to off or full? One wonders how China, Singapore, or Iran would choose to employ this value-neutral system. The technological component of the Internet faith does not fall, but it is weakened. The state may not be able to deploy Austinian sanctions backed by threats over the Internet, but the technology provided by PICS gives it a different arsenal of methods with which to regulate content materially, rather than juridically--by everyday softwired routing practices, rather than by threats of eventual sanction. As for the listener-based software filters, they present even more problems. Journalists studying these programs found that their list of selected sites was problematic and, most importantly, was actually hidden from the users. A close look at the actual range of sites blocked by these apps shows they go far beyond just restricting "pornography." Indeed, some programs ban access to newsgroups discussing gay and lesbian issues or topics such as feminism. Entire domains are restricted, such as HotWired. Even a web site dedicated to the safe use of fireworks is blocked. All this might be reasonable, in a twisted sort of way, if parents were actually aware of what the programs banned. But here's the rub: Each company holds its database of blocked sites in the highest security. Companies fight for market share based on how well they 1997] CORPORA TE LA W SYMPOSIUM upgrade and maintain that blocking database. All encrypt that list to protect it from prying eyes .... 46. Declan B. McCullagh & Brock N. Meeks, Keys to the Klugdom (visited June 24, 1997) <http://www.eff.org/pub/Publications/Decan-McCullagh/cwd.kys.to.th.kingdom.0796.articlc>. 47. See President Remarks, rupra note 44. 48. Geeta Anand, Library OI's limits on 'Net access; Compromise callsforfalier sojftare only on computers used by children, BOSTON GLOBE, Mar. 22, 1997, at Al. 49. Marc Ferranti, Siie-Filtering Issue Goes to Stat Level, INFOWORLD, Apr. 21, 1997, at 60. 50. Communications Privacy and Consumer Empowerment Act, H.R. 1964, 105th Cong. (1997). 51. With a cavalier disregard for the problems that this raises for my thesis, some of the best investigative reporting on, and discussion of, the politics of private technological censorship has been done by the cyberouralist Declan McCullagh and his "Fight Censorship" discussion list. See McCullagh & Meeks, supra note 46. In one sense, this raises the issue that I discussed earlier-the politics of the Internet are up for grabs and the conventional categories of political ideology and theory are much more mutable there. 52. One commentator wrote: 53. Boyle, supra note 9, at 47; BOYLE, supra note 15,at 18-20, 51-61, 162-63; James Boyle, Q: Is Congress tuminag the Internet into an infornation toll rad?, INSIGHT, Jan. 15, 1996; James Boyle, Sold Out, N.Y. TIMES, Mar. 31, 1996, at E15. IV. SAFE HARBORS AND UNINTENDED CONSEQUENCES ' The programs turned out to ban sites ranging from the National Rifle Association to the National Organization for Women, and did so in a way that was often undetectable by their purchasers. Nevertheless, enthusiasm for these programs continues unabated. As I mentioned earlier, President Clinton has promised that the government is working on an Internet V-chip,47 Boston city libraries are installing blocking software on computers accessible to children, and Texas is considering mandating that Internet access companies make copies of such programs available to all their new customers.49 Representative Markey introduced a bill into Congress that would require both the creation of an "E-chip" and a provision for free or "at cost" blocking software.5° In constitutional terms, this raises interesting questions of state action. One of the attractions of the technical solution is often that it allows the state to enlist private parties to accomplish that which it is forbidden to accomplish directly. But this state action problem is merely the constitutional incarnation of the political limitations of the jurisprudence of digital libertarianism--its sole focus on state power, narrowly defined, and its blindness to the technical and economic shaping, rather than the legal sanctioning, of the communications environment. I do not want to overstate the effect of the mindset that I am describing. Not everyone in the digital world thinks this way. Libertarians too, have been worried by the dangers posed by technologically invisible filtering of communication.5' Indeed, one of the most interesting thing about Internet politics is that it has forced libertarians to confront some of the tensions inherent to their own ideas. Finally, other commentators have made the points I make here, though they also lamented the blindness imposed by an entirely libertarian focus.52 Nevertheless, the result of the Supreme Court's decision in Reno 196 UNIVERSITY OF CIMCINNATI LA WREVIEW [Vol.66 v. ACLU will simply be to sharpen the turn to the kinds of filtering devices mentioned here. It is unlikely that this will leave the Internet as free, or the state as powerless, as the digerati seem to believe. 54. NICHOLAS NEGROPONTE, BEING DIGITAL (1995). Seth Finkelstein, Intornet Blocdng Programs and Privatized Censorship, THE ETHICAL SPECTACLE (Aug. 1996) <http://www.spectacle.org/896/finkel.html>. I started this Article convinced that it was about the failures of libertarianism in cyberspace. I now believe that the real question is how cyberspace will change libertarianism. Digital libertarians show occasional willingness to admit that property rights can be "coercive regulation" as well as the well-spring of liberty. Privacy discourse in cyberspace focuses on excesses of private, as well as public, power. All in all, cyberspace is a hard place to maintain the most simplistic type of libertarian definitionalism. V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES I have argued elsewhere that the current government proposals for the "reform" of copyright on the Internet weigh only the costs of cheaper copying rather than its benefits, underestimate the importance of fair use to competition policy and free speech, fail to recognize the unique features of both intellectual property and networked environments, and apply bad economic analysis to an even worse depiction of current law.53 Leaving aside the virtues or vices of these proposals for the moment, I will focus here on the methods by which they were to be implemented. Enforcement is a key problem for any Internet copyright regime. The Internet Trinity I discussed earlier would seem to apply with particular force to the problem of policing copyright on a global distributed network. The technology is resistant to control, the subject matter of the regime is intangible and trivially easy to circulate, and both the content and the people regulated by the regime are frequently beyond the jurisdiction of the sovereign in question. The combination of these circumstances produced a series of warnings that intellectual property law was doomed because neither its conceptual structure nor its enforcement mechanism could survive "being digital."54 The best known of these warnings is also the best written: Seth Finkelstein, Intornet Blocdng Programs and Privatized Censorship, THE ETHICAL SPECTACLE (Aug. 1996) <http://www.spectacle.org/896/finkel.html>. I started this Article convinced that it was about the failures of libertarianism in cyberspace. I now believe that the real question is how cyberspace will change libertarianism. Digital libertarians show occasional willingness to admit that property rights can be "coercive regulation" as well as the well-spring of liberty. Privacy discourse in cyberspace focuses on excesses of private, as well as public, power. All in all, cyberspace is a hard place to maintain the most simplistic type of libertarian definitionalism. 1997] CORPORA TE IA W SYMPOSIUM The riddle is this: if our property can be infinitely reproduced and instantaneously distributed all over the planet without cost, without our knowledge, without its even leaving our possession, how can we protect it? How are we going to get paid for the work we do with our minds? And, if we can't get paid, what will assure the continued creation and distribution of such work? 55. Barlow, sufna note 5. V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES Since we don't have a solution to what is a profoundly new kind of challenge, and are apparently unable to delay the galloping digitization of everything not obstinately physical, we are sailing into the future on a sinking ship. This vessel, the accumulated canon of copyright and patent law, was developed to convey forms and methods of expression entirely different from the vaporous cargo it is now being asked to carry. It is leaking as much from within as without. Legal efforts to keep the old boat floating are taking three forms: a frenzy of deck chair rearrangement, stern warnings to the passengers that if she goes down, they will face harsh criminal penalties, and serene, glassy-eyed denial.55 If one saw these technological transformations as mainly a threat to both the copyright owner and the enforcement power of the state, how would one respond, particularly if one took seriously the difficulties in policing that the Internet Trinity points out? One would try to focus on building the regime into the architecture of transactions in the first place-both technically and economically-rather than policing the transactions after the fact. More concretely, one would want to escape from the practical and legal limitations of a sovereign-citizen relationship. Thus, one might seek out private actors involved in providing Internet services who are not quite as mobile as the flitting and frequently anonymous inhabitants of cyberspace. In this case, the parties chosen were the Internet service providers. One would pin liability on them and leave it up to them to prevent copyright infringement through technical surveillance, tagging, and so on, and to spread the cost of the remaining copyright infringement over all the users of their service, rather than all the purchasers of the product in question. By enlisting these nimbler, technologically savvy players as one's private police, one would also gain another advantage: freedom from some of the constitutional and other restraints that would burden the state were it to act directly. Intrusion into privacy, automatic scrutiny of electronic mail, and curtailment of fair use rights so as to make sure that no illicit content was being carried would occur in the 198 UNIVERSITh OF CINCIj&NA 77 LA WREVIEW private realm, far from the scrutiny of public law. There are advantages to privatizing the Panopticon, it turns out. 56. INFORMATION INFRASTRUCTURE TASK FORCE, INTELLECTUAL PROPERTY AND THE NATIONAL INFORMATION INFRASTRUCTURE: THE REPORT OF THE WORKING GROUP ON INTEL..ECrUAL PROPERTY RIGHTS, 114-24 (1995) ("White Paper"); Boyle, supra note 9, at 58-11I; Niva Elkin-Koren, Copight Low and SocialDialogue on the Information Superihway: The Case Against Copyright Liability ofBullAin Board Operators, 13 CARDOZO ARTS & ENT. L.J. 346 (1993); Sf. Religious Tech. Ctr. v. Netcom On-Une Communication Servs., 907 F. Supp. 1361, 1377 (N.D. Cal. 1995) (discussing the theory that strict liability for Internet service providers "would chill the use of the Internet because every access provider or user would be subject to liability when a user posts an infringing work to a Usenet newsgroup"). V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES Given all these "advantages," it is unsurprising to find that strict liability for on-line service providers became a central feature in the Clinton administration's "White Paper,"56 the bills implementing its ideas, 57 and the United States proposals for the World Intellectual Property Organization (WIPO) treaties in Geneva.' The specifics of the White Paper were relatively simple. On-line service providers were to be made strictly liable for copyright violations committed by their subscribers; in part, this was done by an expansive definition of fixation so that even holding a document in RAM as it was browsed would constitute the creation of a copy. 59 Clearly then, the relatively more stable versions held in a server's disk cache or stored temporarily in its computers would count as copies. The theory also depends on the notion that we should analogize the on-line service provider to an innocent but infringing photo shop and, thus, impose strict liability on the provider as a direct infringer.6" Notably, this theory was rejected by the only court to have faced it squarely.6' In one sense, this strategy is very similar to the use of strict liability elsewhere in the legal system, and of course, it can be understood entirely without reference to the Foucauldian gloss. Yet, the conventional reasons for imposing strict liability are strikingly absent.62 56. INFORMATION INFRASTRUCTURE TASK FORCE, INTELLECTUAL PROPERTY AND THE NATIONAL INFORMATION INFRASTRUCTURE: THE REPORT OF THE WORKING GROUP ON INTEL..ECrUAL PROPERTY RIGHTS, 114-24 (1995) ("White Paper"); Boyle, supra note 9, at 58-11I; Niva Elkin-Koren, Copight Low and SocialDialogue on the Information Superihway: The Case Against Copyright Liability ofBullAin Board Operators, 13 CARDOZO ARTS & ENT. L.J. 346 (1993); Sf. Religious Tech. Ctr. v. Netcom On-Une Communication Servs., 907 F. Supp. 1361, 1377 (N.D. Cal. 1995) (discussing the theory that strict liability for Internet service providers "would chill the use of the Internet because every access provider or user would be subject to liability when a user posts an infringing work to a Usenet newsgroup"). 57. See National Information Infrastructure Copyright Protection Act of 1995, S. 1284, 104th Cong. (1995), H.R. 2441, 104th Cong. (1995). 58. S&e World Intellectual Property Organization Treaty, Dec. 20, 1996, CRNR/DC/94 (visited June 26, 1997) <http://www.wipo.org/eng/diplconf/distrib/94dc.htm>; see also Newsfom WIPO (visited June 26, 1997) <http://www.hrrc.org/wiponews.html> (detailing course of deliberations during the Diplomatic Conference). 59. See Boyle, supra note 9, at 83-94 (discussing MAI Sys. Corp. v. 58. S&e World Intellectual Property Organization Treaty, Dec. 20, 1996, CRNR/DC/94 (visited June 26, 1997) <http://www.wipo.org/eng/diplconf/distrib/94dc.htm>; see also Newsfom WIPO (visited June 26, 1997) <http://www.hrrc.org/wiponews.html> (detailing course of deliberations during the Diplomatic Conference). 57. See National Information Infrastructure Copyright Protection Act of 1995, S. 1284, 104th Cong. (1995), H.R. 2441, 104th Cong. (1995). V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES In the on-line setting, none of these claims is obviously correct. In some cases service providers may be able to prevent illicit copying relatively cheaply without imposing large social costs. On many other occasions, however, it seems that the costs of enforcement may outweigh the benefits. To ensure that illicit copying is not being carried on, service providers may incur, or impose, high transaction costs; they may be forced to impose draconian restrictions on the fair use privileges of their subscribers, for example. (Because Internet service providers would pay for all detected copyright infringements, but would not be forced to internalize the cost to their customers of restricting fair use, the incentives would be asymmetrically anti-consumer.) Leaving aside the efficiency costs of enforcement by service providers, there is also the question of whether they are the cheapest cost avoider. In many cases, the party best situated to avoid the cost of copyright infringement will be the owner of the copyright. Whether by developing technical solutions or by fine-tuning their business plan so as to minimize the incentives to violate copyright in the first place, copyright owners might well be the cheapest cost avoiders. If that is true, it would actually be inefficient to allow them to rely on another party for enforcement of their rights. Beyond the question of the cheapest cost-avoider, remains the question of best loss spreader. Here, too, it is hard to be confident that the Internet service providers are the appropriate parties upon which to impose liability. The economic analysts' mantra is "activities should internalize their full costs." If the costs of a good or activity are not passed on to those who use the good or engage in the activity, then those individuals will make inefficient choices. Thus, for example, if the price of gasoline does not reflect the environmental damage done by gasoline, that damage becomes a negative externality, and gasoline is inefficiently priced relative to its "true" costs. Over what group, then, should the costs (i.e., the copyright owner's foregone profit) of illicit copying be imposed? The inquiry is a fascinating one, with more layers than I can fully explore here. It is complicated by the fact that the costs imposed by the illicit copying of an information good are economically different in some ways from the costs imposed by theft of material goods. V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES Peak Computer, Inc., 991 F.2d 511 (9th Cir.1993)). 60. See id. at 103-04. The alternative, of course, would be to analogize the service provider to a business that rented copy machines with which material could be copied illegally; in that case, the business would be liable only if it was guilty of contributory infringement. be liable only if it was guilty of contributory infringement 61. See Religious Tech. Ctr. v. Netcom On-Line Communication Servs., 907 F. Supp. 1361, 1377 (N.D. Cal. 1995); see also Playboy Enters., Inc. v. Chuckleberry Publications, Inc., 939 F. Supp. 1032 (S.D.N.Y. 1996); Sega Enters., Ltd. v. Maphia, 948 F. Supp. 923 (N.D. Cal. 1996). 62. We impose strict liability on manufacturers of products for a number of reasons-one of which is that we believe the state could not possibly inspect every product and every design in the marketplace. Simply by forcing manufacturers to internalize the costs of injuries caused by their products, we produce CORPORA TE IA W SYMPOSIUM 1997] 199 a strong, private set of incentives that, in turn, encourages internal mechanisms of review and product redesign. See GUIDO CAiARESI, THE COST OF ACCIDENTS: A LEGAL AND ECONOMIC ANALYSIS (1970); see a/so Guido Calabresi, Frst Pary, Third Pary, and Product Liabilip Systems: Can Economic Analysis of Laz Tell UsAnythingAbou Than', 69 IOWA L. REv. 833 (1984); A. MrrCHELL POLINSKY, AN INTRODUCTION TO LAW AND ECONOMICS 97-106 (2d ed. 1989). Plaintiffs become private attorneys general. However, there are striking differences between the familiar example of the use of strict liability in the product liability setting, and the imposition of strict liability on Internet service providers. In product liability, the conventional range of reasons for imposing strict liability on manufacturers and sellers includes a number of assumptions. First, they are generally the cheapest cost avoiders; in other'words, they are best able to respond to liability for damage by making changes that could prevent the damage. Second, they are generally the best loss spreaders; in other words, they are best able to pass the cost of unavoidable or cost- justified damage on to the appropriate group: consumers of the good in question. Last, they are generally in an advantageous position in terms of knowledge and effective power-at least as compared to the relatively powerless individual consumer. See Escola v. Coca Cola Bottling Co., 150 P.2d 436, 440-43 (1944) (TraynorJ., concurring). owners and allow them to reap the benefits of the Internet without fully bearing its costs. 63. See 2JOHN AUSTIN, LECTURES ONJURISPRUDENCE 136 (5th ed. 1885). 64. Foucault, supra note 1, at 104. 65. See National Information Infrastructure Copyright Protection Act of 1995, S. 1284, 104th Cong. §§ 1201, 1202 (1995), H.R. 2441, 104th Cong. §§ 1201, 1202 (1995). 66. See World Intellectual Property Organization Treaty, Dec. 23, 1996, CRNR/DC/94 (visited June 26, 1997) <http://www.wipo.org/eng/diplconf/distrib/94dc.htm>; see also News from WIPO (visited June 26, 1997) <http://www.hrrc.org/wiponews.html> (detailing course of deliberations during the diplomatic conference). 67. See S. 1284, H.R. 2441, § 1203. V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES As a content provider, I can make a rational economic decision to sell my good across some cheap but "leaky" medium, which lowers my costs of advertising and distribution and increases the numbcr of unauthorized copies circulating. I may even believe that some of the unauthorized copies provide a benefit to me-making my word processing program a de facto standard in the industry or establishing my band as the best known and, therefore, increasing the market for future products. But let us leave aside the joys of pointing out that economic analysis depends on questions of interpretation that cannot themselves be decided according to economic criteria. There is, at the very least, strong reason to doubt that users of on- line services, rather than purchasers of the good in question, are the appropriate group over whom the costs of illicit copying should be spread. This would, in fact, actively undermine the competitive incentives to companies to develop their own anticopying methods. Finally, the asymmetry of power and knowledge that occurs when Mrs. McPherson confronts the Buick Motor Company is by no means as clear when Microsoft wants Netcom to do its cnrorcement work. For all of these reasons, imposing strict liability on Internet service providers does look rather different from imposing it on manufacturers ofdefective products. If there is an advantage to this scheme, that advantage redounds mainly to the content providers; such a plan would shift cnforcement costs fiom 200 [Vol.66 UNIVERSITY OF CLACINNATI LA WREVIEW With or without Foucault, however, thinking about the use of strict liability as an enforcement mechanism does illustrate the limitations of the Austinian view of the state's exercise of power. Unsurprisingly perhaps, Austin argued against strict liability and judges under the influence of Austinian reasoning, actually declared that strict liability was not true law.63 My central point here is not the undesirability of strict liability for on-line service providers, though the rationale, legal basis, and constitutionality of such a system seem doubtful to me. Rather, I think that the possible impact of a strict liability system on actual privacy, speech, and discourse, indicates another limitation of the jurisprudence of digital libertarianism. 67. See S. 1284, H.R. 2441, § 1203. V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES Once again, the focus on public, criminal, and sanction-backed acts by states exercising their power direcdy tends to obscure and, therefore, to undervalue the efficacy of efforts that rely on privatized enforcement and surveillance, cost spreading, and the use of "material coercions rather than the physical existence of a sovereign." 64 It is to the latter point that I now turn. One prong of the Clinton administration's plan for copyright on the Internet depended on enrolling private actors to act as enforcement agents in a way that sidestepped the rights, duties, and privileges between citizen and sovereign. The other prong depended on coating technological anticopying devices with the authority of the law in such a way as to change the relative powers of current copyright holders on the one hand, and their customers and future competitors on the other. The two most important provisions are the "circumvention of copyright protection systems" section and the "integrity of copyright management information" section of the NII Copyright Protection Act of 1995.65 Similar provisions were proposed by the United States during the WIPO conference.66 These two provisions seem, initially, to be entirely unobjectionable. The circumvention section imposes civil liability on importers, manufacturers, and distributors of devices the primary purpose or effect of which is to circumvent a copyright protection system. 67 The 67. See S. 1284, H.R. 2441, § 1203. CORPORA TE LA W SYMPOSIUM 1997] management section imposes civil and criminal liability on someone who removes or tampers with copyright management information.6 8 Obviously, technological protections are going to be an important means through which digital intellectual property is safeguarded; these technological protections will include, among other things, the kind of deeply embedded information that the management information section protects. Documents will keep track of how many times they are read and may complain if they are read too much or by the wrong person. Pamela Samuelson calls these texts that "rat" on you.69 Digital books sold to one person may be encoded so that they cannot be read by someone else on another computer. Given the possibility of documents that have the copyright details bound into in every packet of data, and that also check themselves to be sure that no alterations have been made, quotation may be perceived as alteration. 68. See S. 1284, H.R. 2441, § 1203, § 1204. 69. Pamela Samuelson, Widl the Cottghl Office be Obsokle in the TwenY-First Centuy?, 13 CARDOZO ARTS & ENT. LJ. 55, 58 n.18 (1994). 70. See Telecommunications Act of 1996, Pub. L. No. 104-104, § 551, 110 Stat. 56, 140 (to be codified at 47 U.S.C. § 303). 71. See Kristin S. Bums, Proecting the Child. The V-chip rovisions of the TecommunicatonsAct of 1996, 7 DEPAuL-LCAJ. ARTS & ENT. L. 143 (1996); David V. Scott, The V-chip Debate Bloching Television Sex, Vtolence, and the First Amendment, 16 LOy. L.A. ENT. L.J. 143 (1996). UNIVERSITY OF CINCINNA77 LIA WREVIEW [Vol.66 UNIVERSITY OF CINCINNA77 LIA WREVIEW [Vol.66 202 V. PRIVATIZED PANOPTICONS AND LEGALIZED ENCLOSURES (Presumably Internet service providers would also be encouraged to introduce some system of scanning that looked for altered or unauthorized packets of data.) The point about all of this is that there will be a continuing technological struggle between content providers, their customers, their competitors, and future creators. Obviously it will sometimes be in the interest of content providers to make it as difficult as possible for citizens to exercise their fair use rights. They will try to build technological and contractual fences around the material that they provide, not just to prevent its theft, but to prevent it from being used in ways that have not been paid for, even if those uses are privileged under current intellectual property law. They may want to stop their competitors from achieving interoperability, or prevent their customers from selling second-hand versions of their products. The technical means through which to do this can be thought of as digital fences. Sometimes those fences will be used to stop clear violations of existing rights. Sometimes they will be used to enclose the commons or the public domain. Thus, by making it illegal or impractical for me to go around, through, or over the fence, the state adds its imprimatur to an act of digital enclosure. The Intemet Trinity tells us that information wants to be free and that the thick fingers of Leviathan are too clumsy to hold it back. The position is less clear if that information is guarded by digital fences which themselves are backed by a state power maintained through private systems of surveillance and control. 72. See Howard S. Dakoff, Note, The Cloer Chip Proposal" Deciphering the Unfownded Fears that Are WronJlu/y Deraili its Imp/lenotation, 29J. MARSHALL L. REV. 475, 482-84 (1996) (discussing the use of the government's purchasing power to create a de facto encryption system); see also Richard L Field, 1996: Srvey of the rear's Developments in Electronic Cash Law and the Laws Affecting Eectronic Banking in the United States, 46 AM. U.L REV. 967, 993 (1997); Ira S. Rubenstein, Expof Contls on Enqbtion So.ftare 748 PIU/COMM 309 (1996); A. Michael Froomkin, The Metaphor is the Aey: Cqptography, the Clper Chip, and the Constitution, 143 U. PA. L REV. 709 (1995). 73. 47 U.S.C. § 1001-10 (Supp. 1994). VI. A COMMUNICATIONS SAMPLER The tendencies I have been describing here by no means end with the CDA and the NII Copyright Protection Act. In fact, the turn to privatized and technologically-based enforcement to avoid practical and constitutional obstacles seems to be the rule rather than the exception. p Outside of the Internet, the most obvious example of this is the V- chip, a device to enable parents to restrict television programming through a "voluntary" rating system. Although the rating system is voluntary, the device is mandated by § 551 of the Telecommunications Act of 1996.70 The V-chip decodes a set of ratings agreed to by private parties and suggested by a state-convened "private" board. It then blocks programming that is above a ratings threshold set by parents.7' The attractiveness of this hardwired mix of public and private decisions can be judged by the spread of V-chip analogies: President Clinton's "V-chip for the Intemet," and Representative Markey's "E-chip." Why is this device so popular, not just as a device, but as a rhetorical trope? The answer, I think, is partly provided by the characteristics outlined here. The V-chip seems to be merely a neutral facilitator of parental choice. The various acts of coercion involved-the government making the television company insert the thing into the machine, the public- private board choosing which ratings criteria will be available for parents to use--simply disappear into the background. Finally, the distributed privatized nature of the system promises that it might actually work; though admittedly, state administration of the television system poses fewer headaches than state administration of the Internet. Another set of examples is provided by encryption policy. In the digital era, encryption is no longer merely the stuff of spy novels. It provides the walls, the boundaries, and the ways of preventing unauthorized or unwanted entry. Faced with the development of a cryptography industry, which would produce digital walls unbreakable by the state, the government responded by attempting to legislate its own back door. The first proposal was that the encryption of all communications had to be made through a government designed device known as the "Clipper Chip." Your phone, fax, or computer system would encrypt your communication using the algorithm hardwired into CORPORA TE LA W S.MPOSIUM 1997] the Clipper Chip. VI. A COMMUNICATIONS SAMPLER The Clipper Chip utilizes a "key escrow" system under which the government maintains a "back door" key to decrypt all Clipper communications; a key which is supposed to be available only to law enforcement agencies who, most of the time, would have to get judicial approval of their actions. After considerable controversy, use of the Clipper Chip encryption system was declared voluntary for both the government and the private sector. This might seem to be a partial vindication for the digital libertarian position. In fact, however, the Clipper Chip project continues to have considerable influence on the domestic encryption industry because the government has, for the most part, adopted the Clipper Chip and has used its considerable purchasing power to make it a de facto industry standard.72 Although the success of this method may have been undermined by later technological development, the strategy shows the way in which a hardwired regime might be implemented by market power as well as legislative fiat. One of the arguments behind the Clipper Chip was that law enforcement agencies were merely striving to achieve the same level of physically permissible surveillance in a world of encoded transmissions as they currently possessed. With this as a baseline, it was obvious that the material possibility for interception and decryption should be hardwired into the system itself. The same argument was made successfully over digital telephony. Realizing that new telephony technology, such as call forwarding, cellular telephones, and digital communications in general, presented increasing challenges to wire tapping, Congress passed the Communications Assistance for Law Enforcement Act,73 more commonly known as the "Digital Telephony Act." At its heart, the Digital Telephony Act requires telecom- munications companies to make "tappability" a design criteria for the system. Everything recorded by the traditional "pen register" system, as well as a few new categories of information, must be digitally recorded. Under the Digital Telephony Act, information regarding a subscriber's name, address, telephone number, telephone toll billing UNIVERSITY OF CIMNCIX A TI L4 WREVIEW 204 [Vol.66 Vol.66 records, length of service, and the types of services utilized, are now available to the government. 4 , Technologically hardwired protections have also been implemented in order to protect intellectual property as in the Digital Audio Tape (DAT) standard. Unlike compact disks, which until recently were "read- only," digital audio tape technology allows users to make perfect copies of recordings. VI. A COMMUNICATIONS SAMPLER Fearing that this ability would lead to the development of an extensive market for copied tapes, the recording industry pushed for mandatory technological protection, which they received in the Audio Home Recording At of 1992."5 This Act requires all DAT recorders to utilize the "serial copy management system," which allows a first copy to be made onto DAT, but prevents all subsequent copies. These examples suggest a number of conclusions at odds with popular wisdom. Most obviously, they offer a cautionary note to the libertarian techno-optimists who believe that technology always grows free from governmental control and always moves in the direction of greater liberty. Let us lay aside many of the assumptions behind that belief for a moment, such as that governments are generally the greatest threat to daily liberty, or, conversely, that liberty should be defined primarily around the absence of governmental restraint. Even with these qualifications, the idea that the technological changes of the digital revolution are always outside the control of the state seems unproven. In fact, the state is working very hard to design its commands into the very technologies that, collectively, are supposed to spell its demise. Another point needs to be made; there are-whether one likes them or not--strong arguments that the "technologies of freedom" actually require an intensification of the mechanisms of surveillance, public and private, to which we are currently subjected. If the digital technologies enlarge our space for living, both conceptually and practically, the dangers posed by that expansion will prompt the demand-often the very reasonable demand-that the Panopticon be hardwired into the "technologies of freedom."7b 74. 18 U.S.C. § 2703(c)(1)(C) (1994). See Susan Friewald, Uncetain Privacy Communication Attributes Afler the Digital Telephony Act, 69 S. CAL. L. REV. 949 (1996). 75. 17 U.S.C. §§ 1001-1010(1994). 76. See generally ITHILL DE SOiA POOL, TECHNOLOGIES OF FREEDOM (1983). 76. See generally ITHILL DE SOiA POOL, TECHNOLOGIES OF FREEDOM (1983). 74. 18 U.S.C. § 2703(c)(1)(C) (1994). See Susan Friewald, Uncetain Privacy Communication Attributes Afler the Digital Telephony Act, 69 S. CAL. L. REV. 949 (1996). 75 17 U S C §§ 1001 1010(1994) VII. CONCLUSION Looked at in a vaguely Foucauldian light, these examples I have given in this Article seem to point to two conclusions, conclusions which may seem paradoxical. On the one hand, the studies indicate that the CORPORA TE LA W SYMPOSIUM 1997] 0 205 confident assumption that the state cannot regulate cyberspace is definitionally blind to some of the most important ways that some states could, in fact, exert power. The jurisprudence of digital libertarianism could use a lot less John Austin and a lot more Michel Foucault. But one cannot simply limit the analysis to the available avenues of state power. Foucault's Discipline and Punish was not a manual for state officials, but a challenge--in some ways similar to the challenges posed by legal realism and feminism--to the very categories of public and private and to the belief that power begins and ends with the state. p g We need a similar challenge to those categories in cyberspace. If the first conclusion of this study is that the state may actually have more power than the digerati believe, the second conclusion is that the attraction of technical solutions is that they apparently elide the question of power-both private and public-in the first place. The technology appears to be "just the way things are"; its origins are concealed, whether those origins lie in state-sponsored scheme or market-structured order, and its effects are obscured because it is hard to imagine the alternative. Above all, technical solutions are less contentious; we think of a legal regime as coercing, and a technological regime as merely shaping-or even actively facilitating-our choices. In the Lochner era, a strikingly similar contrast was drawn between the coercive nature of public law and the free private world of a market that was merely shaped by neutral, facilitative rules of contract and property. The legal realists did a remarkably good job of pointing out the shortcomings of that picture of the market. If we are to have some alternatives to the jurisprudence of digital libertarianism, we will have to offer a richer picture of Internet politics than that of the coercive (but impotent) state and the neutral and facilitative technology.
https://openalex.org/W2803791743	http://periodicos.ufc.br/rene/article/download/32483/pdf/	English	null	Social representations of imprisoned women on Acquired Immunodeficiency Syndrome	Rev Rene	2,018	cc-by	4,786	1Universidade Federal da Paraíba. João Pessoa, PB, Brazil. DOI: 10.15253/2175-6783.2018193280 www.revistarene.ufc.br Original Article DOI: 10.15253/2175-6783.2018193280 www.revistarene.ufc.br Corresponding author: Tatyana Ataíde Melo de Pinho Corresponding author: Tatyana Ataíde Melo de Pinho Rua Hercílio Alves de Sousa, 249, Bancários. CEP: 58051-290. João Pessoa, PB, Brazil. E-mail: tataide8@hotmail.com Received: Nov. 27st 2017; Accepted: Mar. 14st 2018. Rua Hercílio Alves de Sousa, 249, Bancários. CEP: 58051-290. João Pessoa, PB, Brazil. E-mail: tataide8@hotmail.com Introduction this population, due to the greater vulnerability of the prison environment, characterized by violence, mar ginalization and oppression(7-8). Acquired Immunodeficiency Syndrome (AIDS) emerged in Brazil in 1982 as a mysterious and feared phenomenon, mainly due to its lethality and disfigu ring body characteristics. Today, after more than three decades, it still represents a global and dynamic phe nomenon, highlighted among the infectious diseases that cause more deaths among women(1). Given this scenario, knowledge about HIV and AIDS is referred to in the literature as an important tool for adopting self-care practices(6,9), favoring pre vention and reduction of risk behavior(10), above all, in the prison population, whose deprivation of liberty reinforces the precariousness and fragility of the gua rantee of sexual rights, due to the bureaucratic and structural limitations existing in the prison establish ment(7-8). The epidemic has presented epidemiological and social changes over the last few years. It has ce ased to be a metropolitan disease that originated in the upper classes, which affected people from the ar tistic-cultural milieu and was restricted to risk groups (homosexuals, drug users and sex workers) to be considered an internal disease, with a popular origin, affecting any person and spreading to socially consi dered non-vulnerable individuals, such as heterosexu als, monogamous women, the elderly and children(2). The evidence of gaps in the knowledge of wo men deprived of their freedom in relation to AIDS en courages research that addresses dimensions related to the association between infection and imprison ment. Among the many dimensions, there is highlight to the identification of social representations about AIDS(6), the guarantee of sexual and reproductive ri ghts of these women(8), the analysis of individual, se xual, social and contextual conditions that contribute to the adoption of HIV risk behaviors(9-10), among other aspects, both nationally and internationally. It is noteworthy that women have become in creasingly vulnerable to this infection, accounting for half of those living with AIDS worldwide(3). In addi tion, the steady increase of the female imprisoned po pulation, reaching more than 10.35 million people in prisons around the world(3), has potentiated the vul nerability of contamination by the Human Immunode ficiency Virus (HIV). Representações sociais de mulheres em situação de cárcere sobre a Síndrome da Imunodeficiência Adquirida Tatyana Ataíde Melo de Pinho1, Antonia Oliveira Silva1, Cláudia Jeane Lopes Pimenta1, Maria Adelaide Silva Paredes Moreira1 Objective: to understand the social representations of imprisoned women on Acquired Immunodeficiency Syndrome. Methods: qualitative study conducted with 32 women imprisoned in a rehabilitation center. The Free Association of Words Test was used, with inductive stimuli such as “Human Immunodeficiency Virus” and “Acquired Immunodeficiency Syndrome”. Data were processed by the EVOC software and analyzed in light of the Theory of Social Representations. Results: the elements of the central nucleus of the representation were prevention, fear, death, disease and anguish; the periphery revealed negative aspects regarding the contents. Conclusion: there has been a process of modification in the social representations on the Acquired Immunodeficiency Syndrome with the incorporation of new elements such as the prevention and the reduction of the feeling of imminent death. g Descriptors: Acquired Immunodeficiency Syndrome; Women; Prisoners; Nursing. Objetivo: compreender as representações sociais de mulheres em situação de cárcere sobre a Síndrome da Imunodeficiência Adquirida. Métodos: estudo qualitativo, realizado com 32 mulheres sentenciadas em um centro de reabilitação. Utilizou-se o Teste de Associação de Livre de Palavras, tendo como estímulos indutores “Vírus da Imunodeficiência Humana” e “Síndrome da Imunodeficiência Adquirida”. Dados processados pelo software EVOC e analisados à luz da Teoria das Representações Sociais. Resultados: os elementos do núcleo central da representação foram prevenção, medo, morte, doença e angústia; a periferia revelou facetas negativas quanto aos conteúdos. Conclusão: observou-se início de um processo de modificação nas representações sociais sobre a Síndrome da Imunodeficiência Adquirida, tendo-se a incorporação de novos elementos, como a prevenção e a diminuição do sentimento de morte iminente. p ç ç Descritores: Síndrome de Imunodeficiência Adquirida; Mulheres; Prisioneiros; Enfermagem. Corresponding author: Tatyana Ataíde Melo de Pinho Received: Nov. 27st 2017; Accepted: Mar. 14st 2018. Rev Rene. 2018;19:e3280. Pinho TAM, Silva AO, Pimenta CJL, Moreira MASP Rev Rene. 2018;19:e3280. Methods tral Nucleus Theory, characterized as one of the three approaches of Theory of Social Representations, not limited to the explanatory scope of the process of for mation of representations, but with an emphasis on the cognitive-structural dimension(13). This softwa re provides the organization of the words produced, respecting the hierarchy presented by the frequencies (collective dimension) and mean order of evocation, which concerns the individual dimension, allowing the accomplishment of statistical calculations and the construction of matrices of competitions(12). This is a qualitative study carried out in a fe male penitentiary, in the city of João Pessoa, Paraíba, Brazil, between October and November 2014. The selection of the participants occurred in a non-pro babilistic way, by convenience, using as an inclusion criterion the serving of sentence in a closed system, corresponding to 32 women. Women who were in the process of progression from closed to semi-open regi me were excluded from the study. In the frame of four houses, the upper left qua drant, called the probable central nucleus, is formed by the words that presented the highest frequency and were more readily evoked, constituting the most stable and permanent part of the representation. In this case, the content is connected to the collective memory of the group, giving meaning to the repre sentation. The lower left quadrant, called the contrast zone, is formed by the words that obtained lower fre quencies and lower average order of evocation, de monstrating variations of centrality of representation for specific groups. The words located in this quadrant are the intermediate elements, which can approach the central nucleus or the peripheral elements(12). For data collection, a semi-structured questio nnaire was applied, addressing sociodemographic va riables and issues or images inherent to the field of representation, as well as knowledge or information and attitudes of women in deprivation of liberty re garding HIV and AIDS. The speeches of each subject were identified by the abbreviation “Sub.”, followed by an ordinal number corresponding to the interview order. The analysis of the speeches was based on the Theory of Social Representations, which seeks to identify the problem from the perspective of the in dividual, constituting a theoretical support used to implement and actualize health practices(11). This fra mework allows the understanding of the concepts or phenomena that subjects associate with the disease, which are responsible for the way empirical reality abstracts them and gives them meaning(12). Social representations of imprisoned women on Acquired Immunodeficiency Syndrome Social representations of imprisoned women on Acquired Immunodeficiency Syndrome Introduction The number of women deprived of their liberty living with HIV has increased over the years, thus, the re is a need for investments in sociocultural diagnoses of HIV infection and in prevention and care actions applied in a comprehensive, multisectoral and inte grated way, with the purpose of meeting the specific needs of this population(6). In this sense, it is essen tial to investigate AIDS from the perspective of these women, seeking to understand the ways in which the infection is represented in the prison environment. The relevance expressed in these data was extensively discussed in the Melbourne Declaration, which signaled changes and concerns in the outline of this infection, revealing the emergence of new vulne rable groups, among which are the women deprived of their liberty(3-5). In this sense, despite the efforts of the Brazilian Ministry of Health, HIV/AIDS prevention, diagnosis and care actions in the Brazilian prison sys tem are still not in accordance with the care practice provided to other population groups, which favors the perpetuation of the threatening and destructive image of the infection(6). Through this context, some questions have ari sen: what are the social representations about aids built by imprisoned women? What elements are pre sent in the central and peripheral systems of these re presentations? Therefore, the present study aimed to unders tand the social representations of imprisoned women on Acquired Immunodeficiency Syndrome. The strategies developed for women deprived of liberty must be differentiated, given the need for greater assistance aimed at promoting the health of Rev Rene. 2018;19:e3280. Social representations of imprisoned women on Acquired Immunodeficiency Syndrome Methods The upper right quadrant, called the first peri phery, contains words that present higher frequency and higher average order of evocation. The lower ri ght quadrant, called the second periphery, consists of words of low frequency and less readiness to evoca tion, which form the peripheral elements of represen tation. In this quadrant, there are less frequent and less important cognitions to determine the meaning of representation, but with more relation to social in sertion and more sensitive to the subjects’ immediate practices. These peripheral zones are unstable, allo wing variations of representations, as they express the representational elements associated with daily reality and social practices(12). The Free Association of Words Test was also used, which consists of a type of investigation that is structured from the evocation of answers given by the participants through one or more inductive stimuli, allowing to highlight the semantic universes related to a given object(13). The investigated women were asked to evoke five words that came to mind as they heard the inducing stimuli “Human Immunodeficiency Vi rus” and “Acquired Immune Deficiency Syndrome”. They were then asked to choose the evocation percei ved as the most important(12,14). The representations are organized around the central nucleus, which determines the meaning and the internal organization of the representation, having The data were organized in a specific database to be processed in EVOC software, based on the Cen Rev Rene. 2018;19:e3280. Pinho TAM, Silva AO, Pimenta CJL, Moreira MASP the functions of generating and organizing(12). The first function presents elements responsible for crea ting or transforming the content of the elements of the peripheral system; and the second function seeks to unify and stabilize representation. In this way, a chan ge of the elements that make up the central nucleus can trigger a transformation of the representation of the studied object. In addition to these two functions, there are still two dimensions related to the central nucleus: the functional, linked to the accomplishment of tasks, and the normative, which involves social, affective and ideological dimensions. The peripheral system presents a functional dimension, making it possible to represent the reality of the moment due to the flexibility that regulates and adapts the central system to a concrete situation(12). men, such as anguish, disease, fear, death and preven tion. Methods With regard to the intermediate elements, loca ted in the lower left quadrant, these were: prejudice, sentence and cure; and, in the upper right quadrant, the elements: care, depression, isolation and treat ment, indicating the range of meanings attributed to AIDS and that approach both to the centrality and to the periphery. In the lower right quadrant, called the second periphery or peripheral system, the following terms were found: contempt, pain, condom, poor and trans mission, which translated feelings and emotions expe rienced in the daily life of these women, expressing, in most cases, negative aspects related to suffering against the representational context. Prevention, anguish, disease, fear, and death were the probable central nucleus. The emotional factor is determined by the impending death linked to the disease. The element prevention converged to evocations of care and treatment. Fear appeared to be associated with the fear of falling ill, especially in pri son, due to multiple partners, sharing of objects such as nail pliers and sewing needles; there was also the fear of exclusion, of loneliness. Therefore, fear was linked not only to the risk of death, but also to a social stigma. The study complied with the formal require ments contained in the national and international regulatory standards for research involving human beings. Rev Rene. 2018;19:e3280. Discussion To interpret the results, we should consider the limitations related to the method used, due to the sub jectivity related to the qualitative approach. In the perspective of social representations, cognitions with functional dimensions were obser ved, associating HIV and AIDS with prevention, con doms, transmission and physical pain, as well as with normative dimensions related to death, fear, anguish, pain, social, contempt due to prejudice, mainly for fear of contagion(14). In the intermediate nucleus, the evocations related to the psychosocial contents were grouped, especially with prejudice and the relation of it with depression, isolation, the sentence, cure, care and the treatment. ...Most people discriminate against people with AIDS ... Nobody wants to be close to those who have AIDS; they feel disgusted by the sick person, have these attitudes of withdrawal because of the fear of getting the disease... (Sub. 15). The results of this study evidenced a process of modification in the social representations of AIDS, in corporating elements related to prevention, care and treatment of the disease, which represents evolution in the way the epidemic has been understood, althou gh there are frequent crystallized positions on the in fection and its association with death and suffering(6). Around the central nucleus, we found the pe riphery of the representation of AIDS, which focused mainly on negative feelings, which emerged from the process of thinking about the disease and its so cial consequences. The socio-cognitive contents of a psycho-sociological approach denoted a negative view of the disease, associated with poor, transmissi ble, pain, attitudes of contempt and isolation. This finding is relevant because it points to a representational change in the central system, as it evidences an attempt to overcome the representation of immediate death by the syndrome, seeking strate gies to cope with its condition in an attempt to adapt to the current context of life and health(15). Thus, the representation of AIDS brings to the fore the idea of prevention, however, there are still practices and risk behaviors, such as not using condoms in a relationship based on trust and strong bonds of affection(16). Also, the non-sharing of objects and the need to know the partner with whom they relate are the most used pre vention strategies used by the female prison popula tion(6). The elements that formed the periphery en compassed evocations whose contents linked to suffering were anchored in physical, biological and psychological dimensions. Results Regarding the profile of the participants, there was a prevalence of 75.0% in the age group between 20 and 30 years old, single (50.0%), with up to eight years of study (84.4%) and evangelicals (56.2%). Of the women interviewed, 53.1% were serving their sentences for less than a year, 87.5% declared them selves to be heterosexual, only 3.1% had received an intimate visit, and 90.6% had not used a condom. Negative sentiment domains were defined by the terms death, fear, disease and anguish; and a functional dimension linked to more operational pro cesses, showing proximity to the social object investi gated. This element consists of a set of practices and behaviors, such as the use of condoms, the non-sha ring of supposedly personal objects (pliers, needles, cutlery, sandpaper, dish, absorbent) and abstinence from relationships with suspects. The results showed 128 evocations with 41 di fferent words. The mean frequency was calculated at five and the rang was two and a half. The most fre quent elements were prevention, death, fear, treat ment, disease, depression, care, distress and isolation. The lowest frequencies were related to cure, sentence, condom, prejudice, contempt, pain, poor and trans mission. Thus, the prevention evoked by women see med to point to a representation of AIDS with mea ning generation and internal organization around the need for safe sex, dependent on the use of condoms and the non-sharing of personal objects, but with the disclaimer that trust in the other was still a reason for The first quadrant constituted the probable central nucleus represented by the imprisoned wo Rev Rene. 2018;19:e3280. Social representations of imprisoned women on Acquired Immunodeficiency Syndrome not adopting these practices. You can only get AIDS through sex without a condom, nail plier, blood, nee dle, razor, cutlery, glass ... Even if you have a husband, most of them have a woman in here and do not protect themselves during sex. They do not allow condoms to get in here ... I used it at the beginning, now that I know him, I no longer need to use a condom ... (Sub. 05). I’m married but I do not use a condom. I trust him, I’m already used to him, so I do not use it. The ones who have a naughty husband have to use a condom... (Sub. 15). Discussion Only those who have AIDS can avoid transmission to other people. So, it is very difficult here in pri son, because we live with a lot of people here and we do not know who has the disease. People with AIDS should have a special place, not to join with normal people. This is the only way to prevent and avoid transmission ... (Sub.01). Many people live in my cell and if anyone has AIDS, all the others will get it, so they should stay in a separate cell. I only sit in places when the warmth of the place disappears, I always expect it to cool in order to prevent AIDS. We can also get it by drinking water in the same glass, wearing the same clothes, in a hug, a kiss, in everything. We can know if a person has AIDS; just by looking at them, it is clear to see. The person becomes thin, disabled, with no hair... (Sub.25). Although the imprisoned women have attached to forms of prevention to represent AIDS, contents such as fear, death and anguish are still rooted in col lective emotions, being a product of social represen tations marked at the beginning of the epidemic, but still circulating in the scientific means, in mass com munication, in social media and in popular thought, generating prejudice, stigma and negative represen Rev Rene. 2018;19:e3280. Pinho TAM, Silva AO, Pimenta CJL, Moreira MASP tations(17-19). Moreover, such representations are also anchored in psychosocial dimensions, such as the fear of rejection and contempt that adds to the suffering due to the possibility of prejudice to which these wo men are exposed(2,19-20). Therefore, they often prefer to hide the disease as a form of social resistance not to be discriminated against it. Therefore, it is necessary to rethink ways of comba ting these aspects of the repercussion of the disease that are present in the social representations of AIDS, which are responsible for disseminating frightening meanings in relation to the epidemic, negatively in fluencing the implementation of actions and strate gies for preventing and controlling this injury(15-16). The participants of the present study presen ted scientifically proven knowledge about the forms of HIV transmission, such as sexual intercourse without the use of condoms, contaminated blood and contact with sharps, characterizing the reified universe of re presentations. Discussion On the other hand, they also pointed out elements revealing prejudice, listing items such as cutlery, glass, “seat warmth”, which demonstrates the consensual character of representations. In this sense, one can perceive the presence of negative connotation that is tied to a distorted re presentation, propagated and replicated over time, mainly by the media. The origin of this concept lies in the lack of scientific knowledge about the disease at the beginning of the epidemic, alluding to the re ferences that associated it with certain groups of the population, with attitudes and practices not accepted by society. This set of factors caused negative infor mation to be conveyed by the mass media, reflecting a stereotype of pejorative connotation in relation to the carriers and the possible risk groups, which generated fear in the population and increased prejudice(19). Similar data were evidenced by a study car ried out with 174 imprisoned women, which revea led the existence of social representations related to the beginning of the epidemic in the country, with the presence of misinformation about the disease, which supports discriminatory and prejudiced attitudes(6). Although they recognize the importance of preven tion, women still perceive the infection as an evil that relates to the other, not perceiving themselves at risk. This problem becomes more visible when they main tain stable relationships, with confidence between the partners, since many expressed the lack of the need to use the condom. In the case of women deprived of their liberty, such representations are directly and indirectly in fluenced by the presence of barriers generated by the prison system itself, such as illicit drug use, deficiency of the social support system, high incidence of mental problems and presence of a history of sexual work and physical, sexual, psychological and emotional abuse by intimate partners(6). Associated with this, there are obstacles related to the liberation and resocialization of these women in the community, such as increased sexual risk, increased use of illicit substances and high rates of recidivism(9). It is important to highlight that the “monoga mous relationship” was considered by the partici pants as a form of prevention. This brings up the is sues of trust and fidelity, since, because they believe they are in a stable relationship, they are not at risk of acquiring the infection. Rev Rene. 2018;19:e3280. References prison condition itself, the physical inadequacy of the internal and external spaces, the high population den sity, the precarious hygiene, health and nutrition and the non-availability of condoms(17). 1. Lima TC, Freitas MIP. Caracterização de população com 50 anos ou mais atendida em serviço de referência em HIV/AIDS. Rev Ciênc Méd [Internet]. 2013 [citado 2017 nov. 13]; 22(2):77-86. Disponível em: http://periodicos.puc-campinas. edu.br/seer/index.php/cienciasmedicas/article/ view/2129/1779 1. Lima TC, Freitas MIP. Caracterização de população com 50 anos ou mais atendida em serviço de referência em HIV/AIDS. Rev Ciênc Méd [Internet]. 2013 [citado 2017 nov. 13]; 22(2):77-86. Disponível em: http://periodicos.puc-campinas. edu.br/seer/index.php/cienciasmedicas/article/ view/2129/1779 A study of 1,369 men and women incarce rated in New York City identified that a significant proportion of the prisoners were sexually active and that most of them were not in the marital visitation program. This refers to the presence of sexual rela tionships within the prison environment, which oc curred without the use of condoms, since they were not available to the prison population(10). 2. Oliveira DC. Construction and transformation of social representations of aids and implications for health care. Rev Latino-Am Enfermagem. 2013; 21(esp):276-86. doi: http://dx.doi.org/10.1590/ S0104-11692013000700034 Still today, people perceive HIV infection as if it was due to risky behavior, failing to consider that the modes of transmission of a disease often have more social than individual reasons. Therefore, these issues are not solved by seeking to blame people for actions taken or by labeling individuals as models of behavior, but by understanding the motives that condition people and groups to be more vulnerable to HIV infection(9). 3. Joint United Nations Programme on HIV/AIDS (UNAIDS). Ending AIDS: Progress Towards the 90 – 90 – 90 Targets [Internet]. 2017 [cited 2017 Nov. 13]. Available from: http://www.unaids.org/ sites/default/files/media_asset/Global_AIDS_ update_2017_en.pdf 3. Joint United Nations Programme on HIV/AIDS (UNAIDS). Ending AIDS: Progress Towards the 90 – 90 – 90 Targets [Internet]. 2017 [cited 2017 Nov. 13]. Available from: http://www.unaids.org/ sites/default/files/media_asset/Global_AIDS_ update_2017_en.pdf 4. Walmsley R. World female imprisonment list: women and girls in penal institutions, including pre-trial detainees/remand prisioners [Internet]. 2017 [cited 2017 Nov. 13]. Available from: http:// www.prisonstudies.org/sites/default/files/ resources/downloads/world_female_prison_4th_ edn_v4_web.pdf 4. Walmsley R. World female imprisonment list: women and girls in penal institutions, including pre-trial detainees/remand prisioners [Internet]. 2017 [cited 2017 Nov. 13]. Available from: http:// www.prisonstudies.org/sites/default/files/ resources/downloads/world_female_prison_4th_ edn_v4_web.pdf Conclusion This study evidenced the beginning of a process of modification in the social representations of AIDS, incorporating new elements, such as prevention and reduction of feeling of imminent death. However, the disease still presents a threatening and destructive image, which increases prejudice and stigmatization of the carriers. 5. 5. Seffner F, Parker R. The waste of experience and precariousness of life: contemporary political moment of the Brazilian response to aids. Interface. 2016; 20(57):293-304. doi: http:// dx.doi.org/10.1590/1807-57622015.0459 6. Trigueiro DRSG, Almeida SA, Monroe AA, Costa GPO, Bezerra VP, Nogueira JA. AIDS and jail: social representations of women in freedom deprivation situacions. Rev Esc Enferm USP. 2016; 50(4):554- 61.doi:http://dx.doi.org/10.1590/S0080- 623420160000500003 Discussion In addition, many women are uncomfortable in suggesting condom use for their partner in long-term relationships, as their use is still tied to the idea of betrayal, mistrust and promiscui ty(19). Even with the advances in treatments and the increase in the life expectancy of people living with AIDS, the physical and social suffering inherent to the disease has not changed, that is, intolerance, fear, discrimination and prejudice are still present in the current society, intensified by idealizations on the in fection, which are still frequent in the imaginary of a large part of the population(2). In the case of sexual intercourse occurring wi thin the prison environment, there are other factors increasing the risk of HIV transmission, such as the Social stigma affects the quality of life of wo men, especially in situations of deprivation of liberty. Social representations of imprisoned women on Acquired Immunodeficiency Syndrome Social representations of imprisoned women on Acquired Immunodeficiency Syndrome Collaborations 7. Lopes TC, Pinheiro R. Trajetórias de mulheres privadas de liberdade: práticas de cuidado no reconhecimento do direito à saúde do Centro de Referência de Gestantes de Minas Gerais. Physis. 2016; 26(4):1193-212. doi: http://dx.doi. org/10.1590/s0103-73312016000400007 7. Lopes TC, Pinheiro R. Trajetórias de mulheres privadas de liberdade: práticas de cuidado no reconhecimento do direito à saúde do Centro de Referência de Gestantes de Minas Gerais. Physis. 2016; 26(4):1193-212. doi: http://dx.doi. org/10.1590/s0103-73312016000400007 Pinho TAM contributed to the design concep tion and design, analysis and interpretation of data. Pimenta CJL collaborated with the writing of the ar ticle. Silva AO and Moreira MASP contributed to the critical review of the intellectual content and final ap proval of the version to be published. Rev Rene. 2018;19:e3280. Pinho TAM, Silva AO, Pimenta CJL, Moreira MASP 8. Costa LHR, Alves, JP, Fonseca CEP, Costa FM, Fonseca FF. Gender in the context of sexual and reproductive rights of women deprived of liberty. Enferm Glob [Internet]. 2016 [cited 2017 Nov. 13]; 43:151-63. Available from: http://revistas. um.es/eglobal/article/view/207141/194671 15. Hipolito RL, Oliveira DC, Gomes AMT. Conteúdos simbólicos da qualidade e vida gerados por pessoas que vivem com HIV/aids. Atas Cia [Internet]. 2015 [citado 2017 nov 13]; 484-6. Disponível em: http://proceedings.ciaiq.org/ index.php/ciaiq2015/article/viewFile/111/107 16. Pimenta CJL, Costa IP, Farias MCAD, Costa KNFM, Pereira TLB, Leite ES, et al. HIV/AIDS and seniors: knowledge of elderly about the disease. Int Arc Med. 2016; 9(20):1-7. doi: http://dx.doi. org/10.3823/1891 9. Fogel CI, Gelaude DJ, Carry M, Herbst JH, Parker S, Scheyette A et al. Context of risk for HIV and sexually transmitted infections among incarcerated women in the south: individual, interpersonal, and societal factors. Women Health. 2014; 54(8):694-711. doi: http://dx.doi.org/10.1 080/03630242.2014.932888 17. Pinheiro PNC, Freitas CM, Scopacasa LF, Silva KL, Gubert FA, Luna IT. Adolescent with Human Immunodeficiency Virus - understanding family beliefs and values. Rev Rene. 2017; 18(4):507- 14. doi: http://dx.doi.org/10.15253/2175- 6783.2017000400012 10. Rowell-Cunsolo TL, Szeto B, Sampong SA, Larson EL. Predictors of sexual behavior among men and women in New York City area prisons. Cult Health Sex. 2016; 8:1-14. doi: http://dx.doi.org/10.1080 /13691058.2016.1188420 18. Faria JO, Silva GA. Nursing diagnoses in persons with HIV/aids: an approach based on Horta’s conceptual model. Rev Rene [Internet]. 2013 [cited 2017 Nov. 13]; 14(2):290-300. Available from:http://www.revistarene.ufc.br/revista/ index.php/revista/article/view/821 11. Moscovici F. Representações sociais: investigações em psicologia social. Petrópolis: Vozes; 2012. 12. Moscovici F. O fenômeno das representações sociais. In: Moscovici S. Representações sociais. Petrópolis: Vozes; 2003. p.29-109. 19. 14. Bardin L. Análise de conteúdo. São Paulo: Edições 70; 2011. Collaborations Pimenta CJL, Costa IP, Leite ES, Farias MCAD, Barbosa TL, Maciel EJS, et al. Understanding of the Elderly Attached to the Family Health Strategy about the HIV/AIDS Infection. Int Arc Med. 2015; 8(241):1-7. doi: http://dx.doi.org/10.3823/1840 13. Rosa AS. A rede associativa: uma técnica para captar a estrutura, os conteúdos, e os índices de polaridade, neutralidade e estereotipia dos campos semânticos relacionados com as representações sociais. In: Moreira ASP, Camargo BV, Jesuíno JC, Nóbrega SM, editoras. Perspectivas teórico-metodológicas em representações sociais. João Pessoa: Universitária UFPB; 2005. p. 61-127. 20. Strauss M, Rhodes B, George G. A qualitative analysis of the barriers and facilitators of HIV counseling and testing perceived by adolescents in South Africa. BMC Health Serv Res. 2015; 15:250. doi: http://doi.org/10.1186/s12913-015-0922-0 14. Bardin L. Análise de conteúdo. São Paulo: Edições 70; 2011. Rev Rene. 2018;19:e3280.
https://openalex.org/W2118619151	https://bmcmedicine.biomedcentral.com/counter/pdf/10.1186/1741-7015-7-66	English	null	Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease	BMC medicine	2,009	cc-by	12,437	BioMed Central BioMed Central Open Acc Research article Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease Alessandra Pani1, Antonella Mandas2, Giacomo Diaz1, Claudia Abete1, Pier Luigi Cocco1, Fabrizio Angius1, Annalisa Brundu2, Nico Muçaka2, Maria Elena Pais2, Antonio Saba2, Luigi Barberini3, Cristina Zaru1,4, Manuela Palmas1,4, Paolo F Putzu4, Alessandra Mocali5, Francesco Paoletti5, Paolo La Colla1 and Sandra Dessì2 Address: 1Department of Biomedical Sciences and Technologies, University of Cagliari, Italy, 2Department of Internal Medical Science, University of Cagliari, Italy, 3Department of Cardiovascular and Neurological Science, University of Cagliari, Italy, 4Alzheimer Center, ASL 8, Cagliari, Italy and 5Department of Experimental Pathology and Oncology, University of Florence, Italy Email: Alessandra Pani - pania@unica.it; Antonella Mandas - amandas@medicina.unica.it; Giacomo Diaz - gdiaz@unica.it; Claudia Abete - claudia.abete@gmail.com; Pier Luigi Cocco - pgcox@tiscali.it; Fabrizio Angius - fangius@unica.it; Annalisa Brundu - annabrundu@omeca.it; Nico Muçaka - nico.mucaka@tiscali.it; Maria Elena Pais - mariaelenapais@libero.it; Antonio Saba - anto.sab@alice.it; Luigi Barberini - barberini@unica.it; Cristina Zaru - czaru@tiscali.it; Manuela Palmas - manuela.palmas@tiscali.it; Paolo F Putzu - p.putzu@tin.it; Alessandra Mocali - alessandra.mocali@unifi.it; Francesco Paoletti - francesco.paoletti@unifi.it; Paolo La Colla - placolla@unica.it; Sandra Dessì - sdessi@unica.it * C di h uela Palmas - manuela.palmas@tiscali.it; Paolo F Putzu - p.putzu@tin.it; Alessandra Mocali - alessandra.mocali@un esco Paoletti - francesco.paoletti@unifi.it; Paolo La Colla - placolla@unica.it; Sandra Dessì* - sdessi@unica.it * Corresponding author Published: 2 November 2009 BMC Medicine 2009, 7:66 doi:10.1186/1741-7015-7-66 Received: 27 July 2009 Accepted: 2 November 2009 This article is available from: http://www.biomedcentral.com/1741-7015/7/66 © 2009 Pani 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. Received: 27 July 2009 Accepted: 2 November 2009 © 2009 Pani 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. BMC Medicine Open Access Background temic metabolic dysfunction that beyond the brain affects also other tissues including dermal fibroblasts, which have often been employed as an in vitro model for neuro- logical diseases, particularly AD [11,12]. Indeed, we have recently reported that skin fibroblasts from patients with diagnosis of probable sporadic AD display an imbalance between free cholesterol (FC) and cholesterol esters (CEs) pools to suggest that increased CE levels in these cells may represent an additional peripheral indicator of disease [13,14]. g Alzheimer's disease (AD) is the most common progressive neurodegenerative disease affecting millions of people worldwide. The AD brain is marked by severe neurode- generation like the loss of synapses and neurons, atrophy and depletion of neurotransmitter systems in the hippoc- ampus and cerebral cortex [1,2]. Although we still do not know what starts the AD process, we do know that dam- age to the brain begins as many as 10 to 20 years before the symptoms. To date, AD diagnosis during life, and par- ticularly of early stages of disease, is based on evaluation of multiple parameters obtained through neuropsycho- logical testing [3], conventional imaging [4] as well as the exclusion of other neuropathologies by means of elabo- rate, serial and expensive procedures. Numerous progresses in the discovery, validation, and standardiza- tion of molecular biomarkers in brain or biological fluids of aid in diagnosis at different stages of AD and in the assessment of disease progression, have recently been made [5-7]. In particular, the analysis of amyloid beta- peptides (Aβ) in the liquor and the advancements in func- tional neuroimaging [8,9] have definitely improved the accuracy of AD diagnosis; however, it is worth saying that these procedures are available only in the best academic centres and for a limited number of patients. On the con- trary, in non-specialized hospital units there are several problems to make accurate differential diagnosis of AD over a large number of subjects and also assess whether a mild cognitive impairment (MCI) might reveal early stages of disease or should rather be linked to normal aging. Therefore, a first-line test, even though not as spe- cific as those mentioned above, yet easy to be performed and denoting systemic metabolic alterations would be an useful tool for basic and clinical AD research. These metabolic alterations in dermal fibroblasts prompted us to further investigate cholesterol metabo- lism in freshly isolated (ex vivo) peripheral blood mono- nuclear cells (PBMCs). Abstract Background: Alzheimer's disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimer's disease molecular biomarkers in brain as well as in biological fluids have been made. Among them, those involving lipid metabolism are emerging as potential candidates. In particular, an accumulation of neutral lipids was recently found by us in skin fibroblasts from Alzheimer's disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimer's disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimer's disease patients and from their first-degree relatives. Methods: Blood samples were obtained from 93 patients with probable Alzheimer's disease and from 91 of their first-degree relatives. As controls we utilized 57, cognitively normal, over-65 year- old volunteers and 113 blood donors aged 21-66 years, respectively. Data are reported as mean ± standard error. Statistical calculations were performed using the statistical analysis software Origin 8.0 version. Data analysis was done using the Student t-test and the Pearson test. Results: Data reported here show high neutral lipid levels and increased ACAT-1 protein in about 85% of peripheral blood mononuclear cells freshly isolated (ex vivo) from patients with probable sporadic Alzheimer's disease compared to about 7% of cognitively normal age-matched controls. Page 1 of 12 (page number not for citation purposes) BMC Medicine 2009, 7:66 http://www.biomedcentral.com/1741-7015/7/66 http://www.biomedcentral.com/1741-7015/7/66 A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimer's disease blood samples was also observed. Additionally, correlation analyses reveal a negative correlation between high density lipoprotein-cholesterol and cognitive capacity, as determined by Mini Mental State Examination, as well as between high density lipoprotein-cholesterol and neutral lipid accumulation. We observed great variability in the neutral lipid-peripheral blood mononuclear cells data and in plasma lipid analysis of the subjects enrolled as Alzheimer's disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimer's disease patients. Conclusion: We suggest that neutral lipid-peripheral blood mononuclear cells and plasma high density lipoprotein-cholesterol determinations might be of interest to outline a distinctive metabolic profile applying to both Alzheimer's disease patients and asymptomatic subjects at higher risk of disease. Page 2 of 12 (page number not for citation purposes) Background These cells are an ideal source for investigation due to their critical roles in immune response, metabolism, and communication with other cells and extracellular matrices almost everywhere in the human body, as well as for their ease of collection [15]. Moreover, starting from the notion that family history of AD-like dementia is considered a high-risk factor for the development of this disease [16,17] and with the aim to identify sensitive metabolic traits in support to diagnosis of probable AD, we have analyzed lipid metabolism in freshly isolated (ex vivo) PBMCs, and in plasma of AD patients and of their cognitively normal first degree rela- tives (AD-FDR). As controls, we utilized cognitively normal over-65-year- old subjects (control 1) and blood donors aged 21-66 years (control 2). High levels of neutral lipids (NL), mainly CEs, in PBMCs and low high density lipoprotein- cholesterol (HDL-C) levels in plasma have been found in about 90% of patients with probable sporadic AD exam- ined. As expected, a great variability in the NL-PBMCs data and in plasma lipid analysis of the subjects enrolled as AD-FDR was found; however, about 30% of them dis- played a peripheral metabolic cholesterol pattern similar to that of the AD patient in each one of their families. Recently, an interesting blood test for early AD diagnosis based on the expression of eighteen signaling plasma pro- teins was reported [10]. Waiting for the assay validation by other research groups, these findings lend further sup- port to the hypothesis that AD patients suffer from a sys- Page 2 of 12 (page number not for citation purposes) BMC Medicine 2009, 7:66 http://www.biomedcentral.com/1741-7015/7/66 or over-weight subjects have intentionally been excluded from the present study. Methods Participants p Blood samples were obtained from patients with possible or probable AD and from their FDR (see data reported in Table 1 and Table 2, respectively). Subjects were enrolled at the Alzheimer Center, USL 8, Cagliari, and at the Geri- atric Unit of the University Hospital, Cagliari (Italy) fol- lowing informed written consent from all individuals or, when necessary, from their legal guardians, under local institutional review board supervision and with approval by local Ethical Committees. Routine clinical and labora- tory evaluation, including magnetic resonance imaging, was performed to exclude other causes of cognitive impairment. Patient evaluation included medical history, physical and neurological examinations, and laboratory blood tests to rule out metabolic causes of dementia (thy- roid hormones, Vitamin B12, etc.), and neuroimaging (computer tomography and/or magnetic resonance) of the brain. In addition, all patients underwent neuropsy- chological tests, including the clinical dementia rating scale (CDR), the global deterioration scale (GDS), the Clock Drawing Test (CDT) and the Mini-Mental State Examination (MMSE) corrected for degree of schooling and age. Patients had CDR score 3-5, GDS score 6-7, CDT score < 12, and MMSE score < 24. Patients with neoplastic or hematological disorders, recent infections, severe hepatic or renal failure, myocardial infarction or cranial trauma in the previous six months, or who had received statins, antineoplastic, corticosteroid, or immunosuppres- sive drug treatment or surgery were not included in the present study. Additionally, 170 individuals: 57 volun- teers (control 1) aged between 66-87 years, with no cogni- tive impairment, as established by clinical interview and by a normal CDR, GDS and MMSE score, and 113 blood donors (control 2), enrolled at the Transfusion Center, USL7, Iglesias, Italy, aged between 20-66 years (table 2), with no personal or family history of neurological or psy- chiatric disorders, served as controls. Given the influence of malnutrition on cholesterol metabolism, either under- [3H]Thymidine-incorporation studies At the time points considered, cells were incubated in trip- licate for 6 h with [3H]thymidine (5 μCi/ml). After incu- bation, the cells were rinsed twice with phosphate buffered saline (PBS), and DNA was precipitated by add- ing 5% trichloroacetic acid at 4°C. Cells were digested with 1 M NaOH at room temperature. Aliquots were assayed for [3H]thymidine radioactivity and protein con- tent. Isolation of PBMCs Blood samples were centrifuged at 2200 rpm for 15 min to separate plasma. Plasma was removed, transferred to centrifuge tubes and stored at -80°C until analysis. The buffy coat was collected and peripheral blood mononu- clear cells (PBMCs) were isolated by density gradient cen- trifugation (Lymphoprep; density, 1.077 g/L; Nycomed Pharma, Oslo, Norway) at 1200 rpm for 10 minutes at 20°C, and washed twice with Hanks balanced salt solu- tion (HBSS). These freshly isolated (ex vivo) PBMCs were utilized for the experiments. Where indicated, purified PBMCs were seeded onto 24-well plates at 1.0 × 104/mL in RPMI 1640 medium with the standard supplements: (10% fetal calf serum (FCS), 200 mM L-glutamine, and 100 U/mL penicillin-streptomycin) and growth-stimu- lated with 10 μg/ml phytohemagglutinin (PHA; Sigma) for 24, 48 and 72 hours. The trypan blue exclusion test was used to assess cell viability. Neutral lipid staining p g To visualize the degree of cytoplasmic neutral lipid accu- mulation, freshly isolated PBMCs were washed three times with PBS, and fixed by soaking in 10% formalin. Cells were then treated with isopropyl alcohol (60%), washed, and stained with oil red O (ORO) (a lipid-solu- ble dye which stains NL, including CE, but not FC). They appear as bright red spots in the cytoplasm, and are then counterstained with Mayer's hematoxylin. After staining, cells were imaged using a Leitz inverted-phase microscope fitted with a digital camera. At least two different fields per sample were imaged and analyzed. The red intensity was scored on a semi-quantitative scale (from 0 to 4) by two blinded observers: 0 indicated no staining; 1, rare positive cells or staining barely visible at low power (×200); 2, focal staining or faint diffuse staining clearly visible at low power; 3, multifocal staining or moderate diffuse staining; and 4, intense diffuse staining. There was significantly high correlation between the scores of the two observers (r2 = 0.96; P = 0.000). For convenience, scores from only one observer were used for cell mean-score calculation. In some experiments ORO intensity was determined using Image J software (National Institutes of Health, United States). Red intensity was expressed as mean pixels ± SE/ We initially determined the content of NL in ex vivo PBMCs from AD patients and age-matched controls (con- trol 1) by staining cells with Oil Red O (ORO), a lyso- chrome fat-soluble dye widely used for demonstrating the presence of NL (CE and TG), which appear as bright red spots in the cytoplasm, as described above, in Materials and Methods. Cell mean ORO ± SE are reported in Table 1. As shown in Table 3, NL in control 1 PBMCs were gen- erally absent (score 0) (49% female and 55% male) or very low (score 1) (38% female and 40% male) to indicate that intracellular lipids in the elderly are maintained at low levels, possibly by a cyclic process, as originally reported by Goldstein and Brown [18]. By contrast, NL concentration in AD cells was high, since the majority of AD-PBMCs (about 70% female and 81% male) presented ORO staining levels with a score from 2 to 4. Cellular lipid synthesis and efflux To determine the rate of lipid synthesis, PBMCs were incu- bated for 6 h with 5 μCi/ml sodium [14C]acetate (DuPont ± NEN; specific radioactivity 50 mCi/mmol). At 6, 18 and 36 h of incubation, cells were harvested, washed with PBS, and total lipids (TL) in cells and medium extracted with cold acetone. Lipid subclasses (FC, CE and triglycerides Table 1: Characteristics of control 1 subjects and Alzheimer's disease patients. Control 1 AD Total Female Male Total Female Male Number 57 37 20 93 67 26 Age (mean ± SE) 77.2 ± 0.8 77.0 ± 1.0 77.5 ± 1.3 77.2 ± 0.7 77.9 ± 0.9 77.1 ± 1.3 t-test (C1 vs AD) P = 0.944 P = 0.534 P = 0.206 MMSE 28.2 ± 0.2 27.9 ± 1.3 28.6 ± 0.3 18.7 ± 0.6 18.9 ± 0.6 18.3 ± 1.3 t-test (C1 vs AD) P = 0.000 P = 0.000 P = 0.000 ORO (mean ± SE) 0.6 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 2.6 ± 0.1 2.7 ± 0.1 2.5 ± 0.2 t-test (C1 vs AD) P = 0.000 P = 0.000 P = 0.000 MMSE = Mini Mental State Examination AD = Alzheimer's Disease ORO = Oil Red O Table 1: Characteristics of control 1 subjects and Alzheimer's disease patients. http://www.biomedcentral.com/1741-7015/7/66 BMC Medicine 2009, 7:66 Table 2: Characteristics of control 2 and Alzheimer's disease-First-degree relatives subjects. Control 2 AD-FDR Total Female Male Total Female Male Number 113 30 83 91 66 25 Age (mean ± SE) 44.7 ± 0.9 42.2 ± 1.7 45.6 ± 1.1 46.6 ± 1.4 45.5 ± 1.4 47.3 ± 2.6 t-test (C2 vs. AD-FDR) P = 0.229 P = 0.174 P = 0.480 ORO (mean ± SE) 0.5 ± 0.1 0.7 ± 0.1 0.4 ± 0.1 1.4 ± 0.1 1.5 ± 0.1 1.5 ± 0.2 t-test (C2 vs. AD-FDR) P = 0.000 P = 0.027 P = 0.000 AD-FDR = Alzheimer's Disease - First Degree Relatives ORO = Oil Red O Table 2: Characteristics of control 2 and Alzheimer's disease-First-degree relatives subjects. AD-FDR = Alzheimer's Disease - First Degree Relatives ORO = Oil Red O AD-FDR = Alzheimer's Disease - First Degree Relatives ORO = Oil Red O cm2 of triplicate wells and obtained by utilizing four dif- ferent selected regions of interest (ROIs). Cellular lipid synthesis and efflux [TG]) were separated by thin layer chromatography (TLC TLC on kiesegel plates (Merck, Darmstadt, Germany) using a solvent system containing n-heptane/isopropyl ether/formic acid (60:40: 2, by vol.) and the incorpora- tion of [14C]acetate into these different lipid fractions in cells and medium was measured in a Liquid Scintillation Counter. Efflux of [14C]TL from the cells into the medium at each time point was expressed as the percentage of the radioactivity recovered in medium/total radioactivity (cells + medium). Lipid profiles in freshly isolated PBMCs from AD patients and elderly controls Characteristics of 93 AD patients (mean age 77.2 ± 0.7, 67 female and 26 male) and 57 controls (mean age 77.2 ± 0.8, 37 female and 20 male) enrolled for this study are given in Table 1. Statistical analysis Data are reported as mean ± standard error (SE). Statistical calculations were performed using the statistical analysis software Origin 8.0 version (Microcal, Inc, Northampton, MA, USA). Data analysis was done using the Student t-test and the Pearson test. A value of P < 0.05 was considered to be statistically significant. Results Total cholesterol (TC) and HDL-C content was deter- mined in plasma by using routine colorimetric enzymatic procedures (Sclavo Diagnostics International S.r.l. Sovi- cille, Italy). Page 4 of 12 (page number not for citation purposes) Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients In previous studies we demonstrated that, in normal PHA- stimulated PBMCs, CE synthesis increases at least 12 hours before the start of DNA synthesis, indicating that cholesterol esterification is involved in the regulation of cell-cycle progression in these cells [19-22]. In the current study we quantified DNA synthesis and NL in PHA-stim- ulated control 1 and AD PBMCs. DNA synthesis was measured by 3H-thymidine incorporation in the two cell groups cultured in parallel under identical conditions. In PHA-stimulated control 1 cells, DNA synthesis increased at 48 h of culture, reached a peak at 72 h and then declined. In AD PBMCs, increased incorporation of thy- midine into DNA was already detected 24 h after PHA addition, reaching a peak at 48 h, indicating that PBMCs Changes in 3H-thymidine incorporation were associated with parallel changes in the content of NL. Figure 2A shows ORO images of PHA-stimulated PBMCs from one AD patient (85 years old) and one control 1 (84 years old), representing 20 AD and 20 control 1, respectively. In AD PBMCs, ORO staining was even evident at 0 h, and steadily increased at 48-72 h. Conversely, control 1 cells became ORO-positive at 48 h after PHA stimulation, and still were increasing at 72 h (Figure 2B). Also of interest is that cluster formation in AD PBMCs, indicative of cell activation, is even evident before PHA-stimulation (0 h), while it became manifest in control 1 only at 48 h after stimulation. Table 3: Distribution of ORO grading in control 1 subjects and Alzheimer's disease patients. Score Control 1 AD Total Female Male Total Female Male 0 29 18 11 1 1 0 1 22 14 8 13 8 5 2 6 5 1 21 14 7 3 0 0 0 41 32 9 4 0 0 0 17 12 5 total 57 37 20 93 67 26 ORO = Oil Red O AD = Alzheimer's Disease Plasma lipid profile in control 1 subjects and AD patients We next measured TC and HDL-C levels in plasma col- lected from each blood sample after PBMCs separation. As shown in Figure 3, the levels of HDL-C in AD patients were significantly reduced, in both absolute and relative (% HDL-C/TC) terms. TC was moderately lower in AD patients as compared to subjects of control 1, but this was not statistically significant. Neutral lipid staining The intensity of ORO staining in PBMCs positively and significantly correlated with the severity of the cognitive alteration, as determined by the MMSE scores in the AD population (r = -0.419; P = 0.000) (Figure 1). No significant correlation between MMSE and ORO staining was found in control 1 (r = 0.105; P = 0.435). Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1741-7015/7/66 BMC Medicine 2009, 7:66 MMSE vs ORO intensity in PBMCs from control 1 and AD Figure 1 MMSE vs ORO intensity in PBMCs from control 1 and AD. Pearson correlation test revealed a significant inverse cor- relation between these variables in AD patients but not in control 1. MMSE vs ORO intensity in PBMCs from control 1 and AD Figure 1 MMSE vs ORO intensity in PBMCs from control 1 and AD. Pearson correlation test revealed a significant inverse cor- relation between these variables in AD patients but not in control 1. freshly isolated from AD patients grow at a faster rate than those from control 1 (data not shown). Page 5 of 12 (page number not for citation purposes) Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two different fields per sample were imaged. Red ORO inten- sity was measured in these two fields using NIH Image J soft- ware. Panel A shows ORO staining images for one control 1 (84 years old; ORO score 0 at 0 time) and one AD (85 years old; ORO score 3 at 0 time), which are representative of about thirty subjects for each group. Panel B shows red intensity expressed as mean pixels ± SE/cm2, as described in Materials and Methods. P < 0.01, compared with control 1 by Student's t test. ! " # $ % & ' ( ) + , ' - . / ( + 0 1 & 2 ) + 0 % 3 4 % 3 + ' % 2 5 / 6 7 8 9 9 : ; 8 < = > ? @ A B C > D E = ? D Plasma lipid profile in control 1subjects and AD patients Figure 3 Plasma lipid profile in control 1subjects and AD patients. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.116; HDL P = 0.000; %HDL/TC P = 0.002; TC female P = 0.227; D HDL female P = 0.000; % HDLC/TC female P = 0.002; TC male P = 0.221; HDL male P = 0.003; %HDLC/TC male, P = 0.297 of AD vs the corresponding con- trol 1 by Student's t test. Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients Red ORO inten- sity was measured in these two fields using NIH Image J soft- ware. Panel A shows ORO staining images for one control 1 (84 years old; ORO score 0 at 0 time) and one AD (85 years old; ORO score 3 at 0 time), which are representative of about thirty subjects for each group. Panel B shows red intensity expressed as mean pixels ± SE/cm2, as described in Materials and Methods. P < 0.01, compared with control 1 by Student's t test. PBMCs from control 1 subjects and AD patients. Immediately after separation, cells were incubated with PHA for the time periods indicated. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two different fields per sample were imaged. Red ORO inten- sity was measured in these two fields using NIH Image J soft- ware. Panel A shows ORO staining images for one control 1 (84 years old; ORO score 0 at 0 time) and one AD (85 years old; ORO score 3 at 0 time), which are representative of about thirty subjects for each group. Panel B shows red intensity expressed as mean pixels ± SE/cm2, as described in Materials and Methods. P < 0.01, compared with control 1 by Student's t test. 0-1 score at all time points assessed. We also noted i) a moderate but not significant rise in FC incorporation (data not shown) in cells with 2-4 ORO score, ii) a statis- tically significant rise in TG, and iii) highly significant increases in CE incorporation (Figure 6B). The fractional rate of lipid efflux (% of [14C]lipid in medium/total [14C]lipid) was significantly lower in 1-4 scores compared to 0 score, at all time points (Figure 6C). 0-1 score at all time points assessed. We also noted i) a moderate but not significant rise in FC incorporation (data not shown) in cells with 2-4 ORO score, ii) a statis- tically significant rise in TG, and iii) highly significant increases in CE incorporation (Figure 6B). The fractional rate of lipid efflux (% of [14C]lipid in medium/total [14C]lipid) was significantly lower in 1-4 scores compared to 0 score, at all time points (Figure 6C). Lipid profiles in plasma and PBMCs of control 2 subjects and AD-FDR It is well known that individuals who have had a late- onset AD case in their family may run an increased risk of developing AD themselves [16,17]. Therefore, we meas- ured TC and HDL-C levels in plasma and carried out ORO staining in PBMCs from 91 FDR (mean age 46.6 ± 1.4; 66 female and 25 male) of the above AD patients (AD-FDR). We then compared data with those obtained from 113 age-matched blood donors (control 2) (mean age 44.7 ± 0.9; 30 female and 83 male) (Table 2). A high degree of variability in the ORO data and in plasma lipid analysis of the subjects enrolled as AD-FDR was observed (Table 2 and 4). However, 44% of PBMCs from AD-FDR had ORO staining levels scoring 2-3, as compared to only 9.7% of PBMCs from controls 2 (Table 4). PBMCs was significantly greater in AD compared to con- trol 1 (Figures 5A and 5B). Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients @ A B C > D E = ? D 7 ! " # $ % & ' ( ) + , ' - . / ( + 0 1 & 2 ) + 0 % 3 4 % 3 + ' % 2 5 / 6 7 8 9 9 : ; 8 < = > ? @ A B C > D E = ? D Plasma lipid profile in control 1subjects and AD patients Figure 3 Plasma lipid profile in control 1subjects and AD patients. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.116; HDL P = 0.000; %HDL/TC P = 0.002; TC female P = 0.227; D HDL female P = 0.000; % HDLC/TC female P = 0.002; TC male P = 0.221; HDL male P = 0.003; %HDLC/TC male, P = 0.297 of AD vs the corresponding con- trol 1 by Student's t test. Plasma lipid profile in control 1subjects and AD patients Figure 3 Plasma lipid profile in control 1subjects and AD patients. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.116; HDL P = 0.000; %HDL/TC P = 0.002; TC female P = 0.227; D HDL female P = 0.000; % HDLC/TC female P = 0.002; TC male P = 0.221; HDL male P = 0.003; %HDLC/TC male, P = 0.297 of AD vs the corresponding con- trol 1 by Student's t test. Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients Figure 2 Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients. Immediately after separation, cells were incubated with PHA for the time periods indicated. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two different fields per sample were imaged. Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients Statistical analysis revealed that ORO staining levels negatively correlated with HDL- C levels in AD patients (r = -0.378; P = 0.000), but not in control 1 (Figures 4A and 4B). No significant correlation was found between plasma TC and MMSE in AD patients as well as in control 1 (Figure 4C). Additionally, Western blotting with DM10 antibody, used to monitor ACAT-1 protein, revealed that ACAT-1 protein content, in ex vivo Table 3: Distribution of ORO grading in control 1 subjects and Alzheimer's disease patients. Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1741-7015/7/66 BMC Medicine 2009, 7:66 Plasma lipid profile in control 1subjects and AD patients Figure 3 Plasma lipid profile in control 1subjects and AD patients. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.116; HDL P = 0.000; %HDL/TC P = 0.002; TC female P = 0.227; D HDL female P = 0.000; % HDLC/TC female P = 0.002; TC male P = 0.221; HDL male P = 0.003; %HDLC/TC male, P = 0.297 of AD vs the corresponding con- trol 1 by Student's t test. TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC 0-1 score at all time points assessed. We also noted i) a moderate but not significant rise in FC incorporation (data not shown) in cells with 2-4 ORO score, ii) a statis- tically significant rise in TG, and iii) highly significant increases in CE incorporation (Figure 6B). The fractional rate of lipid efflux (% of [14C]lipid in medium/total [14C]lipid) was significantly lower in 1-4 scores compared to 0 score, at all time points (Figure 6C). Lipid profiles in plasma and PBMCs of control 2 subjects d AD FDR Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients Figure 2 Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients. Immediately after separation, cells were incubated with PHA for the time periods indicated. Lipid profiles in PHA-stimulated PBMCs of control 1 subjects and AD patients TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients Figure 2 Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients. Immediately after separation, cells were incubated with PHA for the time periods indicated. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two different fields per sample were imaged. Red ORO inten- sity was measured in these two fields using NIH Image J soft- ware. Panel A shows ORO staining images for one control 1 (84 years old; ORO score 0 at 0 time) and one AD (85 years old; ORO score 3 at 0 time), which are representative of about thirty subjects for each group. Panel B shows red intensity expressed as mean pixels ± SE/cm2, as described in Materials and Methods. P < 0.01, compared with control 1 by Student's t test. ! " # $ % & ' ( ) * + , ' - . / ( + 0 1 & 2 ) + 0 % 3 4 % 3 + ' % 2 5 / 6 7 8 9 9 : ; 8 < = > ? @ A B C > D E = ? D 7 8 9 9 : ; 8 < = > ? Cellular cholesterol metabolism and efflux Interest- ingly, no correlation was found between total plasma cholesterol and MMSE in AD patients, indicating that, contrary to what has been reported in several other stud- ies, hypercholesterolemia per se does not appear to be a risk factor for AD [36-38] 0 200 400 600 800 1000 Ͳ ! " # # $ ACAT-1 protein expression in control 1 and AD PBMCs Figure 5 ACAT-1 protein expression in control 1 and AD PBMCs. Western blotting with DM10 was used to monitor ACAT-1 protein content. The anti-ACAT-1 DM10 antibody specifically recognized a single protein band from PBMC extracts with an apparent molecular weight of about 50 kDa. No other protein signal(s) was detectable. A. Representative ACAT-1 immunoblot. B. Results of densitometric analysis by Scion Image software of ACAT-1 protein immunoreactivity relative to β-actin in 20 AD patients and 20 control 1. Data values are represented as mean ± SE P = 0.000 vs control 1. ACAT 1 protein expression in control 1 and AD PBMC Figure 5 ACAT-1 protein expression in control 1 and AD p p g ACAT-1 protein expression in control 1 and AD PBMC W bl i i h DM10 d ACAT 1 protein expression in control 1 and AD PBMCs. Western blotting with DM10 was used to monitor ACAT-1 protein content. The anti-ACAT-1 DM10 antibody specifically recognized a single protein band from PBMC extracts with an apparent molecular weight of about 50 kDa. No other protein signal(s) was detectable. A. Representative ACAT-1 immunoblot. B. Results of densitometric analysis by Scion Image software of ACAT-1 protein immunoreactivity relative to β-actin in 20 AD patients and 20 control 1. Data values are represented as mean ± SE P = 0.000 vs control 1. not shown). Interestingly, three unaffected relatives of comparable age (two brothers and one sister) of three AD patients did not have relevant accumulation of neutral lip- ids in their PBMCs, nor had lower levels of HDL-C (data not shown). When PBMCs were stimulated with PHA, about half the PBMCs exhibited ORO kinetics similar to those of AD patients, while the other half were similar to controls 2 (Figures 8A and 8B). Cellular cholesterol metabolism and efflux Given the role of HDL in preventing cell damage by removing excess cholesterol from cells, it is conceivable that low HDL-C level in AD patients is indicative of decreased removal of lipids, mainly cholesterol, from tis- sues. To verify this possibility we therefore quantified FC, CE and TG syntheses, as well as lipid efflux, in PBMCs from twenty subjects, four for each ORO score group (0- 4), chosen at random from the enrolled AD patients and control 1 subjects. Total lipid synthesis (TLS) and efflux values were obtained from the sum of [14C]acetate incor- porated into FC, CE and TG fractions separated by TLC, in cells and medium. As shown in Figure 6A, TLS was slightly higher in cells with 2-4 ORO score compared to cells with TC and HDL-C levels did not significantly change in FDR plasma (Figure 7). However, HDL-C levels inversely corre- late with ORO scores, even though not significantly (data Page 6 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1741-7015/7/66 BMC Medicine 2009, 7:66 DL-C vs ORO intensity and MMSE vs TC in PBMCs from control 1 and AD. A. HDL-C vs ORO intensity; B. %HDL-C vs RO intensity; C. MMSE vs TC gure 4 DL-C vs ORO intensity and MMSE vs TC in PBMCs from control 1 and AD. A. HDL-C vs ORO intensity; B HDL-C vs ORO intensity; C. MMSE vs TC. The test revealed a significant inverse correlation between HDL-C and HDL/TC vs ORO in AD patients but not in control 1. No correlation was observed between MMSE vs TC in both group y y; y; g HDL-C vs ORO intensity and MMSE vs TC in PBMCs from control 1 and AD. A. HDL-C vs ORO intensity; B. %HDL-C vs ORO intensity; C. MMSE vs TC. The test revealed a significant inverse correlation between HDL-C and %HDL/TC vs ORO in AD patients but not in control 1. No correlation was observed between MMSE vs TC in both groups. http://www.biomedcentral.com/1741-7015/7/66 BMC Medicine 2009, 7:66 ACAT-1 protein expression in control 1 and AD PBMCs Figure 5 ACAT-1 protein expression in control 1 and AD PBMCs. Western blotting with DM10 was used to monitor ACAT-1 protein content. The anti-ACAT-1 DM10 antibody specifically recognized a single protein band from PBMC extracts with an apparent molecular weight of about 50 kDa. No other protein signal(s) was detectable. A. Representative ACAT-1 immunoblot. B. Cellular cholesterol metabolism and efflux In agreement with these data, ACAT-1 Western blotting analysis revealed that about 50% of ex vivo FDR PBMCs show a significant increase in ACAT-1 protein levels compared to controls 2 (Figures 9A and 9B). Cellular cholesterol metabolism and efflux A moderate reduction in TC was also observed by us in AD patients. Additionally, correlation analyses revealed a positive cor- relation between HDL-C and MMSE, as well as between HDL-C and CE accumulation, as determined by ORO staining in AD patients. Although the significance, if any, of the observed correlation between low HDL-C levels and AD risk remains to be established, our data indicate that the low HDL-C levels in the plasma of AD patients may be a consequence of a decreased removal rate of cho- lesterol from tissues, as a result of the increased entrap- ment of CEs by cells. In reality, the fractional rate of lipid efflux (% of 14C-lipid in medium/total14C-lipid) was sig- nificantly lower in those cells with high ORO scores com- pared to those with low scores. It therefore seems that low HDL-C levels, coupled with lipid accumulation in periph- eral cells, represent an additional index of AD. Interest- ingly, no correlation was found between total plasma cholesterol and MMSE in AD patients, indicating that, contrary to what has been reported in several other stud- ies, hypercholesterolemia per se does not appear to be a Ͳ 0 200 400 600 800 1000 Ͳ ! " # # $ terol homeostasis and, more specifically, in raft organiza- tion may also have a great influence on AD development and progression [26-28]. In particular, CEs, rather than FC, seem to be directly correlated with Aβ production. It has, in fact, been reported that Aβ generation is modu- lated by the activity of acyl-coenzyme A: cholesterol acyl- transferase (ACAT), the enzyme that catalyses the formation of CEs inside cells [29,30]. Inhibitors of ACAT were reported to strongly reduce Aβ production in cell and AD animal-based models [31,32]. In depth, it was reported that in a mouse model of AD the ACAT inhibitor CP-113,818 reduced by 90% amyloid pathology as com- pared to transgenic mice which received placebo [32]. Cellular cholesterol metabolism and efflux Results of densitometric analysis by Scion Image software of ACAT-1 protein immunoreactivity relative to β-actin in 20 AD patients and 20 control 1. Data values are represented as mean ± SE P = 0.000 vs control 1. Ͳ 0 200 400 600 800 1000 Ͳ ! " # # $ terol homeostasis and, more specifically, in raft organiza- tion may also have a great influence on AD development and progression [26-28]. In particular, CEs, rather than FC, seem to be directly correlated with Aβ production. It has, in fact, been reported that Aβ generation is modu- lated by the activity of acyl-coenzyme A: cholesterol acyl- transferase (ACAT), the enzyme that catalyses the formation of CEs inside cells [29,30]. Inhibitors of ACAT were reported to strongly reduce Aβ production in cell and AD animal-based models [31,32]. In depth, it was reported that in a mouse model of AD the ACAT inhibitor CP-113,818 reduced by 90% amyloid pathology as com- pared to transgenic mice which received placebo [32]. Findings reported here show significantly higher neutral lipid levels (mainly CE) and increased ACAT-1 protein amounts in at least 85% of PBMCs freshly isolated (ex vivo) from patients with probable sporadic AD as com- pared to cognitively normal age-matched controls. These data are in keeping with and extend our preliminary find- ings showing higher NL levels in AD PBMCs [33], and fur- ther support the hypothesis that intracellular CE levels are systemically increased in AD-patients. That said, at least in theory, one might expect that cholesterol levels in the plasma HDL fraction, which is responsible for removing the excess of cholesterol from cells, will undergo a decrease [34]. In accordance with this, a recent study of middle-aged adults reports a link between low HDL-C and memory loss. Decreasing HDL-C was associated with memory decline over a five-year follow-up period; while no link was found between TC and TG plasma concentra- tions and memory deficit or decline [35]. Similarly, we found a significant reduction, both in absolute and rela- tive terms, in HDL-C levels in plasma from AD blood sam- ples as compared to age-matched controls. Page 8 of 12 (page number not for citation purposes) Cellular cholesterol metabolism and efflux Findings reported here show significantly higher neutral lipid levels (mainly CE) and increased ACAT-1 protein amounts in at least 85% of PBMCs freshly isolated (ex vivo) from patients with probable sporadic AD as com- pared to cognitively normal age-matched controls. These data are in keeping with and extend our preliminary find- ings showing higher NL levels in AD PBMCs [33], and fur- ther support the hypothesis that intracellular CE levels are systemically increased in AD-patients. That said, at least in theory, one might expect that cholesterol levels in the plasma HDL fraction, which is responsible for removing the excess of cholesterol from cells, will undergo a decrease [34]. In accordance with this, a recent study of middle-aged adults reports a link between low HDL-C and memory loss. Decreasing HDL-C was associated with memory decline over a five-year follow-up period; while no link was found between TC and TG plasma concentra- tions and memory deficit or decline [35]. Similarly, we found a significant reduction, both in absolute and rela- tive terms, in HDL-C levels in plasma from AD blood sam- ples as compared to age-matched controls. A moderate reduction in TC was also observed by us in AD patients. Additionally, correlation analyses revealed a positive cor- relation between HDL-C and MMSE, as well as between HDL-C and CE accumulation, as determined by ORO staining in AD patients. Although the significance, if any, of the observed correlation between low HDL-C levels and AD risk remains to be established, our data indicate that the low HDL-C levels in the plasma of AD patients may be a consequence of a decreased removal rate of cho- lesterol from tissues, as a result of the increased entrap- ment of CEs by cells. In reality, the fractional rate of lipid efflux (% of 14C-lipid in medium/total14C-lipid) was sig- nificantly lower in those cells with high ORO scores com- pared to those with low scores. It therefore seems that low HDL-C levels, coupled with lipid accumulation in periph- eral cells, represent an additional index of AD. Discussion 0 1 2 3 4 0 500 0 1000 0 1500 0 2000 0 * * 14C-acetate incorporated into TL (cpm/10 4 cells) ORO score 6 h 18 h 36 h * 0 1 2 3 4 0 2000 4000 6000 8000 * * * * * 14C-acetate incorporated into CE (cpm/10 4 cells) ORO score 6h 18h 36h * 0 1 2 3 4 0 10 20 30 40 * * * * * * * * * % of TL efflux ORO score 6 h 18 h 36 h * 0 1 2 3 4 0 500 0 1000 0 1500 0 2000 0 * * * 14C-acetate incorporated into TL (cpm/10 4 cells) ORO score 6 h 18 h 36 h * ORO score 0 1 2 3 4 0 2000 4000 6000 8000 * * * * * 14C-acetate incorporated into CE (cpm/10 4 cells) ORO score 6h 18h 36h * 0 1 2 3 4 0 14C- ORO score 0 1 2 3 4 0 10 20 30 40 * * * * * * * * * % of TL efflux OROscore 6 h 18 h 36 h * ORO score 0 1 2 3 4 0 10 20 30 40 * * * * * * * * * % of TL efflux ORO score 6 h 18 h 36 h * 0 1 2 3 4 0 10 20 30 40 * * * * * * * * * % of TL efflux ORO score 6 h 18 h 36 h * Lipid synthesis and efflux in PBMCs with different ORO scores Figure 6 Lipid synthesis and efflux in PBMCs with different ORO scores. Freshly isolated PBMCs (four for each ORO score group) were plated at a density of 10,000 cell/cm2 in six well plates and incubated with PHA for the time periods indicated. Total cellular lipid synthesis (A) and cholesterol ester synthesis (B) were evaluated by incubating cells for six hours in medium containing [14C]acetate at a final concentration of 2 μCi/ml. After incubation, cells were washed with PBS and lipids extracted with acetone. Lipid subclasses were separated out by thin layer chromatography (TLC) and [14C]acetate incorporation into the various lipid fractions was measured. Discussion Cholesterol is essential for the organization of liquid- ordered microdomains (rafts) in the plasma membrane, which serve as a scaffold for the proper assembling and functioning of a variety of membrane-associated proteins, including β- and γ-secretases [23,24] that are involved in the Aβ formation. Progressive accumulation of Aβ in the brain is considered a pathogenetic event common to all forms of AD [25]. This suggests that alterations in choles- Page 8 of 12 (page number not for citation purposes) BMC Medicine 2009, 7:66 http://www.biomedcentral.com/1741-7015/7/66 efflux in PBMCs with different ORO scores nd efflux in PBMCs with different ORO scores. Freshly isolated PBMCs (four for each ORO score at a density of 10,000 cell/cm2 in six well plates and incubated with PHA for the time periods indicated. ynthesis (A) and cholesterol ester synthesis (B) were evaluated by incubating cells for six hours in medium tate at a final concentration of 2 μCi/ml. After incubation, cells were washed with PBS and lipids extracted subclasses were separated out by thin layer chromatography (TLC) and [14C]acetate incorporation into the ns was measured. Efflux (C) from the cells into the medium at each time point was expressed as the per- vity in medium/total radioactivity (cells + medium). Data shown are the mean ± SE of four subjects from P < 0.05. Discussion Efflux (C) from the cells into the medium at each time point was expressed as the per- centage of radioactivity in medium/total radioactivity (cells + medium). Data shown are the mean ± SE of four subjects from each score group, P < 0.05. Page 9 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1741-7015/7/66 BMC Medicine 2009, 7:66 Plasma lipid profile in control 2 and AD-FDR Figure 7 Plasma lipid profile in control 2 and AD-FDR. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.784; HDL P = 0.154; %HDL/TC P = 0.04; TC female P = 0.185; D HDL female P = 0.304; % HDLC/TC female P = 0.05; TC male P = 0.490; HDL male P = 0.215; %HDLC/TC male, P = 0.316 of AD-FDR vs the corresponding control 2 by Stu- dent's t test. TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC Ͳ Table 4: Distribution of ORO grading in control 2 and Alzheimer's disease-First-degree relatives subjects. Score Control 2 AD-FDR Total Female Male Total Female Male 0 70 15 55 30 21 9 1 32 10 22 20 13 7 2 10 4 6 21 14 7 3 1 1 0 19 17 2 4 0 0 0 1 1 0 total 113 30 83 91 66 25 ORO = Oil Red O AD-FDR = Alzheimer's Disease - First Degree Relatives Table 4: Distribution of ORO grading in control 2 and Alzheimer's disease-First-degree relatives subjects. TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC -- TC HDL-C HDL-C/TC Ͳ However, although it is generally accepted that alterations in cholesterol metabolism are a critical event in AD, it is still unclear whether they are the cause or the effect of dis- ease and, actually, we cannot completely exclude the pos- sibility that changes in lipid metabolism may be due to factors other than AD. Abbreviations ACAT: Acyl-coenzyme A: Cholesterol AcylTransferase; AD: Alzheimer's Disease; AD-FDR: Alzheimer's Disease - First Degree Relatives; Aβ: Amyloid Beta-peptides; CDR: Clini- cal Dementia Rating scale; CDT: Clock Drawing Test; CEs: Cholesterol Esters; FC: Free Cholesterol; FCS: Fetal Calf Serum; FDR: First Degree Relatives; GDS: Global Deterio- ration Scale; HBSS: Hanks Balanced Salt Solution; HDL-C: High Density Lipoprotein - Cholesterol; MCI: Mild Cogni- tive Impairment; MMSE: Mini Mental State Examination; NL: Neutral Lipid; NL-PBMCs: Neutral Lipid - Peripheral Blood Mononuclear Cells; ORO: Oil Red O; PBMCs: Peripheral Blood Mononuclear Cells; PBS: Phosphate Buffered Saline; PHA: PhytoHemaAgglutinin; ROIs: Regions Of Interest; SE: Standard Error; TG: Triglycerides; TL: Total Lipid; TLC: Thin Layer Chromatography; TLS: Total Lipid Synthesis. Discussion Dementia occurs late in life, but it is increasingly recognized that there is a long preclinical phase characterized by progressive neuropathological changes that become clinically detectable with age. The "life-long" view of dementia contributes to stress the importance of risk factors in midlife [39]. Individuals who have had a late-onset AD case in their family may run an increased risk of developing AD themselves [17,18], thus the analysis of lipid metabolism in these subjects could be a suitable biochemical approach to monitor changes in cholesterol homeostasis before any cognitive decline might occur. We observed great variability in the ORO data and in plasma lipid analysis of the subjects enrolled as AD-FDR. However, 44% of PBMCs from AD-FDR had ORO staining levels that scored from 2 to 3, as compared to only 9.7% of PBMCs from controls. Plasma lipid profile in control 2 and AD-FDR Figure 7 Plasma lipid profile in control 2 and AD-FDR. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.784; HDL P = 0.154; %HDL/TC P = 0.04; TC female P = 0.185; D HDL female P = 0.304; % HDLC/TC female P = 0.05; TC male P = 0.490; HDL male P = 0.215; %HDLC/TC male, P = 0.316 of AD-FDR vs the corresponding control 2 by Stu- dent's t test. Plasma lipid profile in control 2 and AD-FDR Figure 7 Plasma lipid profile in control 2 and AD-FDR. Data are expressed as a mean ± SE of the enrolled subjects. TC P = 0.784; HDL P = 0.154; %HDL/TC P = 0.04; TC female P = 0.185; D HDL female P = 0.304; % HDLC/TC female P = 0.05; TC male P = 0.490; HDL male P = 0.215; %HDLC/TC male, P = 0.316 of AD-FDR vs the corresponding control 2 by Stu- dent's t test. and frontotemporal dementias; ii) asymptomatic individ- uals with decreased plasma HDL-C and increased PBMC lipids will really go on towards Alzheimer's disease over time. Conclusion Overall, the assessment of cholesterol ester metabolism in PBMCs ex vivo by means of a reliable assay that can be easily and quickly performed might be a very useful bio- chemical tool in support to AD diagnosis. In addition, the fact that some AD-FDR tend to display a metabolic choles- terol pattern similar to that of AD patients, may suggest that the determination of PBMC ORO staining and plasma HDL-C levels may be useful for identification of a subset of midlife subjects who might run a major risk of developing AD; and the identification of incipient AD- related cognitive impairment would be the basis for initi- ation of treatment with drugs that might slow or stop the degenerative process, such as ACAT inhibitors [29-31]. Above all it remains to be assessed whether: i) changes in NL are also present in PBMCs from patients with non-AD neuropathologies such as for instance vascular, Lewy body Competing interests Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ ! " # $ % & ' % ( ! " % ) ) & # ' * + , - % . . ! " # $ % & ' / ! " # $ % & " ' ( ) " ' * + * " ) & , Ͳ - , . & , Ͳ - , . / / / / 0 1 2 3 4 1 5 6 7 8 Ͳ 9 8 : ; < = > > ? @ = A B C D E F G H I D J K L E H M N C O F H M B P 7 8 Ͳ 9 8 : 6 Ͳ Ͳ Ͳ ! " # $ % & ' % ( ! " % ) ) & # ' * + , - % . . ! " # $ % & ' / < ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects Figure 9 ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects. Western blotting with DM10 was used to monitor the ACAT-1 protein content. The anti- ACAT-1 DM10 antibody specifically recognized a single pro- tein band from cell extracts of PBMCs, with an apparent molecular weight of 50 kDa. No other protein signal(s) was detectable. A. Representative immunoblot of ACAT-1 of two control 2; two AD-FDR with ORO score between 0 and 1 (FDR-1) and two AD-FDR with ORO score between 2-3 (FDR-2). B. Neutral lip control 2 a Figure 8 Panel B shows red inten- sity expressed as mean pixels ± SE/cm2 of triplicate wells obtained by utilizing four created different regions of interest (ROIs). P < 0.01, compared with control 2 by Student's t test. p j g Neutral lipid accumulation in PHA-stimulated on the manuscript and participated in the design. All authors read and approved the final manuscript. Competing interests p g This study was supported by grants from Fondazione Regione Autonoma della Sardegna and Fondazione Monte dei Paschi di Siena. The authors state that there are no conflicts of interest that may have influenced this work and that some findings in this study have been the subject Page 10 of 12 (page number not for citation purposes) Page 10 of 12 (page number not for citation purposes) BMC Medicine 2009, 7:66 http://www.biomedcentral.com/1741-7015/7/66 ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects Figure 9 ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects. Western blotting with DM10 was used to monitor the ACAT-1 protein content. The anti- ACAT-1 DM10 antibody specifically recognized a single pro- tein band from cell extracts of PBMCs, with an apparent molecular weight of 50 kDa. No other protein signal(s) was detectable. A. Representative immunoblot of ACAT-1 of two control 2; two AD-FDR with ORO score between 0 and 1 (FDR-1) and two AD-FDR with ORO score between 2-3 (FDR-2). B. Results of densitometric analysis by Scion Image software of ACAT-1 protein. Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ ! " # $ % & ' % ( ! " % ) ) & # ' + , - % . . ! " # $ % & ' / on the manuscript and participated in the design. All authors read and approved the final manuscript. Acknowledgements Th h i h h k ll h l bl d d AD i Neutral lipid accumulation in PHA-stimulated PBMCs from control 2 and AD-FDR subjects Figure 8 Neutral lipid accumulation in PHA-stimulated PBMCs from control 2 and AD-FDR subjects. Immedi- ately after separation cells were incubated with PHA for the indicated time periods. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two dif- ferent fields per sample were imaged. Red ORO intensity was measured in these two fields by using NIH Image J software. Competing interests Panel A shows images of ORO staining of one control 2 (47 years old) and two AD-FDR one (48 years old) with ORO score 0 and the other one (37 years old) with ORO score 3 at 0 time. These images are representative of at least 30 con- trol 2, 15 AD-FDR with ORO score between 0 and 1, and 15 with ORO score between 2 and 3. Panel B shows red inten- sity expressed as mean pixels ± SE/cm2 of triplicate wells obtained by utilizing four created different regions of interest (ROIs). P < 0.01, compared with control 2 by Student's t ! " # $ % & " ' ( ) " ' + * " ) & , Ͳ - , . & , Ͳ - , . / / / / 0 1 2 3 4 1 5 6 7 8 Ͳ 9 8 : ; < = > > ? @ = A B C D E F G H I D J K L E H M N C O F H M B P 7 8 Ͳ 9 8 : 6 ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects Figure 9 ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects. Western blotting with DM10 was used to monitor the ACAT-1 protein content. The anti- ACAT-1 DM10 antibody specifically recognized a single pro- tein band from cell extracts of PBMCs, with an apparent molecular weight of 50 kDa. No other protein signal(s) was detectable. A. Representative immunoblot of ACAT-1 of two control 2; two AD-FDR with ORO score between 0 and 1 (FDR-1) and two AD-FDR with ORO score between 2-3 (FDR-2). B. Results of densitometric analysis by Scion Image software of ACAT-1 protein. References 1. Govaerts L, Schoenen J, Bouhy D: Pathogenesis of Alzheimer's disease: molecular and cellular mechanisms. Rev Med Liege 2007, 62:209-216. 2. Giannakopoulos P, Kövari E, Gold G, von Gunten A, Hof PR, Bouras C: Pathological substrates of cognitive decline in Alzheimer's disease. Front Neurol Neurosci 2009, 24:20-29. 2. Giannakopoulos P, Kövari E, Gold G, von Gunten A, Hof PR, Bouras C: Pathological substrates of cognitive decline in Alzheimer's disease. Front Neurol Neurosci 2009, 24:20-29. Acknowledgements The authors wish to thank all the volunteers, blood donors, AD patients and their relatives, who agreed to enter this study. The technical assistance given by Anna Saba and Noemi Ruggeri and the excellent English revision done by Edward J. Steeden are much appreciated. of patent application no.PCT/IT2007/000110 "Methods for the diagnosis of proliferative and/or conformational diseases" (S. Dessì, P. La Colla, and A. Pani). Neutral lip control 2 a Figure 8 Neutral lipid accumulation in PHA-stimulated PBMCs control 2 and AD-FDR subjects Figure 8 Neutral lipid accumulation in PHA-stimulated Neutral lipid accumulation in PHA-stimulated PBMCs from control 2 and AD-FDR subjects Figure 8 Neutral lipid accumulation in PHA-stimulated PBMCs from control 2 and AD-FDR subjects. Immedi- ately after separation cells were incubated with PHA for the indicated time periods. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two dif- ferent fields per sample were imaged. Red ORO intensity was measured in these two fields by using NIH Image J software. Panel A shows images of ORO staining of one control 2 (47 years old) and two AD-FDR one (48 years old) with ORO score 0 and the other one (37 years old) with ORO score 3 at 0 time. These images are representative of at least 30 con- trol 2, 15 AD-FDR with ORO score between 0 and 1, and 15 with ORO score between 2 and 3. Panel B shows red inten- sity expressed as mean pixels ± SE/cm2 of triplicate wells obtained by utilizing four created different regions of interest (ROIs). *P < 0.01, compared with control 2 by Student's t test. Neutral lipid accumulation in PHA stimulated PBMCs from control 2 and AD FDR subjects Figure 8 Neutral lipid accumulation in PHA-stimulated PBMCs from control 2 and AD-FDR subjects. Immedi- ately after separation cells were incubated with PHA for the indicated time periods. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two dif- ferent fields per sample were imaged. Red ORO intensity was measured in these two fields by using NIH Image J software. Panel A shows images of ORO staining of one control 2 (47 years old) and two AD-FDR one (48 years old) with ORO score 0 and the other one (37 years old) with ORO score 3 at 0 time. These images are representative of at least 30 con- trol 2, 15 AD-FDR with ORO score between 0 and 1, and 15 with ORO score between 2 and 3. 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Dessi S, Batetta B, Pani A, Spano O, Sanna F, Putzolu M, Bonatesta R, Piras , Pani P: Role of cholesterol synthesis and esterification in the growth of CEM and MOLT4 lymphoblastic cells. Biochem J 1997:603-608. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 12 of 12 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge J 23. Simons K, Ehehalt R: Cholesterol, lipid rafts, and disease. J Clin Invest 2002, 110:597-603. Authors' contributions Panza F, Capurso C, D'Introno A, Colacicco AM, De Candia D, Capurso A, Solfrizzi V: Total cholesterol levels and the risk of mild cognitive impairment and Alzheimer's disease. J Am Ger- iatr Soc 2007, 55(1):133-135. ( ) 37. Pregelj P: Involvement of cholesterol in the pathogenesis of Alzheimer's disease: role of statins. Psychiatr Danub 2008, 20(2):162-167. J p 15. 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https://openalex.org/W4376603037	https://www.revista.ueg.br/index.php/revelli/article/view/12961/9146	Portuguese	null	O USO VARIÁVEL DO /R/ EM CODA EXTERNA	Revelli	2,022	cc-by	6,449	O USO VARIÁVEL DO /R/ EM CODA EXTERNA: CONTRIBUIÇÕES DA SOCIOLINGUÍSTICA APLICADA AO ENSINO THE VARIABLE USE OF THE /R/ IN EXTERNAL CODA: CONTRIBUTIONS OF APPLIED SOCIOLINGUISTICS TO TEACHING FREITAS, Rosely Lopes de FERREIRA, Jannaina Reis VIEIRA, Marília Resumo: O presente artigo, ancorado em uma perspectiva decolonial, dedica-se a refletir sobre propostas interventivas de ensino de língua materna, desenvolvidas no âmbito do Subprojeto do PIBID/Língua Portuguesa. Nessa perspectiva, observa-se que as normas culta e popular revelam muitas semelhanças e, para evidenciar tais similitudes, foram elaboradas quatro atividades em que se enfatiza o uso variável de /R/ em coda externa no Português Brasileiro, cuja aplicação foi idealmente planejada para o oitavo do Ensino Fundamental. A teia teórica da Sociolinguística aplicada ao ensino é complementada com cartas do Atlas Linguístico do Brasil (CARDOSO et al, 2014) e com as contribuições de Monaretto (2002). As atividades propostas estão fundamentadas nos contínuos de Bortoni-Ricardo (2004), bem como na gramática de três eixos (VIEIRA, 2017), com o objetivo de descrever o uso variável do fenômeno supramencinado. Os resultados corroboram que o trabalho com a reflexão linguística permite que o aluno possa compreender os fenômenos linguísticos, aprimorando sua competência comunicativa. Palavras-chave: /R/. Sociolinguística. Ensino. corroborate that the work with linguistic reflection allows the student to understand linguistic phenomena, improving his communicative competence. corroborate that the work with linguistic reflection allows the student to understand linguistic phenomena, improving his communicative competence. Keywords: /R/. Sociolinguistics. Teaching. Palavras-chave: /R/. Sociolinguística. Ensino. Abstract: This article, anchored in a decolonial perspective, is dedicated to reflecting on interventional proposals for mother tongue teaching, within the scope of the PIBID/Portuguese Language Subproject. In this perspective, it is observed that the cultured and popular norms reveal many similarities and, to highlight such similarities, four activities were elaborated in which the variable use of /R/ in external coda in Brazilian Portuguese is emphasized, whose application was ideally planned for the eighth of elementary school. The theoretical web of Sociolinguistics applied to teaching is complemented with letters from the Linguistic Atlas of Brazil (CARDOSO et al, 2014) and with the contributions of Monaretto (2002). The proposed activities are based on the Bortoni-Ricardo ushers (2004), as well as on the three-axis grammar (VIEIRA, 2017), in order to describe the variable use of the phenomenon mentioned above. The results REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 1 1 corrobo linguisti corrobor li i ti 1 Introdução Por um longo período no Brasil, o estudo formal foi destinado, especificamente, às camadas prestigiadas socioeconomicamente, tendo como objetivo primordial a aprendizagem do Latim, devido seu valor social da época. Com o decorrer do tempo, a disciplina Língua Portuguesa foi inserida nos currículos pedagógicos, entretanto, esse ensino ainda privilegiava uma norma linguística totalmente distante do Português Brasileiro. Com o advento das pesquisas sociolinguísticas no território brasileiro, os estudiosos passaram a estudar e a compreender que a norma linguística brasileira era muito divergente das normas impostas pelos compêndios gramaticais. Porém, devido diversos fatores (incluindo os políticos, sociais, culturais, pedagógicos e outros), os resultados dessas pesquisas não chegavam/chegam nas escolas e até mesmo nas Universidades. Dessa forma, foi devido os esforços da de Bortoni-Ricardo (e orientando(a)s), que se propôs um liame dos estudos sociolinguísticos ao ensino, denominado Sociolinguística Educacional. Outra estudiosa que se dedica a estudar/orientar diversos trabalhos sobre a variabilidade linguística brasileira nos diferentes níveis linguísticos e aliá-los ao ensino de Língua Portuguesa nos ensinos fundamentais e médios é a Profa. Dra Silvia Rodrigues Vieira, professora do Departamento de Letras Vernáculas da Faculdade de Letras da Universidade Federal do Rio de Janeiro (UFRJ). Dentre várias obras da estudiosa, destacamos aqui a organização de dois livros: Gramática, variação e ensino: diagnose e propostas pedagógicas (2018) e Variação, gêneros textuais e ensino de Português: da norma culta à norma-padrão (2019). Este foi organizado também pela pesquisadora Monique Débora Lima. Dessa forma, vários trabalhos, como os de Dorneles (2011), ratificam a variabilidade do –r no Português Brasileiro (PB), podendo ser realizado como tepe, glotal e até mesmo sofrer um apagamento total. No que se refere ao apagamento dessa variável, REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 2 pesquisad pesquisadores como Oliveira (1983) e Linares, Peixoto e Moreira (2008) chegaram à conclusão de que ele é mais recorrente em verbos do que em substantivos e em coda externa do que interna. Além disso, é imprescindível salientar que o apagamento do /R/ em coda externa é um fenômeno muito antigo no PB, sendo, inicialmente, associado as variedades populares, porém, hoje ele é frequente na fala de indivíduos de diferentes estratos sociais (CALLOU, MORAES E LEITE, 1998; LINARES, PEIXOTO E MOREIRA, 2008). Apesar de ter uma ampla literatura linguística a respeito da variabilidade do /r/ no PB, nota-se uma escassez de trabalhos sobre esse tema direcionado ao ensino. 1 Introdução Por esse motivo, objetivamos apresentar uma proposta pedagógica com o intuito de que os alunos reflitam/percebam que (a) as normas cultas e populares possuem semelhanças e diferenças e ambas se diferenciam da norma-padrão, (b) uso variável de /R/ em nomes e verbos no infinitivo, (c) o apagamento do /R/ em coda externa em infinitivos verbais é um fenômeno gradual, por isso, não sofre estigmatização como os fenômenos descontínuos, diferente do /R/ retroflexo, (d) variabilidade do /R/ em infinitivos verbais em dois contextos de fala (reunião e entrevista) e escrita (redação). Com base no exposto, este artigo, resultante das atividades desenvolvidas no âmbito do núcleo de Língua Portuguesa do PIBID, com oito alunos bolsistas CAPES em uma escola pública do estado de Goiás, expõe uma sequência didática, orientada pelos contínuos de urbanização, oralidade-letramento e monitoração estilística (BORTONI- RICARDO, 2004), que procura descrever o uso variável de /R/ em coda externa de nomes e verbos em excertos orais e escritos, pautando-se na perspectiva do uso no PB. Dessa maneira, esse trabalho é voltado para pesquisadores, docentes de Língua Portuguesa e para todos que se interessarem pelo assunto. Por fim, este trabalho está dividido em 5 etapas: introdução, fundamentação teórica, propostas pedagógicas, análise das propostas realizadas, considerações finais e referências. REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 3 REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 3 O O REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 5 Os objetivos da disciplina de Língua Portuguesa começaram a mudar a partir dos anos 1950, quando houve uma progressiva transformação social e, principalmente, devido às possibilidades de acesso à escola: agora, as pessoas provenientes de camadas desprestigiadas também tinham direito a estudar (SOARES, 2002). No decorrer do século XX, as políticas linguísticas em favor do Português Brasileiro (doravante PB) passam a se consolidar Desse modo, o advento da Sociolinguística no Brasil contribuiu significativamente para a descrição da realidade linguística do PB, contribuindo para a documentação de variedades cultas e populares (BAGNO, 2002). Nessa perspectiva, o professor pode se instrumentalizar acerca das diferenças entre as variedades linguísticas faladas pelos alunos de classes populares e a variedade de prestígio, possibilitando metodologias de ensino voltadas para o respeito linguístico. Além disso, o desenvolvimento de estudos descritivos do PB propiciou uma nova maneira de tratar a oralidade, a escrita e o texto no ensino (SOARES, 2002). No que diz respeito à BNCC e ao tratamento da variação linguística, são elencadas, aqui, algumas considerações primordiais: a discussão acerca da variação linguística, variedades prestigiadas/desprestigiadas e preconceito linguístico e o tratamento dado ao tema nos livros didáticos; o uso de gravações e textos em diferentes variedades linguísticas, a fim de identificar características regionais, urbanas e rurais. É relevante, em primeiro momento, postular uma série de problemáticas no que concerne ao ensino de Língua Portuguesa, tais como: confusão entre norma-padrão e norma culta; distinção entre norma, modalidade e registro e, posteriormente, sobre os contínuos de variação linguística e os três eixos para o ensino da gramática. Essas questões foram apresentadas por Bagno (2002), Martins, Vieira e Tavares (2014), Bortoni-Ricardo (2004) e Vieira (2019). Segundo Bagno (2002), norma-padrão não é uma língua, mas sim um modelo, um ideal e um padrão de comportamento linguístico baseado na obra de escritores consagrados. Já a norma culta é uma língua real, baseada na fala de falantes cultos (BAGNO, 2002). De acordo Vieira (2019), essa homogeneidade proposta pelos discursos normativos estabelece uma generalidade de regras de comportamento social para toda e REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 4 qualquer qualquer qualquer situação comunicativa, ocasionando uma equação equivocada: “norma-padrão = Língua Portuguesa” e, consequentemente, o surgimento dos mitos linguísticos. Nesse viés, é imprescindível guiar as orientações normativas para que a padronização seja efetivamente sensível aos contínuos de modalidade (do maior ao menor grau de oralidade/fala-letramento/escrita) e registro (do mais ao menos formal/monitorado)” (VIEIRA, 2019). Ademais, segundo Martins, Vieira e Tavares (2014), é muito importante que o professor estabeleça a distinção entre norma, modalidade e registro, haja vista que, devido a algumas práticas pedagógicas não esclarecedoras, os alunos são levados a acreditar em algumas assertivas, tais como: a variação de registro (mais formal) é exclusiva da norma culta e da modalidade escrita. Nesse sentido, é preciso assumir que tanto falantes de normas cultas, quanto falantes de normas populares variam quanto ao grau de formalidade. Ademais, é preciso dissociar a ideia de que a modalidade falada é intrínseca ao registro informal e de que a modalidade escrita está relacionada exclusivamente ao registro formal. Nessa perspectiva, Bortoni-Ricardo (2004) propõe três contínuos para explicar a variação no PB: (1) contínuo de urbanização, (2) contínuo de oralidade-letramento e (3) contínuo de monitoração estilística. No contínuo de urbanização, há duas extremidades: de um lado, estão situados os falares rurais mais isolados, representados por falares típicos/estigmatizados do polo rural, denominados traços descontínuos. Do outro lado, estão situados os falares urbanos, suscetíveis à influência de agentes padronizadores da língua (mídia, imprensa e escola) e que apresentam traços linguísticos presentes na variedade culta, denominados traços graduais. No contínuo de oralidade-letramento, denominam-se eventos de letramento as falas permeadas por um texto escrito; já os eventos de oralidade são aqueles que não têm interferência da língua escrita. Por último, o contínuo de monitoração estilística está relacionado desde interações espontâneas até interações planejadas que exigem muita atenção do falante. De forma bastante simplificada, Vieira (2017) propõe os seguintes eixos: (1) Ensino de gramática e atividade reflexiva, (2) Ensino de gramática e produção de sentidos e, (3) Ensino de gramática, variação e normas. REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 5 3 O apagamento de /R/ em coda externa no PB 3 O apag 3 O apagamento de /R/ em coda externa no PB E-202203 6 popular. popular. popular. Sobre a classe morfológica, chegou-se à conclusão de que esse fenômeno é mais recorrente nos verbos (67,12%) do que em nomes (32,88%). Com ênfase nessas evidências, objetiva-se que os alunos observem as semelhanças entre as normas culta e popular, compreendendo que a primeira não é algo intacto, homogêneo ou acabado, mas flexível e suscetível à variação de acordo com os diversos contextos comunicativos. Abaixo, são expostas as propostas e intervenção pedagógicas que visam ao tratamento do uso variável do /R/ em coda externa, desenvolvidas no oitavo ano do Ensino Fundamental em uma escola pública do estado de Goiás. 3.1.1 Propostas pedagógicas Nesta seção, serão descritas as três atividades propostas com base no fenômeno fonético-fonológico em estudo, o /R/ em coda externa no PB. 1 Os excertos a e b foram retirados do livro Educação em língua materna – a Sociolinguística na sala de aula, de Bortoni-Ricardo (2004). Já o excerto c foi retirado de uma entrevista do corpus Sociolinco (BERNARDES, 2020). 3 O apagamento de /R/ em coda externa no PB 3 O apagamento de /R/ em coda externa no PB Segundo Dorneles (2011), a realização do –r no PB parece ser o fenômeno que apresenta maior quantidade de variação dos alofones no que se refere ao ponto de articulação, podendo ser produzido como tepe, glotal e até mesmo sofrer um apagamento total. Monaretto (2002) afirma que, quando o /R/ está presente no início da palavra (rato) e em início de sílaba, precedido por consoante (honra), a forma preferida é o /R/ forte (fricativa velar ou vibrante alveolar). Entretanto, de acordo com a linguista, com o passar do tempo, houve um crescimento do uso da fricativa velar em detrimento do uso da vibrante alveolar na fala do Sul do Brasil. Em relação ao /R/ pós-vocálico, a pesquisadora percebeu que a variante predominante é o /R/ fraco, de forma mais precisa, o tepe. Nessa conjuntura, Monaretto (2002) constatou que, na posição de coda, o tepe prevalece (60%), em comparação às demais variantes: apagamento (25%), vibrante alveolar (9%), retroflexo (5%) e (1%) fricativa velar. Todavia, por meio de uma comparação de três amostras (1970, 1989 e 1999), a autora diagnosticou que, em um intervalo de 30 anos, aproximadamente, o tepe diminuiu e o apagamento aumentou em Porto Alegre (especialmente em infinitivos verbais e no futuro do subjuntivo). De acordo com ela, esse processo se encontra em estágios mais avançados em outros lugares do Brasil. Para Callou, Moraes e Leite (1998), o apagamento do /R/ em coda externa é um fenômeno muito antigo no PB, considerado como uma característica dos falares incultos, mas, com o decorrer do tempo, expandiu-se e hoje é comum na fala de pessoas pertencentes a vários estratos sociais. Além disso, a autora pontua também que, no século XVI, nas peças de Gil Vicente, esse fenômeno era usado para representar a fala dos escravos. Oliveira (1983) atesta que o /R/ em coda externa é mais apagado (70,83%) do que o interno (17,59%) e, quanto à classe morfológica, esse fenômeno é mais apagado em verbos (81,59%) do que em nomes (26,09%). Nesse segmento, Linares, Peixoto e Moreira (2008) compararam o apagamento do /R/ em coda externa, nas variedades culta e popular e concluíram que esse fenômeno é recorrente nas falas dos informantes, respectivamente, 62,82% e 88,05%. Ademais, os autores acreditam que esse fenômeno está mais presente na fala dos falantes de nível REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 7 3.1.1 Proposta I É importante que o docente mencione que existem diferenças linguísticas entre as normas culta e popular, mas que também há semelhanças. Nesse viés, é necessário mencionar que o apagamento do /R/ em coda externa em verbos é recorrente na variedade culta, mas que, na escrita monitorada, ele precisa ser mantido. A seguir, é descrita a proposta II, como continuidade à sequência de atividades sobre o /R/ em coda externa no PB. 3.1.2 Proposta II a) “D a) “De uns tempo pra cá, ninguém qué roçá mais, Num certo ponto, eu dô razão, eu mesmo fui um desses que saí da roça por causa disso, né? Que eu não tinha terreno de meu, morava dependente de oto, de fazendeiro. Fazenderos não dão cuié de chá mesmo, né? Tem que plantá, planta, tem que parti à meia, ota hora à terça, né?” (BORTONI- RICARDO, 1985, p. 99). b) “O qu’eu tô comprendenu de poco tempo pra cá é negoçu de reporti. Qu’eu cumpanho nutiça, reporti de rádio e televisão, que agora qu’eu tô aprendenu, nunca tinha usado nem televisão, que a gente morava na roça, e mesmo aqui né? mesmo aqui, é de pocos tempo pra cá que os menino deu conta de comprá um rádio” (BORTONI- RICARDO, 1985, p. 225). c) “aqui se você precisá de um ortopedista, se você precisá de um gastro então, imagina, eu acho, eu duvido que já tenha tido um gastro aqui em Goiás e assim... hoje em dia, adultos e crianças com a péssima alimentação que fazem... um gastro acaba sendo essencial a gente tem estrutura física, às vezes mas não tem estrutura humana pra podê atendê todo mundo” (BERNARDES, 2020 – Corpus Sociolinco).  Obs.: O professor deve explicou a diferença entre os fenômenos graduais e descontínuos (BORTONI-RICARDO, 2004). É importante que o docente mencione que existem diferenças linguísticas entre as normas culta e popular, mas que também há semelhanças. Nesse viés, é necessário mencionar que o apagamento do /R/ em coda externa em verbos é recorrente na variedade culta, mas que, na escrita monitorada, ele precisa ser mantido. A seguir, é descrita a proposta II, como continuidade à sequência de atividades sobre o /R/ em coda externa no PB. 3.1.1 Proposta I A partir da premissa de que os contínuos de contínuos de urbanização, oralidade- letramento e monitoração estilística “são instâncias distintas da variação linguística e, respectivamente, estão ligadas aos conceitos de norma (s), modalidade (oral ou escrita) e registro (formal ou informal)” (LIMA, p. 125, 2017), na presente atividade, serão abordados os eixos I e III (VIEIRA, 2019), com base na proposta de situar a fala dos informantes nos três contínuos (BORTONI-RICARDO, 2004) e de verificar as convergências/divergências entre elas. Atividade: Veja a seguir, três excertos de fala retirados de entrevistas desenvolvidas para um estudo científico1. Tente descrever a qual camada social pertencem os informantes em cada caso. Em seguida, sublinhe os fenômenos linguísticos que se “distanciam” da norma ensinada na escola e verifique se há semelhanças ou diferenças nas falas. 1 Os excertos a e b foram retirados do livro Educação em língua materna – a Sociolinguística na sala de aula, de Bortoni-Ricardo (2004). Já o excerto c foi retirado de uma entrevista do corpus Sociolinco (BERNARDES, 2020). REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 7 a) “De uns tempo pra cá, ninguém qué roçá mais, Num certo ponto, eu dô razão, eu mesmo fui um desses que saí da roça por causa disso, né? Que eu não tinha terreno de meu, morava dependente de oto, de fazendeiro. Fazenderos não dão cuié de chá mesmo, né? Tem que plantá, planta, tem que parti à meia, ota hora à terça, né?” (BORTONI- RICARDO, 1985, p. 99). b) “O qu’eu tô comprendenu de poco tempo pra cá é negoçu de reporti. Qu’eu cumpanho nutiça, reporti de rádio e televisão, que agora qu’eu tô aprendenu, nunca tinha usado nem televisão, que a gente morava na roça, e mesmo aqui né? mesmo aqui, é de pocos tempo pra cá que os menino deu conta de comprá um rádio” (BORTONI- RICARDO, 1985, p. 225). c) “aqui se você precisá de um ortopedista, se você precisá de um gastro então, imagina, eu acho, eu duvido que já tenha tido um gastro aqui em Goiás e assim... hoje em dia, adultos e crianças com a péssima alimentação que fazem... um gastro acaba sendo essencial a gente tem estrutura física, às vezes mas não tem estrutura humana pra podê atendê todo mundo” (BERNARDES, 2020 – Corpus Sociolinco).  Obs.: O professor deve explicou a diferença entre os fenômenos graduais e descontínuos (BORTONI-RICARDO, 2004). 3.1.2 Proposta II Partindo do pressuposto de que os alunos compreenderam os contínuos de variação linguística, bem como o que caracteriza os fenômenos descontínuos e graduais, REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 8 a presen a present a presente etapa tem como objetivo levá-los a refletir acerca do uso variável de /R/ em nomes e verbos no infinitivo, em uma perspectiva regional. Para isso, propõe-se a análise de cartas linguísticas, o que motivaria a compreensão do fenômeno sob um viés interdisciplinar com a Geografia. A esse respeito, é importante salientar que, conforme as pesquisas sociolinguísticas acerca do PB, o apagamento do /R/ em coda externa em infinitos verbais é mais frequente em verbos do que em nomes. Também é significativo acentuar também que o apagamento do /R/ em coda externa em infinitivos verbais é um fenômeno gradual, por isso, não sofre estigmatização como os fenômenos descontínuos. Além disso, é importante mencionar que há variantes de /R/ estigmatizadas, como por exemplo, a retroflexa (/R/ caipira), e que tal fato se deve a fatores ideológicos e sociais. Vale a pena ressaltar que esse mesmo fenômeno, quando associado a um falante de inglês americano, é visto como status. Atividade: Observe abaixo, nas três Cartas linguísticas retiradas do Alib (Atlas linguístico do Brasil), as ocorrências do /R/ nas diversas regiões do nosso país e reflita sobre acerca dessas ocorrências, levando em consideração as seguintes questões: a) O apagamento do /R/ é mais frequente em nomes ou em verbos? b) Em quais regiões o /R/ retroflexo é mais presente? b) Em quais regiões o /R/ retroflexo é mais presente? b) Em quais regiões o /R/ retroflexo é mais presente? c) Na sua opinião a produção do /R/ retroflexo é mais estigmatizado do que o apagamento do /R/ em verbos no infinitivo? Por quê? REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 9 REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 9 9 Figura 1: Carta F04 C2 do ALiB Fonte: Cardoso et al (2014, p. 101). Figura 2: Carta F04 C2 do ALiB Fonte: Cardoso et al (2014, p. 103). Figura 3: Carta F04 C4 do ALiB Figura 1: Carta F04 C2 do ALiB Figura 1: Carta F04 C2 do ALiB g Fonte: Cardoso et al (2014, p. 101). Figura 2: Carta F04 C2 do ALiB Fonte: Cardoso et al (2014, p. 101). Fonte: Cardoso et al (2014, p. 101). Fonte: Cardoso et al (2014, p. 101). Figura 2: Carta F04 C2 do ALiB Figura 2: Carta F04 C2 do ALiB REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 10 Figura 2: Carta F04 C2 do ALiB Fonte: Cardoso et al (2014, p. 103). Figura 3: Carta F04 C4 do ALiB Fonte: Cardoso et al (2014, p. 103). Fonte: Cardoso et al (2014, p. 103). Figura 3: Carta F04 C4 do ALiB Figura 3: Carta F04 C4 do ALiB Figura 3: Carta F04 C4 do ALiB Fonte: Cardoso et al (2014, p. 105). Por fim, há que se considerar a proposta III, descrita a seguir, teórica e metodologicamente. Fonte: Cardoso et al (2014, p. 105). Fonte: Cardoso et al (2014, p. 105). Fonte: Cardoso et al (2014, p. 105). Por fim, há que se considerar a proposta III, descrita a seguir, teórica e metodologicamente. 2 O primeiro trecho foi retirado do livro Educação em língua materna – a sociolinguística na sala de aula, de Bortoni-Ricardo (2004). O segundo trecho foi retirado de uma entrevista do corpus Sociolinco, referente à dissertação de Bernardes (2020). O terceiro trecho foi retirado do site https://catracalivre.com.br/educacao/leia-algumas-das-redacoes-que-foram-nota-mil-no-enem-2016/, acesso em 10 de outubro de 2021. 3.1.3 Proposta III A atividade III tem o propósito de motivar os alunos a refletirem acerca do apagamento ou não do /R/ em coda externa dos infinitos verbais em diferentes gêneros textuais. Para isso, serão disponibilizados dois fragmentos de entrevistas: uma com um falante que emprega a norma culta e outra com um falante que utiliza a norma popular. Em seguida, será fornecido um fragmento de um texto dissertativo-argumentativo. Na sequência, os alunos deverão preencher uma tabela e calcular a proporção de uso/apagamento do /R/ em coda externa nos infinitivos verbais presentes nos três excertos, o que estimula, mais uma vez, um trabalho interdisciplinar, dessa vez, com a Matemática. Atividade 1: Observe, a seguir, três trechos com altos níveis de monitoração estilística: (a) uma reunião da associação de moradores de um bairro, (b) uma entrevista e (c), uma REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 11 11 proposta de texto dissertativo-argumentativo do Enem. Os dois primeiros fragmentos são classificados como evento de oralidade. Já o terceiro é categorizado como evento de letramento. No que diz respeito ao contínuo de urbanização, o primeiro enunciador se enquadra no polo rural, já os outros dois se situam no polo urbano. Nesse sentido, percebe-se que há diferenças entre as falas, mas também há semelhanças, por exemplo: o apagamento do /R/ em coda externa dos infinitivos verbais nas duas entrevistas. Com base nisso, analise a ocorrência desse fenômeno nos três trechos2 e, logo após, preencha a tabela. 3 S1 refere-se ao sujeito entrevistado e, D1, ao documentador. (a) Fala monitorada de um falante que emprega a norma popular: Presidente: Bem gente, tratano da distribuição das fossa, primeiro que/ quero avisar que nóis recebemos só cinquenta fossa, mais vamo recebê mais. Antão, nóis tamo propono dois critero pa distribuição: o primeiro é que só vai recebê aquelas pessoa que tá mermo precisando de u’a fossa e segundo é a ordi de inscrição nessa lista que nós fizemo. O que que vocês acha? (...) Presidente: Pra vocês tê toda informação, é preciso participa das reunião... É muito bom a gente só recramá. Presidente: Pra vocês tê toda informação, é preciso participa das reunião... É muito bom a gente só recramá. (b) Fala monitorada de um falante que emprega a norma culta: (b) Fala monitorada de um falante que emprega a norma culta: S1: cobrar D1: cobrá e incentivá, porque eu vi porque eu quase não trato aqui, por isso que você me fala e eu também não sei, eu quase não vou em medico também mas eu vejo, por exemplo, tem aquela F. lá perto... depois eu vi parece que abriu uma outra ali pra baixo, uma outra clínica ali pra baixo não sei não, a outra é lá em cima é... na rua e cima... aí falaram que tavam vindo uns médicos diferentes pra atendê. S1: eu não sei na rua de cima eu sei que atendê lá o doutor Budim, que é um excelente médico, mas é um clínico, às vezes por uma questão específica, o que ele vai poder fazê é te encaminhá pra um especialista. (c) Escrita monitorada de um falante que emprega a norma culta: (b) Fala monitorada de um falante que emprega a norma culta: S13: aqui, se você precisá de um ortopedista, se você precisá de um gastro, então, imagina, eu acho eu duvido que já tenha tido um gastro aqui em Goiás e, assim, hoje em dia, adultos e crianças com a péssima alimentação que fazem, um gastro acaba sendo essencial. A gente tem estrutura física às vezes, mas não tem estrutura humana pra podê atende. D1: eu acho que um pouco das pessoas também incentivar né? REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 12 12 S1: cobrar (c) Escrita monitorada de um falante que emprega a norma culta: Tema: Caminhos para combater a intolerância religiosa no Brasil Tema: Caminhos para combater a intolerância religiosa no Brasil Larissa Cristine Ferreira (2016) Larissa Cristine Ferreira (2016) Larissa Cristine Ferreira (2016) Título: Orgulho Machadiano Título: Orgulho Machadiano Brás Cubas, o defunto-autor de Machado de Assis, diz em suas “Memórias Póstumas” que não teve filhos e não transmitiu a nenhuma criatura o legado da nossa miséria. Talvez hoje ele percebesse acertada sua decisão: a postura de muitos brasileiros frente a intolerância religiosa é uma das faces mais perversas de uma sociedade em desenvolvimento. Com isso, surge a problemática do preconceito religioso que persiste intrinsecamente ligado à realidade do país, seja pela insuficiência de leis, seja pela lenta mudança de mentalidade social. Segundo pesquisas, a religião afro-brasileira é a principal vítima de discriminação, destacando-se o preconceito religioso como o principal impulsionador do problema. Infere-se, portanto, que a intolerância religiosa é um mal para a sociedade brasileira. Sendo assim, cabe ao Governo Federal construir delegacias especializadas em crimes de ódio contra religião, a fim de atenuar a prática do preconceito na sociedade, além de aumentar a pena para quem o praticar. Ainda cabe à escola criar palestras sobre as religiões e suas histórias, visando a informar crianças e jovens sobre as diferenças religiosas no país, diminuindo, assim, o preconceito religioso. REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 13 13 Atividade 2: a) Como se pode observar no exercício anterior, o apagamento do /R/ em coda externa infinitos verbais é um fenômeno gradual, isto é, está presente tanto na fala de falantes rurais, quanto na fala de falantes urbanos. Entretanto, percebe-se que, em textos escritos monitorados, o seu comportamento é diferente. Nesse sentido, analise todos os /R/ finais dos infinitivos verbais dos três excertos da atividade anterior e coloque- os no quadro a seguir.4 Quadro 1: Infinitivos verbais Infinitivos verbais Fala monitorada de um falante que emprega a variedade popular do PB sem /R/ com /R/ Fala monitorada de um falante que emprega a variedade culta do PB sem /R/ com /R/ Escrita monitorada de um falante que emprega a variedade culta do PB sem /R/ com /R/ Fonte: Adaptado de Bortoni-Ricardo (2004). REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 14 4 Os exercícios da atividade 2 foram inspirados no livro Educação em língua materna – a sociolinguística na sala de aula (BORTONI-RICARDO, 2004).  Compare a frequência de infinitivos verbais realizados com /R/ com a frequência de infinitos verbais realizados sem /R/. Você verá que houve mais ocorrências sem /R/ do que com /R/.  Compare a frequência de infinitivos verbais realizados com /R/ com a frequência de infinitos verbais realizados sem /R/. Você verá que houve mais ocorrências sem /R/ do que com /R/. Na próxima seção, serão tecidas algumas análises a respeito de cada proposta de atividade descrita, de modo a expor os objetivos a serem alcançados com cada uma delas. (c) Escrita monitorada de um falante que emprega a norma culta: Quadro 1: Infinitivos verbais Infinitivos verbais Fala monitorada de um falante que emprega a variedade popular do PB sem /R/ com /R/ Fala monitorada de um falante que emprega a variedade culta do PB sem /R/ com /R/ Escrita monitorada de um falante que emprega a variedade culta do PB sem /R/ com /R/ Fonte: Adaptado de Bortoni-Ricardo (2004). b) Depois que você preencher o quadro, deve realizar alguns cálculos simples: b) Depois que você preencher o quadro, deve realizar alguns cálculos simples: REVELLI Vol 14 2022 Tema Livre  Some todas as ocorrências de infinitivos verbais;  Some todas as ocorrências de infinitivos verbais sem /R/;  Some todas as ocorrências de infinitivos verbais com /R/;  Divida o total de ocorrências de infinitivos verbais sem /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais sem /R/;  Divida o total de ocorrências de infinitivos verbais com /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais com /R/; 4 Os exercícios da atividade 2 foram inspirados no livro Educação em língua materna – a sociolinguística na sala de aula (BORTONI-RICARDO, 2004).  Some todas as ocorrências de infinitivos verbais;  Some todas as ocorrências de infinitivos verbais sem /R/;  Some todas as ocorrências de infinitivos verbais com /R/;  Divida o total de ocorrências de infinitivos verbais sem /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais sem /R/;  Divida o total de ocorrências de infinitivos verbais com /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais com /R/;  Some todas as ocorrências de infinitivos verbais;  Some todas as ocorrências de infinitivos verbais sem /R/;  Some todas as ocorrências de infinitivos verbais com /R/;  Divida o total de ocorrências de infinitivos verbais sem /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais sem /R/;  Divida o total de ocorrências de infinitivos verbais com /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais com /R/;  Divida o total de ocorrências de infinitivos verbais com /R/ pelo total de infinitivos verbais. Assim você encontrará a frequência de infinitivos verbais com /R/;  C de infinit 4 Análise das propostas didáticas sobre /R/ em coda A proposta I oportuniza a observação e reflexão acerca do uso da variável /R/ em nomes e verbos no infinitivo em uma visão regional, viabilizando a análise das Cartas linguísticas uma perspectiva interdisciplinar com a Geografia. Ao concretizar essa atividade, são executados: a) o eixo I, no que tange à comparação/reflexão dos dados de apagamento do /R/ nos verbos e nomes, b) o eixo II, devido à problematização do liame entre variação e prestígio e c) o eixo III, devido às cartas que expõem dados de variação regional do /R/ em coda externa no PB. No que toca à proposta II, no final da atividade, observa-se a variação do /R/ em coda externa em infinitivos verbais, tanto em eventos de oralidade monitorada (entrevista), quando nos de escrita monitorada (texto dissertativo-argumentativo). Tais reflexões permitem que os alunos compreendam que a aceitação/não-aceitação de determinados fenômenos está intrinsicamente relacionada a fatores de ordem cultural, mesmo que de forma inconsciente, de acordo com a seguinte proposta: A proposta III contribui para que os discentes entendam que: (1) a norma culta não é homogênea, haja vista que a variação é inerente a língua; (2) a norma culta se difere da gramática normativa; (3) a norma culta falada monitorada difere da norma culta escrita monitorada; (4) as normas vernaculares e cultas possuem fenômenos parecidos e (5) monitoramos mais ou menos o nosso discurso, de acordo o contexto comunicativo. Tal atividade motiva a análise de três contextos que exigem maior grau de monitoração estilística, haja vista que os dois informantes modulam sua fala em virtude de estarem participando de uma entrevista. Também seria oportuno explorar mais três abordagens: falas espontâneas e contextos de escrita menos monitorada, a fim de que os REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 15 estudant estudante estudantes pudessem averiguar como se configura o uso do /R/ finais dos infinitos verbais em contextos discursivos menos monitorados. estudantes pudessem averiguar como se configura o uso do /R/ finais dos infinitos verbais em contextos discursivos menos monitorados. 5 Considerações finais Ao longo deste artigo, é possível mensurar o quanto a colonialidade afetou o uso da língua materna no Brasil e como a noção de inferioridade linguística reverbera no ensino Língua Portuguesa. Em outras palavras, muitas práticas pedagógicas tradicionais são embasadas em normas que não representam o PB, nem no que tange à norma popular, nem no que diz respeito à norma culta. Tal cenário fragiliza a identidade linguística brasileira, gerando o mito de que o brasileiro não sabe falar português. A Sociolinguística revela, com base em dados robustos de uso, que o PB real difere daquela língua que é postulada pela gramática tradicional; que a norma culta não é homogênea e que os falantes não monitoram sua fala em todos os contextos discursivos; que as normas populares apresentam traços específicos, mas não são totalmente divergentes das normas cultas e que estas também variam na modalidade escrita, conforme o gênero textual. Além dessa série de postulados, a Sociolinguística também mostra que, é necessário aprender a norma culta, a fim de adequar a performance linguística diante de certas exigências comunicativas, haja vista que a aprendizagem dessa norma nos proporciona, de certa forma, maiores oportunidades à ascensão social. Além disso, é destacável que o arcabouço teórico da Sociolinguística fortalece as práticas pedagógicas, contribuindo para que os alunos estejam aptos a refletir sobre sua própria língua e a adquirir uma ampla gama de recursos comunicativos, para os diversos contextos discursivos. Neste artigo, enfocou-se o uso do /R/ em coda externa dos infinitivos verbais (traço gradual), mas, antes disso, foi desenvolvida uma reflexão mais ampla acerca da sistematicidade da variação linguística. Nesse sentido, tal abordagem se faz relevante, contribuindo para um ensino decolonial e interdisciplinar. Por meio das propostas aventadas, é possível descortinar dogmas e proporcionar uma abordagem científica nas aulas de língua materna no Ensino Fundamental. Referências REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 16 16 LIMA, M. D. A. O. O quadro pronominal: atividades lúdicas para o ensino de gramática e variação. In: VIEIRA, S. R. Gramática variação e ensino: diagnose e propostas pedagógicas. Rio de Janeiro: Letras UFRJ, 2017. P. 107-143. LINARES, A. B. B; PEIXOTO, C. R; MOREIRA. T. Apagamento do /R/ em final de palavras: um estudo comparativo entre falantes do nível culto e do nível popular. Disponível em <http://www.leffa.pro.br/tela4/Textos/Textos/Anais/CELSUL_VIII/apagamento_do_r.p df> LINARES, A. B. B; PEIXOTO, C. R; MOREIRA. T. Apagamento do /R/ em final de palavras: um estudo comparativo entre falantes do nível culto e do nível popular. Disponível em palavras: um estudo comparativo entre falantes do nível culto e do nível popular. Disponível em <http://www.leffa.pro.br/tela4/Textos/Textos/Anais/CELSUL_VIII/apagamento_do_r.p df> Disponível em <http://www.leffa.pro.br/tela4/Textos/Textos/Anais/CELSUL_VIII/apagamento_do_r.p df> MARTINS, Marco Antonio; VIEIRA, Silvia Rodrigues; TAVARES, Maria Alice. Contribuições da sociolinguística brasileira para o ensino de português. Ensino de português e sociolinguística. São Paulo: Contexto, p. 9-35, 2014. MONARETTO, Valéria Neto de Oliveira. A vibrante pós-vocálica em Porto Alegre. EdiPUCRS. Porto Alegre: 2002. OLIVEIRA, M. A. de. Phonological variation and change in Brazilian Portuguese: the case of the liquids. 1983. 270f. (Doutorado em Linguística) – University of Pennsylvania, Philadelphia. OLIVEIRA, L. F. CANDAU, V. M. F. Pedagogia Decolonial e educação anti-racista e intercultural no Brasil. Educação em Revista. Belo Horizonte: UFMG. v. 26, 2010. p. 15-40. SILVA, F. P.; BALTAR, Paula; LOURENÇO, Beatriz. Colonialidade do saber, dependência epistêmica e os limites do conceito de democracia na América Latina. Revista de estudos e pesquisas sobre as Américas, v. 12, n. 1, 2018. p. 68-87. VIEIRA, S. R. Gramática, variação e ensino. Editora Edgard Blücher, 2017. VIEIRA, Silvia Rodrigues. Para Uma Norma-Padrão Flexível no Contexto Escolar: Contribuições dos Estudos Sociolinguísticos, p. 243 -264. In: Dimensões e Experiências em Sociolinguística. São Paulo: Blucher, 2019 SOARES, M. Português na escola: história de uma disciplina curricular. In: BAGNO, M. Linguística da norma. São Paulo: Loyola, 2002, p. 155-177. AMORIM, F. S. Ensino do português brasileiro: por uma pedagogia descolonial. Revista WebSOCIODIALETO, v. 05, n. 14, p. 111-138, 2014. BAGNO, M. Língua, história e sociedade: breve retrospecto da norma-padrão brasileira. In: BAGNO, M. Linguística da norma. São Paulo: Loyola, 2002, p. 179-199. BERNARDES, Patricia M. A variação de segunda pessoa do singular na Cidade de Goiás: você e cê sob um olhar sociolinguístico. 2019. Dissertação (Mestrado em Língua, Literatura e Interculturalidade) - Câmpus Cora Coralina, Universidade Estadual de Goiás, Goiás, 2019. BRASIL. Base Nacional Comum Curricular: Ensino Fundamental. Brasília: MEC/Secretaria de Educação Básica, 2018. BORTONI-RICARDO, Stella Maris. Educação em língua materna. Parábola Ed., 2004. BORTONI-RICARDO, Stella Maris. Nós cheguemu na escola, e agora?: sociolinguística & educação. Parábola, 2005. CALLOU, Dinah; LEITE, Yonne; MORAES, João A. Apagamento do /R/ final no Dialeto Carioca: um Estudo em Tempo Aparente e em Tempo Real. DELTA vol.14, número especial, p. 61-72, 1998. Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-44501998000300006. Acesso em 13 ago. 2011. CARDOSO, S. et al. Atlas linguístico do Brasil, v. 2 (Cartas linguísticas). Londrina: EDUEL, 2014. DORNELES, Darlan Machado. Um estudo variacionista sobre o apagamento do /R/na fala rio branquense. Disponível em <https://www.recantodasletras.com.br/trabalhos- academicos-de-letras/3876612> FERREIRA, L. C. Orgulho machadiano. Disponível em: < https://catracalivre.com.br/educacao/leia-algumas-das-redacoes-que-foram-nota-mil-no- enem-2016/>. FARACO, Carlos Alberto. Norma culta brasileira: construção e ensino. In: ZILLES, Ana Maria S.; FARACO, Carlos Alberto (Orgs.). Pedagogia da variação linguística: língua, diversidade e ensino. São Paulo: Parábola, 2015. p. 19-30. REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 17 LIMA, M e ariaçã REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 18 REVELLI, Vol. 14. 2022. Tema Livre ISSN 1984-6576. E-202203 18 18
https://openalex.org/W1904015299	https://periodicos.uem.br/ojs/index.php/ActaSciAnimSci/article/download/18219/pdf	English	null	<b>Morphology and expression of genes related to skeletal muscle growth in juveniles of pirarucu (<i>Arapaima gigas</i>, Arapaimatidae, Teleostei)</b> - doi: 10.4025/actascianimsci.v35i3.18219	Acta Scientiarum. Animal Sciences	2,013	cc-by	6,317	Fernanda Regina Carani, Bruno Oliveira Da Silva Duran, Tassiana Gutierrez De Paula, Warlen Pereira Piedade and Maeli Dal-Pai-Silva Fernanda Regina Carani, Bruno Oliveira Da Silva Duran, Tassiana Gutierrez De Paula, Warlen Pereira Piedade and Maeli Dal-Pai-Silva Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista, Distrito de Rubião Junior, s/n, 18618-970, Botucatu, São Paulo, Brazil. Author for correspondence. E-mail: fcarani@gmail.com ABSTRACT. Skeletal muscle growth in the pirarucu (Arapaima gigas) is highly interesting to fish farmers because it provides information about how the mechanism in muscle mass increase, characteristic of the species, is regulated. Pirarucu has specific muscle growth that highlights the species’s significance and commercial value. Current research evaluates the morphology and the growth-related gene expression in the red and white skeletal muscles of the pirarucu. Muscle samples were collected from the lateral anterior region and frozen in liquid nitrogen. Histological sections were performed and stained by HE for morphological analysis. Red and white muscle samples were used to determine MyoD, myogenin, and myostatin genes expression by Real-time Polymerase Chain Reaction. Although MyoD and myogenin were not statistically different in the two types of muscles, myostatin was significantly higher in the white rather than in the red muscle. Results show the muscle growth characteristics of the species and may be helpful for improving aquaculture management programs. Keywords: muscle fibre morphology, myogenic regulatory factors, myostatin, skeletal muscle growth, Arapaima gigas, real time PCR. http://www.uem.br/acta ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Acta Scientiarum Doi: 10.4025/actascianimsci.v35i3.18219 http://www.uem.br/acta ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Acta Scientiarum Doi: 10.4025/actascianimsci.v35i3.18219 http://www.uem.br/acta ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Doi: 10.4025/actascianims Morfologia e expressão de genes relacionados ao crescimento do músculo esquelético em juvenis de pirarucu (Arapaima gigas, Arapaimatidae, Teleostei) RESUMO. A caracterização do crescimento muscular no pirarucu (Arapaima gigas) é de elevado interesse para os piscicultores, pois fornece informações de extrema importância sobre como esse mecanismo é regulado e permite o rápido aumento na massa muscular característico da espécie. O pirarucu possui um crescimento muscular típico, o que destaca sua importância e seu valor comercial. O objetivo do presente trabalho foi avaliar a morfologia e a expressão de genes relacionados ao crescimento da musculatura esquelética vermelha e branca do pirarucu. As amostras de músculo vermelho e branco foram obtidas da região lateral anterior superficial e profunda, respectivamente, e estas foram congeladas em nitrogênio líquido. Para análise morfológica, cortes histológicos obtidos em criostato foram submetidos à coloração com HE. A expressão dos genes MyoD, miogenina e miostatina foi feita por PCR em tempo real após transcrição reversa (RT-qPCR). Em relação à expressão de MyoD e miogenina, não houve diferença estatística na comparação entre os músculos; por outro lado, a expressão da miostatina foi significativamente maior no músculo branco, em comparação com o músculo vermelho. Estes resultados refletem as características de crescimento muscular do pirarucu e podem ser úteis na tentativa de melhorar as condições de criação e a sobrevivência da espécie. Palavras-chave: morfologia das fibras musculares, fatores reguladores miogênicos, miostatina, crescimento muscula Arapaima gigas, PCR em tempo real. Acta Scientiarum. Animal Sciences Morphological analysis Histological transversal sections of red and white muscle fibre (10 μm thick) were obtained in a -20°C cryostat and stained with Haematoxylin-Eosin (BANCROFT; STEVEN, 1990). Fish Experiment was approved by the Ethics Committee of the Bioscience Institute, UNESP, Botucatu, São Paulo State, Brazil (Protocol N. 72/07-CEEA). Specimens of pirarucu (Arapaima gigas) (n=7), weighting 47.94 ± 3.64 g and two months old, were euthanized with MS-222 (Tricaine Methanensulfonate-SIGMA®). White and red muscles were isolated from deep and surface layers, respectively, close to the head and near the lateral line (Figure 1). Muscle samples were immediately frozen in liquid nitrogen and stored at -80°C. Myostatin (mstn) is a member of the Transforming Growth Factor beta (TGF-β) superfamily of growth and differentiation factors. It is known for its potent abilities to negatively regulate mammalian skeletal muscle growth (LEE, 2004). Myostatin in mammals is mainly restricted to skeletal muscles (McPHERRON et al., 1997), adipose tissues (GONZALEZ-CADAVID et al., 1998), mammary glands (JI et al., 1998) and cardiac muscles (SHARMA et al., 1999). Due to its wide expression, its function in fish may be more diverse and may influence many other tissues (DELGADO et al., 2008; HELTERLINE et al., 2007; KO et al., 2006; KOCABAS et al., 2002; MACCATROZZO et al., 2001; OSTBYE et al., 2001; RESCAN et al., 2001; ROBERTS; GOETZ, 2001; RODGERS et al., 2001; XU et al., 2003; XUE et al., 2006). Mutations in the myostatin gene result in a ‘double- muscling’ phenotype in many cattle breeds and mice (KAMBADUR et al., 1997; MCPHERRON et al., 1997), whereas a similar muscle increase pattern has also been observed in pigs (JI et al., 1998), chickens (KOCAMIS et al., 1999) and fish (ACOSTA et al., 2005; LEE et al., 2009; MEDEIROS et al., 2009). Figure 1. Pirarucu specimen (Arapaima gigas C.). Muscle sample area is indicated. Figure 1. Pirarucu specimen (Arapaima gigas C.). Muscle sample area is indicated. Figure 1. Pirarucu specimen (Arapaima gigas C.). Muscle sample area is indicated. Introduction hypertrophic muscle growth (ROWLERSON; VEGGETTI, 2001). Proliferating myoblasts are aggregated to the fibre surface and generate new myotubes. When they separate, they give rise to new muscle fibres in a process called hyperplasia (DAL PAI- SILVA et al., 2003a and b; JOHNSTON et al., 2000). Muscle growth in fish depends on the proliferation and differentiation of myogenic precursor cells (termed satellite cells or myoblasts) (JOHNSTON et al., 2000; ROWLERSON; VEGGETTI, 2001). When these cells are activated, they proliferate and differentiate and their nuclei are internalized by existing fibres, which characterize Hypertrophy and hyperplasia are regulated by the sequential expression of Myogenic Regulatory Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 Acta Scientiarum. Animal Sciences 220 Carani et al. Carani et al. Factors (MRFs), myoD, myf-5, myogenin, and mrf4. They are transcriptional factors that bind to a specific DNA sequence, termed E-box (5’-CANNTG-3’), found in the promoter region of many muscle-specific genes (JOHANSEN; OVERTURF, 2005; RESCAN, 2001; SABOURIN; RUDNICKI, 2000; WATABE, 2001). MyoD and myf-5 control the determination of myogenic lineage and regulate myoblast activation and proliferation (GOULDING et al., 1994). Myogenin and mrf4 act in the myoblast differentiation stage in which myotubes fuse to form new myofibres (GROBET et al., 1997). Acta Scientiarum. Animal Sciences Quantitative PCR (qPCR) Skeletal muscle fibres in the pirarucu are organized in distinct compartments: the surface red layer, immediately under the skin, composed of slow-twitch oxidative fibres, and the deep white layer, which predominates in the myotome, is formed by fast-twitch glycolytic fibres. Morphological analysis of skeletal muscle in the pirarucu showed a similar muscle fibres pattern within the compartments; fibres were polygonal or round in shape, multinucleate, and with peripheral nuclei. The muscles had fibres of different sizes separated by a connective tissue, usually called endomysium, and arranged in fascicules by a thick connective septum (Figure 2). Further, the surface red muscle in the pirarucu appeared as a large layer in the lateral line region across the whole body (not shown). MyoD, myogenin, and myostatin genes expression analyses were performed in an ABI 7300 Real Time PCR System (Applied Biosystems, Foster City CA USA). Data from Power SYBR Green PCR amplicons were collected with ABI 7300 System SDS software v. 1.4 (Applied Biosystems, Foster City CA USA). Fluorescence signal baseline and threshold were set manually for each detector (myoD, myogenin, myostatin and 18S rRNA), generating a threshold cycle (Ct) for each sample. Relative quantification was obtained by the Comparative Ct Method (ΔΔCt Method), which required an amplification validation, showing that efficiencies of target and reference genes were similar (LIVAK; SCHMITTGEN, 2001). All PCR efficiencies in current study were measured and found adequate (slope < 0.1). Differences between Ct values were calculated for each mRNA by taking mean Ct of duplicate reactions and subtracting mean Ct of duplicate reactions for the reference gene mRNA (ΔCt = Cttarget gene – Ctreference gene). All samples were then normalized to ΔCt rate of a calibrator sample to obtain a ΔΔCt rate (ΔCttarget – ΔCtcalibrator). Red muscle (which had the lowest relative expression, i.e., highest Ct value, for all genes) was chosen as calibrator sample to evaluate putative target gene differential mRNA expression between the two muscle types. The Comparative Ct method (ΔΔCt) calculated relative quantifications in relation to calibrator sample concentrations (2-ΔΔCt), expressed in arbitrary units and normalized for the endogenous reference gene (18S rRNA). Therefore, by using the 2-ΔΔCt method, data were recorded as the fold-change in gene expression normalized with the reference gene and relative to the calibrator sample (LIVAK; SCHMITTGEN, 2001). Dissociation melting curves confirmed the specific amplification of the cDNA target and the absence of nonspecific amplification products. Maringá, v. 35, n. RNA extraction and RT-PCR Total RNA was extracted from muscle samples using TRIzol® Reagent (Invitrogen, Carlsbad CA USA) protocol. Purity and yield were determined by measuring absorbance of aliquots at 260 and 280 nm and total RNA integrity was confirmed by inspecting the electrophoresis pattern of 28S and 18S ribosomal RNA. Total RNA was incubated with DNase I - Amplification Grade (Invitrogen Life Technologies, Carlsbad CA USA) to remove any genomic DNA present and two micrograms of RNA were reverse transcribed using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City CA USA). These samples were PCR amplified with specific primers pairs to myoD, myogenin and myostatin genes, designed from Ictalurus furcatus sequences (Accession n. AY562555, AY540993 and AY540992, respectively), available in GenBank database (http://www.ncbi.nlm. nih. gov). A set of primers designed from the 18S ribosomal RNA consensus fish sequences was used to amplify a segment of 18S rRNA gene in pirarucu muscle samples (TOM et al., 2004), used as reference gene to normalize the relative quantification results. The Amazonian pirarucu (Arapaima gigas) is an economically important large-size fish for aquaculture projects. It grows fast and the adult reaches up to 3 m in length and 250 kg in weight (SOUZA; VAL, 1990). It has been bred under intensive conditions, with satisfactory production results, reaching 10 kg in just one year (IMBIRIBA, 2001; PEREIRA-FILHO et al., 2003). Muscle morphology and myoD, myogenin, and myostatin genes expression in red and white muscle of pirarucu were analyzed to understand the cellular and molecular mechanisms involved in regulating fish muscle growth. Current study is a contribution towards fish aquaculture since improved production rates are directly related to muscle growth dynamics of fish species, in particular aquaculture species. Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 Acta Scientiarum. Animal Sciences Statistical Analysis All PCR products were sequenced and used to design primer pairs for the qPCR analysis with Primer Express® software (Applied Biosystems, Foster City CA USA): myoD (forward) 5’ CCA GCC CCA GGT CCA ACT, (reverse) 5’ ACA CGT TGG GCC ATT GAA A; myogenin (forward) 5’ AGG CTA CCC AAG GTG GAG ATC, (reverse) 5’TGC AGC CGC TCG ATG TAC T; myostatin (forward) 5’ CGA AGT ACA TGC ACC TGC AGA A, (reverse) 5’ CGT GGG TTG GCC TTG TTT AC; 18S rRNA (forward) 5’ TAC CAC ATC CAA AGA AGG CAG, (reverse) 5’ TCG ATC CCG AGA TCC AAC TAC. Unpaired t-Test compared differences in myoD and myogenin mRNA levels between the calibrator sample (red muscle) and the white muscle samples. Welch’s corrected unpaired t-Test compared differences in myostatin mRNA expression levels for the two groups. The statistical significance level was set at 5% (p < 0.05) for all analyses. GraphPad InStat v. 3.01 software for Windows for the two tests (1998, GraphPad Software, San Diego CA USA) were employed. Skeletal muscle growth in pirarucu 221 Statistical Analysis Discussion Quantitative analysis showed that estimated myoD mRNA levels were not statistically different (p > 0.05) in the pirarucu’s red and white muscles (Figure 3). Similarly, estimated myogenin mRNA levels were not different (p > 0.05) between the two muscle types (Figure 4). Contrastingly, estimated myostatin mRNA levels showed a striking difference (p < 0.01) between muscle types (Figure 5). Data are expressed as mean ± SEM, represented as arbitrary units. Morphological analysis of red and white muscles showed a typical muscle fibre pattern – polygonal or round, multinucleated, and with peripheral nuclei. The two muscles had fibres of different sizes separated by connective tissue, usually called endomysium, and arranged in fascicules by a thick connective septum (Figure 2). The fibre arrangement is similar to that of other teleost fish and depends on fish environment requirements (AGUIAR et al., 2005; DAL PAI et al., 2000; FERNANDEZ et al., 2000), fish species, developmental growth stage and swimming behaviour movement (ANDO et al., 1992; DAL PAI-SILVA et al., 2003a and b). The presence of a thicker connective tissue of perimysium in the pirarucu’s skeletal muscle may provide a support structure for the maintenance of the whole body and allows the transmission of contraction force from muscle fibres to the axial skeleton and caudal fin. These integrated and simultaneous events generate the body wave motion and propulsion movement, extremely important during swimming behaviour of large-size fish (SÄNGER; STOIBER, 2001) such as the pirarucu. Figure 3. Relative MyoD mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas) by Real-time PCR. No significant differences between groups were reported by unpaired t-Test (p > 0.05). Myogenin mRNA levels in red and white muscle (A. gigas) MyoD mRNA levels in red and white muscle (A. gigas) MyoD mRNA levels in red and white muscle (A. gigas) Figure 3. Relative MyoD mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas) by Real-time PCR. No significant differences between groups were reported by unpaired t-Test (p > 0.05). Muscle fibres in the pirarucu are distributed into distinct layers, surface red and deep white, similar to those of most fish species (AGUIAR et al., 2005; DAL PAI-SILVA et al., 1996; SÄNGER; STOIBER, 2001). Discussion White muscle is a widely studied compartment in fish because it comprises most of the fillet and is related to the fast-twitch function and to the greater power outputs required for escape responses and predation behaviour (JOHNSTON, 2006). Although the red muscle in the pirarucu is less important for aquaculture production, a thick layer of red muscle fibres along the whole body lateral line may be directly involved with the breathing behaviour. In fact, it acts as the main source of slow-oxidative fibres for the continuous and sustained swimming movements during aerial respiratory behaviour (CARANI et al., 2008). Myogenin mRNA levels in red and white muscle (A. gigas) Acta Scientiarum Animal Sciences Figure 4. Relative myogenin mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas). No significant differences between groups were reported by unpaired t-Test (p > 0.05). Figure 5. Relative myostatin mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas) by Real-time PCR. Welch’s corrected unpaired t-Test shows significant differences between groups (p < 0.01). MSTN mRNA levels in red and white muscle (A. gigas) (A. gigas) ( g g ) Figure 4. Relative myogenin mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas). No significant differences between groups were reported by unpaired t-Test (p > 0.05). MSTN RNA l l i d d hi l ( g g ) Figure 4. Relative myogenin mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas). No significant differences between groups were reported by unpaired t-Test (p > 0.05). MSTN mRNA levels in red and white muscle (A. gigas) MSTN mRNA levels in red and white muscle (A. gigas) MSTN mRNA levels in red and white muscle (A. gigas) During postnatal muscle growth in fish, hyperplasia is mainly regulated by the Myogenic Regulatory Factor myoD, which stimulates myoblast proliferation and posterior myotube formation (GOULDING et al., 1994; RESCAN, 2001). Current study failed to detect any difference in myoD gene expression between red and white muscles. The above results are similar to myoD gene expression studies in other fish species at different development stages, such as those found in the rainbow trout (Oncorhynchus mykiss) (JOHANSEN; OVERTURF, 2005). However, many other studies Figure 5. Relative myostatin mRNA expression in red (RM) and white (WM) muscles of pirarucu (Arapaima gigas) by Real-time PCR. Quantitative PCR (qPCR) 3, p. 219-226, July-Sept., 2013 Figure 2. Transversal sections of red (A) and white (B) muscles from juvenile pirarucu (Arapaima gigas); Polygonal fibres (F); Endomysium (arrowheads); Connective septum (); Scale: 20 μm. (HE). Figure 2. Transversal sections of red (A) and white (B) muscles from juvenile pirarucu (Arapaima gigas); Polygonal fibres (F); Endomysium (arrowheads); Connective septum (); Scale: 20 μm. (HE). Acta Scientiarum. Animal Sciences Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 222 MyoD, myogenin and myostatin mRNA expression Quantitative analysis showed that estimated myoD mRNA levels were not statistically differen (p > 0.05) in the pirarucu’s red and white muscle (Figure 3). Similarly, estimated myogenin mRNA leve were not different (p > 0.05) between the two musc types (Figure 4). Contrastingly, estimated myostat mRNA levels showed a striking difference (p < 0.01 between muscle types (Figure 5). Data are expressed mean ± SEM, represented as arbitrary units. Figure 3. Relative MyoD mRNA expression in red (RM) an white (WM) muscles of pirarucu (Arapaima gigas) by Real-tim PCR. No significant differences between groups were reporte by unpaired t-Test (p > 0.05). Myogenin mRNA levels in red and white muscle (A. gigas) MyoD mRNA levels in red and white muscle (A. gigas) 222 Carani et al. Acta Scientiarum. Animal Sciences Discussion Welch’s corrected unpaired t-Test shows significant differences between groups (*p < 0.01). Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 Acta Scientiarum. Animal Sciences Skeletal muscle growth in pirarucu 223 fast muscles. Red muscle exhibited low levels of myostatin mRNA content, whereas myostatin expression was extremely high in white muscle. The myostatin expression pattern is analogous to that observed in adult mammals, where myostatin mRNA levels are higher in fast-glycolytic fibres than in slow ones (CARLSON et al., 1999; MATSAKAS et al., 2006). Based on these results and on the observation that satellite cells express myostatin and are quiescent (MCCROSKERY et al., 2003), it has been suggested that the normal function of myostatin in postnatal muscle is to maintain satellite cells in a quiescent, undifferentiated state (MANCEAU et al., 2008). compare myoD expression in fish muscles with no similarity between results (DELALANDE; RESCAN, 1999; TAN; DU, 2002; ZHANG et al., 2006). Discordant results, corroborated by those in current analysis, presume that myoD gene expression in fast and slow muscles may reflect differences in regulating the growth mechanisms of these two types of muscle fibre depending on fish species. In the pirarucu, the similar myoD gene expression in fast and slow muscles seems to be related to the intense satellite cell proliferation involved with intense hyperplasia and hypertrophy mechanisms occurring in both muscles during this growth stage. These events may contribute towards the great muscle mass increase that occurs in the species. Current results show that since the pirarucu’s red muscle has low myostatin mRNA levels, intense satellite cell proliferation and hyperplasia are indicated. In contrast, white muscles have high myostatin mRNA levels and may indicate that it is acting on the white muscle fibre inhibiting satellite cell proliferation. This event disagrees with the high myoblast proliferation rate and fibre hyperplasia that occur at this development stage, regardless of the slow/fast phenotype. Although satellite cells are highly activated, a large population of myotubes has already been formed and their next step is to differentiate into new myofibres. The differentiation process is well characterized and is controlled by the Myogenic Regulatory Factor myogenin (GROBET et al., 1997). The idea that myostatin is regulating the differentiation process by controlling myogenin action is well supported by studies that show myogenin as a probable major target of endogenous myostatin (JOULIA et al., 2003). Discussion Myogenin is characterized by regulating the later stages of muscle fibre formation and growth, which culminate in myoblast fusion, myotube formation, and finally, adult muscle fibre differentiation (GROBET et al., 1997; SCHMALBRUCH; LEWIS, 2000). Current investigation showed that myogenin gene expression had a similar pattern to myoD expression: there was no difference between red and white muscles. This may have been related to the intense process of muscle fibre differentiation occurring during the pirarucu’s growth stage. The fibres, primarily similar to undifferentiated myoblasts, which are at first stimulated by myoD expression to proliferate, now have to fuse and form multinucleated myotubes that differentiate into muscle fibres. In fact, these later processes are mainly regulated by myogenin. Similar myogenin expression levels in red and white muscles may be attributed to the intense muscle fibre formation rate and to the differentiation that occur in a highly activated state in both muscles in the pirarucu’s initial stage. Conclusion Current results suggest that myoD, myogenin, and myostatin modulate the equilibrium between satellite cells proliferation and differentiation during postnatal muscle growth in the initial growth phase of the pirarucu. Future research within the study of muscle growth-related genes throughout the growth phases are of great interest within the context of the fast body development that occurs in this large freshwater ﬁsh during such a short period. Few studies are extant that analyze myostatin expression in slow and fast muscles separately. According to Roberts and Goetz (2001), myostatin mRNA is higher in the red muscle from the adult brook trout, king mackerel, and yellow perch. It is also predominantly expressed in the white muscle from the little tunny and equally expressed in both muscle types in mahi-mahi (ROBERTS; GOETZ, 2001). stbye et al. (2001) demonstrated that myostatin mRNA in salmon is detectable in the red muscle only. Furthermore, Patruno et al. (2008) have shown high levels of myostatin in red and low levels in white muscle of adult sea bass. Nonetheless, there were no significant differences in juveniles. Current study shows a wide difference in myostatin expression between the pirarucu’s slow and Acta Scientiarum. Animal Sciences References 205-211, 2008. CARLSON, C. J.; BOOTH, F. W.; GORDON, S. E. Skeletal muscle myostatin mRNA expression is fibre-type specific and increases during hindlimb unloading. American Journal of Physiology, v. 277, n. 2, p. 601-606, 1999. HELTERLINE, D. L. I.; GARIKIPATI, D.; STENKAMP, D. L.; RODGERS, B. D. Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish. HELTERLINE, D. 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Marine Biotechnology, v. 7, n. 6, p. 576-587, 2005. DAL PAI-SILVA, M.; DAL PAI, V.; MOTA, D. L.; RODRIGUES, A. C. Histochemical study of muscle fibre types in Synbranchus marmoratus Boch, 1795. Annals of Anatomy, v. Acknowledgements Current research was funded by FAPESP Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo, Process 2007/59584-0 and 2007/58896-9) and by CNPq Foundation (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Processes 304070/2009- Acta Scientiarum. Animal Sciences Maringá, v. 35, n. 3, p. 219-226, July-Sept., 2013 224 Carani et al. DELGADO, I.; FUENTES, E.; ESCOBAR, S.; NAVARRO, C.; CORBEAUX, T.; REYES, A. E.; VERA, M. I.; ALVAREZ, M.; MOLINA, A. Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus). Comparative Biochemistry and Physiology B, v. 151, n. 2, p. 197-202, 2008. 2 and 474084/2009-4). 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https://openalex.org/W2143595798	https://www.frontiersin.org/articles/10.3389/fpsyg.2014.01581/pdf	English	null	Collective screening tools for early identification of dyslexia	Frontiers in psychology	2,015	cc-by	15,174	INTRODUCTION has shown that inattention symptoms are associated with weak- nesses in naming speed and working memory (Shanahan et al., 2006; Lui and Tannock, 2007; Marzocchi et al., 2008; Arnett et al., 2012), especially working memory tasks requiring manipulation of information with a higher demand on the central executive mechanisms (Kasper et al., 2012) and mainly those involving visu- ospatial manipulations (Martinussen et al., 2005; Willcutt et al., 2005; Castellanos et al., 2006). In sum, there is an emergent con- sensus that inattention per se is associated with RN and working memory deﬁcits and, hence, is a risk factor for dyslexia regard- less of the presence or absence of PA impairment (Shanahan et al., 2006; Shaywitz and Shaywitz, 2008; Arnett et al., 2012). Dyslexia is a neurologically based learning disorder with a genetic origin (Vellutino et al., 2004; Shaywitz et al., 2008; Peterson and Pennington, 2012) deﬁned as “difﬁculties in accuracy or ﬂuency of reading that are not consistent with the person’s chrono- logical age, educational opportunities, or intellectual abilities” (Snowling and Hulme, 2012, p. 594); prevalence in the gen- eral population varies from 5 to 17% according to language, orthography transparency and with the criteria used to deﬁne low-achievement on reading ability (Ziegler and Goswami, 2005; Shaywitz et al., 2008). There is a great body of evidence since Bradley and Bryant’s (1983) study that phonological processing abilities, mainly phonological awareness (PA),rapid naming (RN), and verbal working memory (VWM), are the strongest early pre- dictors of literacy acquisition (Fletcher et al., 2002; Fuchs et al., 2012) whose difﬁculties are considered by many as the primary causeof dyslexia(Ramus,2003;Vellutinoet al.,2004; Snowlingand Hulme, 2012). However, there is also cogent evidence that RN can be an independent risk factor with separate and unique predictive power (Wolf and Bowers, 1999; Boada et al., 2012). Additionally, children with attention deﬁcit hyperactivity disorder (ADHD) fre- quently show reading problems and a growing body of research In an attempt to capture the unexpected nature of reading dif- ﬁculties emphasized in the deﬁnitions of dyslexia, the traditional clinical approaches have sought to operationalize its identiﬁca- tion/diagnostic in terms of an IQ-achievement discrepancy, which requires reading scores of one or one-and-one-half standard devi- ation below standard scores on IQ for a child to be considered learning-disabled (Fuchs and Fuchs, 2006). However, the IQ- discrepancy model has been frequently criticized for two main reasons. Olga V. C. A. Andrade1, Paulo E. Andrade1* and Simone A. Capellini 2* Olga V. C. A. Andrade1, Paulo E. Andrade1* and Simone A. Capellini 2* Paulo E. Andrade1* and Simone A. Capellini 2* School of Elementary and Secondary Education “Colégio Criativo,” Marília, Brazil Sciences, São Paulo State University “Júlio de Mesquita Filho” – Faculdade de Filosoﬁa e Ciências/Universidade Estadual Olga V. C. A. Andrade1, Paulo E. Andrade1* and Simone A. Capellini 2* 1 Department of Pedagogical Studies, School of Elementary and Secondary Education “Colégio Criativo,” Marília, Brazil 2 Department of Speech and Hearing Sciences, São Paulo State University “Júlio de Mesquita Filho” – Faculdade de Filosoﬁa e Ciências/Universidade Estadual Paulista, Marília, Brazil Current response to intervention models (RTIs) favor a three-tier system. In general,Tier 1 consists of evidence-based, effective reading instruction in the classroom and universal screening of all students at the beginning of the grade level to identify children for early intervention. Non-responders to Tier 1 receive small-group tutoring in Tier 2. Non- responders to Tier 2 are given still more intensive, individual intervention in Tier 3. Limited time, personnel and ﬁnancial resources derail RTI’s implementation in Brazilian schools because this approach involves procedures that require extra time and extra personnel in all three tiers, including screening tools which normally consist of tasks administered individually. We explored the accuracy of collectively and easily administered screening tools for the early identiﬁcation of second graders at risk for dyslexia in a two-stage screening model. A ﬁrst-stage universal screening based on collectively administered curriculum-based measurements was used in 45 7 years old early Portuguese readers from 4 second-grade classrooms at the beginning of the school year and identiﬁed an at-risk group of 13 academic low-achievers. Collectively administered tasks based on phonological judgments by matching ﬁgures and ﬁgures to spoken words [alternative tools for educators (ATE)] and a comprehensive cognitive-linguistic battery of collective and individual assessments were both administered to all children and constituted the second-stage screening. Low-achievement on ATE tasks and on collectively administered writing tasks (scores at the 25th percentile) showed good sensitivity (true positives) and speciﬁcity (true negatives) to poor literacy status deﬁned as scores ≤1 SD below the mean on literacy abilities at the end of ﬁfth grade. These results provide implications for the use of a collectively administered screening tool for the early identiﬁcation of children at risk for dyslexia in a classroom setting. ORIGINAL RESEARCH ARTICLE bli h d 23 J 2015 published: 23 January 2015 doi: 10.3389/fpsyg.2014.01581 Reviewed by: Kathy Ellen Green, University of Denver, USA Mohamed L. Seghier, University College London, UK Mohamed L. Seghier, University College London, UK Layne Kalbﬂeisch, George Mason University, USA Layne Kalbﬂeisch, George Mason University, USA Layne Kalbﬂeisch, George Mason University, USA Layne Kalbﬂeisch, George Mason University, USA Reviewed by: Kathy Ellen Green, University of Denver, USA Mohamed L. Seghier, University College London, UK Collective screening tools for early identiﬁcation of dyslexia Olga V. C. A. Andrade1, Paulo E. Andrade1* and Simone A. Capellini 2* 1 Department of Pedagogical Studies, School of Elementary and Secondary Education “Colégio Criativo,” Marília, Brazil 2 Department of Speech and Hearing Sciences, São Paulo State University “Júlio de Mesquita Filho” – Faculdade de Filosoﬁa e Ciências/Universidade Estadual Paulista, Marília, Brazil Keywords: language, phonological processing, reading, writing, children, early literacy Correspondence: Correspondence: Paulo E. Andrade, Department of Pedagogical Studies, School of Elementary and Secondary Education “Colégio Criativo,” Rua das Saﬁras, 45, Marília, São Paulo, Brazil e-mail: paulo_sustain@yahoo.com; Simone A. Capellini, Department of Speech and Hearing Sciences, São Paulo State University “Júlio de Mesquita Filho” – Faculdade de Filosoﬁa e Ciências/Universidade Estadual Paulista, Avenida Hygino Muzzy ﬁlho, 737 Bairro: Mirante, 17.525-000 – Marília, São Paulo, Brazil Brazil e-mail: sacap@uol.com.br e-mail: sacap@uol.com.br www.frontiersin.org INTRODUCTION An important rationale underlying this trade-off is that FNs mean the number of children actually at risk who will not receive the intervention they need, whereas FPs mean the number of children not at risk who will receive extra instruction (Fletcher et al., 2002; Gilbert et al., 2012). Although FPs are of concern because they imply increased costs to already costly interven- tions, the FNs are much less desirable (Fletcher et al., 2002; Barth et al., 2008). Researchers have currently favored RTI models of three tiers (Fuchs and Fuchs, 2006, 2007; Davis et al., 2007; Fletcher and Vaughn, 2009). In general, Tier 1 consists of evidence-based, effec- tive reading instruction in the general education classroom and universal screening of all students at the beginning of the school year to identify children for early intervention. Those identiﬁed as at-risk have their progress monitored for a short time (e.g., 5 weeks) to conﬁrm/disconﬁrm the risk status. Non-responders to Tier 1 are referred to Tier 2 which consists in small-group tutoring of three to ﬁve students for 20–40 min daily, 3 to 4 times per week over 15- to 20-week sessions (Fuchs and Fuchs, 2007; Fletcher and Vaughn, 2009). Non-responders to Tier 2 are deemed unexpected low-achievers and then are referred to Tier 3, the most inten- sive form of instruction and already considered special education (Fuchs and Fuchs, 2007; Fletcher and Vaughn, 2009), where they are given still more intensive, individual, and increased interven- tion (45–60 min or up to 1.5 h per day) with a more specialized teacher (Fuchs and Fuchs, 2007; Fletcher and Vaughn, 2009). Since universal screening involves all students in a school it is imperative “that the tool can be quickly administered with ade- quate sensitivity and speciﬁcity” (Fletcher and Vaughn, 2009, p. 32). Traditionally, RTI approaches implement screening using curriculum-based measurements (CBMs), typically a single word reading measure administered individually at the beginning of the school year (Fuchs and Fuchs, 2006; Fletcher and Vaughn, 2009). The selection of a critical cutoff score to deﬁne at-risk perfor- mance vary from more conservative cut-points, such as 1 to 2 standard deviation (SD) below the mean, to more liberal, such as 25th and 30th percentiles, which correspond approximately 0.68 SD and 0.5 SD, respectively (Catts et al., 2001; Fletcher et al., 2002; Fuchs and Fuchs, 2006; Davis et al., 2007; Fletcher and Vaughn, 2009). INTRODUCTION Firstly, even if a student show clear difﬁculties since the early days of learning to read, he/she must wait until the third January 2015 \| Volume 5 \| Article 1581 \| 1 www.frontiersin.org Collective identiﬁcation of dyslexia Andrade et al. year or more (typically until the ﬁfth grade) so that student’s achievement is sufﬁciently low and the discrepancy between IQ and achievement becomes large enough to allow the diagnostic and, hence, the student can be considered eligible for interven- tion. Secondly, research has failed to ﬁnd qualitative differences in reading performance and cognitive abilities between low-achievers who fulﬁll the requirement of discrepancy and low-achievers who do not, but according to discrepancy model only the ﬁrst ones will receive the intervention they need. Therefore, the discrepancy model is deemed unfair because it deprives of necessary assistance those children who are as needy and deserving as those given the label (Fuchs and Fuchs, 2006, p. 96). For these and other rea- sons, in the reauthorization of the Individuals With Disabilities Education Improvement Act allowed practitioners to use a new, alternative, model of identiﬁcation called “response to interven- tion”(RTI), an evidence-based multi-tiered system of preventative intervention and progress monitoring in which all children are ﬁrst provided with research-based instruction and universal screen- ing of students at risk for dyslexia, whose progress is frequently monitored (Fuchs and Fuchs, 2006, 2007; Fletcher and Vaughn, 2009). hand, occur when some children perform well on the screening test and are not identiﬁed as at risk, but who are actually at risk and soon will become struggling readers. Sensitivity is the term used to designate the proportion of TPs whereas speciﬁcity refers to the proportion of TNs a given screening system is capable to identify. Sensitivity is calculated by dividing the number of TPs by the sum of TPs and FNs [Sensitivity = TP/(TP+FN)] whereas speciﬁcity is obtained by dividing the number of TNs by the sum of TNs and FPs [Speciﬁcity = TN/(TN+FP);Vellutino et al., 2008; Fuchs et al., 2012]. Since FPs and FNs are inherent in all screening measures (Fletcher et al., 2002), then accuracy is necessarily a result of the trade-off between sensitivity and speciﬁcity (Fuchs et al., 2012; Gilbert et al., 2012). Therefore, accuracy is the proportion of TPs and TNs to the whole sample (N) calculated by dividing the sum of TPs and TNs by the whole sample [Accuracy = (TP+TN)/N]. INTRODUCTION Stricter cut-points decrease the probability of FPs while increase the probability of FNs, whereas more liberal cut-points tend to produce the opposite, i.e., to decrease FNs (producing more TPs) while increase FPs (Fletcher et al., 2002; Davis et al., 2007; Barth et al., 2008). Although FNs are a less desirable error than FPs because it excludes children in need of intervention, an inﬂated proportion of TPs implies in delivering costly preven- tion to students who do not need it thus stressing the already scarce school resources (Davis et al., 2007; Barth et al., 2008; Fuchs et al., 2012). While it has been suggested that an acceptable sensi- tivity and speciﬁcity should be around 90 and 85%, respectively (Vellutino et al., 2008), research has shown that 1-time universal screening has produced an unacceptably high proportion of FPs, ranging from 20 to 60% (Vellutino et al., 2008), whereas speci- ﬁcity can range from 20 to ∼60% when sensitivity is held constant (Johnson et al., 2009, 2010). It is generally agreed that universal screening for at-risk stu- dents is a key component of RTI models which plays a crucial role, both in terms of prevention and identiﬁcation, for a successful implementation of RTI (Davis et al., 2007; Fletcher and Vaughn, 2009; Fuchs et al., 2012; Gilbert et al., 2012). An accurate clas- siﬁcation should yield a high percentage of true positives (TPs; children correctly identiﬁed as at risk, i.e., who will indeed show persistent difﬁculties in learning to read) and a high percentage of true negatives (TNs children correctly identiﬁed as not at risk, i.e., who will not have reading difﬁculties). Although the ideal would be to have a classiﬁcation accuracy of 100% of sensitivity and speciﬁcity, no screening system (one or more screening tools used to identify at risk children) reaches these ideal values so that “false positives (FPs) and false negatives (FNs) are inherent to any assessment device and are inevitably linked” (Fletcher et al., 2002, p. 55). FPs result from incorrect identiﬁcation of children who score below the cut score on the screening instrument, but are not actually at risk and soon become good readers. Frontiers in Psychology \| Educational Psychology INTRODUCTION FNs, on the other In order to maximize the number of TPs while limiting the number of FPs it has been proposed that screening for at-risk stu- dents should be made in a two-stage process (Catts et al., 2001; Compton et al., 2010; Fuchs et al., 2012). The ﬁrst-stage consists January 2015 \| Volume 5 \| Article 1581 \| 2 Frontiers in Psychology \| Educational Psychology Andrade et al. Collective identiﬁcation of dyslexia of a universal screening based on a brief individual assessment (normally a measure of reading ﬂuency) using a more lenient cut point (e.g., 30th percentile) in order to maximize TPs, thus excluding most of the TNs from the costly Tier 2 intervention. Children identiﬁed as at-risk in the ﬁrst stage have their progress monitored during a period of about ﬁve or 6 weeks of primary intervention, after which a second second-stage screening consist- ing of a more thorough assessment is conducted, so that only those identiﬁed as at-risk in the second-stage screening are referred to secondary intervention (Compton et al., 2010; Fuchs et al., 2012). For instance, Compton et al. (2010) used the Word Identiﬁcation and Word Attack subtests of the Woodcock Reading Mastery Tests and the Sight Word Efﬁciency and Phonemic Decoding Efﬁciency subtests of the Test of Word Reading Efﬁciency in the ﬁrst-stage, universal screening of at-risk students in a sample of 355 children in the fall of ﬁrst grade. They found that the use of phonemic decoding efﬁciency (the number of nonsense words accurately decoded in 45 s) as the ﬁrst-stage screening tool and 6 weeks of progress monitoring with word identiﬁcation ﬂuency (WIF) and dynamic assessment as the second-stage screening eliminated 43.4% of FPs of the sample, being the most efﬁcient of the four two-stage models tested. Educational resources in Brazil, however, are still much scarcer than in developed countries, either in time, personnel, and ﬁnan- cial resources. This set of unfavorable factors seriously derails RTI’s implementation in Brazilian schools and makes developing strategies to overcome these limitations an obligatory aim. The present investigation is a pilot study in which we explored the utility of two-stage screening models based solely on collective screening tools in a non-clinical school sample of 45 Brazilian sec- ond graders. INTRODUCTION Curriculum-based assessments (CBAs) carried out in the classroom for all students 2 weeks after the start of the school year were used as the universal screening of the ﬁrst stage. As part of the second stage we administered a cognitive-linguistic proto- col (Capellini et al., 2012) and a set of collective screening tools based on phonological judgments by matching ﬁgures and ﬁgures to spoken words as well, hereinafter referred to as alternative tools for educators (ATEs). The Capellini et al. (2012; CS&S) protocol was developed in Brazil with the purpose to identify the cognitive- linguistic proﬁle of Brazilian children in the ﬁrst stages of reading. ATE tasks were also developed in Brazil for the collective assess- ment of phonological abilities by the ﬁrst author (Andrade et al., 2011). Firstly, we performed a principal components analysis (PCA) factor using a varimax rotation including all measures of the C&S protocol and ATE tasks to examine the factor structure of the CS&S protocol and check whether results actually suggest its proposed cognitive-related dimensions (e.g., literacy, phono- logical processing, VWM, and visual and visuospatial working memory). PCA also provided the extent and nature of rela- tionships between ATE tasks and the cognitive and linguistic measures. Composite scores based on summed raw scores of writing, reading, phonological and naming abilities were cre- ated and used to obtain the percentiles and cutoff points to identify low-achievers, as well as to compare performances of low- achiever and non low-achiever on cognitive-linguistic measures and ATE tasks. Although tasks with larger standard deviation will be weighted more when summing raw scores, we used this procedure in place of summed z-scores for its practical value for educators. More recently, Fuchs et al. (2012) have tested ﬁve two-stage screening models in 783 children from 42 ﬁrst-grade classrooms in 16 schools. In the ﬁrst stage, the six lowest scores on WIF and rapid letter naming of each class (∼30th percentile) were identiﬁed as at-risk, totaling 252 students (32% of the sample), of whom only 195 remained until the end of the study. Then, the authors tested two types of screening tool for their second- stage screening. One type was a cognitive battery administered in the early fall consisting of multiple tests measuring de cogni- tive dimensions of RN, phonological processing, oral language comprehension, and non-verbal reasoning. INTRODUCTION The second type of a second-stage screening consisted of 18 weeks of WIF assess- ments from which authors modeled December and May reading outcomes. From the combination of these two types of second- stage screening the researchers derived ﬁve models of second stage-screening. The two models based solely on December and May reading performance resulted in the two lowest speciﬁci- ties (26.5 and 43.9%, respectively). Logistic regression analyses showed that the two models derived from the combination of the cognitive measures with WIF May and December intercepts, respectively, were the two most accurate models to predict ﬁfth- grade reading comprehension when sensitivity was held constant at 91.7%, with speciﬁcities of 68.4% for the May intercept and of 67.3% for the December intercept. The most interesting ﬁnd- ing, however, was that the model based solely on the cognitive measures showed a comparable ﬁt, with a speciﬁcity of 67.3%. This model is less expensive and can be implemented already in the ﬁrst 2 months of the school year, being more parsimonious and, hence superior. The use of a second-stage screening based only on the cognitive measures reduced the number of students in need of tutoring from 195 identiﬁed in the ﬁrst stage to 65, thus eliminating 66.7% of FPs. From the 65 students identiﬁed as at risk in the second stage 32 were FPs and 33 were identiﬁed as dyslexics in ﬁfth grade, what represented signiﬁcant savings for school districts. Finally, composites scores were also used to run regressions analysis in order to explore the predictive power of the collective tools (ATE tasks and C&S writing composite scores) at the end of ﬁfth grade and to run logistic regression analysis in order to obtain the sensitivity, speciﬁcity, and overall accuracy of differ- ent combinations of these collective screening tools in the early identiﬁcation of poor literacy status at the end of ﬁfth grade level. MATERIALS AND METHODS PARTICIPANTS This prospective, longitudinal study was carried out with a non- referred school-based sample of 69 early Portuguese readers, corresponding to all students enrolled in four second-grade class- rooms of elementary education (as per the grade distinctions in the Brazilian education system) in an upper-middle class private school. However, considering children who could not partici- pate because exclusionary factors (sensory deﬁcits, neurological abnormality, known syndrome, etc.) and those who were granted permission by their parents to participate in this study, we were January 2015 \| Volume 5 \| Article 1581 \| 3 www.frontiersin.org Collective identiﬁcation of dyslexia Andrade et al. left with 45 students, of which 41 were right-handed. Partici- pants were native speakers of Brazilian Portuguese and had no major sensorimotor handicaps (deafness, blindness) or pervasive neurodevelopmental disorders (psychosis, autism) or inadequate command of their native language. Age was calculated at the onset of test administration ranging from 6 to 8 years (average: 7 years and 2 months, SD: 4 months). 2004, p. 367). Accordingly, all participants received curricular pedagogical activities specially designed for the present screen- ing procedure and collectively administered to assess performance on core academic achievement areas. For purposes of this study, however, our main focus will be on assessments of reading and writing skills (Fuchs and Fuchs, 2006; Fletcher and Vaughn, 2009). Assessments of literacy abilities in this diagnostic assessment included: Although this sample size is small when it comes to a study on screening students at risk for a disorder, it is still a reasonable one for a pilot study if we take into account that we wanted a sample with certain speciﬁc characteristics, including homogene- ity with respect to age, socioeconomic status, and pedagogical approaches. Additionally, we argue that a good, effective screen- ing system should show consistent results with either relatively small samples or large samples. In fact, RTI approaches usu- ally adopt a normative cut point, referenced to the classroom, school, district, or nation, below which students are designated at-risk (see Fuchs and Fuchs, 2006; Fuchs et al., 2012). Typically, RTI systems employ universal screening whereby all children in a school are assessed at the beginning of the school year with a critical norm-referenced cut-point on certain measures (usu- ally ≤25th percentile). Curriculum-based assessments Students were asked to recall all the letters of the alphabet in writ- ten form. There was no prompting or assistance. Scores were out of 26: the number of correct letters the child wrote down. The task was untimed. Academic achievements were used as a ﬁrst-stage universal screen- ing and evaluated during the four ﬁrst weeks after the start of the school year by the school’s pedagogical team. The school to which belonged the participants of the present study adopts a procedure which in Brazil is called “diagnostic assessment” (Luckesi, 2005), i.e., a comprehensive set of curriculum-based tests applied at the beginning of all grades and at the outset of a unit of study, aimed at evaluating the student’s learning achievements and needs. Accord- ingtoLuckesi(2005)assessmentsintheschoolshouldchangefrom being purely summative to be eminently “diagnostic,” i.e., assess- ments systematically built in to the curriculum with the aim of identifying the strengths and weaknesses of the students in order to make decisions about further teaching strategies and/or speciﬁc interventions, if necessary. Reading tasks (individually administered) (i) Reading ﬂuency: Number of words read aloud and audibly by the student in 1 min from a list of 70 words. If reading of all 70 words last less than 1 min the number of words correctly read was estimated for 1 min. Thus, the maximum score depended ultimately on a child’s reading speed. (ii) Reading accuracy: In the same trial of reading ﬂuency the number of words read aloud correctly from the same list of 70 words (untimed), with a maximum score of 70. (iii) Reading non-words: Number of non-words read aloud cor- rectly from a list of 10 (untimed). Maximum score of 10. Therefore, the diagnostic assessment adopted in the present paper is somewhat similar to the CBM methods (Fuchs and Fuchs, 2006; Fletcher and Vaughn, 2009), also referred to as CBAs (Dombrowski et al., 2004), used in the RTI approaches “to tailor instructional–intervention decisions to more appropri- ately suit the learning needs of the student” (Dombrowski et al., BEHAVIORAL MEASURES The Cognitive-Linguistic Protocol (CS&S) was developed in Brazil with the aim of producing an effective instrument to identify the cognitive-linguistic proﬁle in the ﬁrst stages of reading acquisition of Brazilian children. The CS&S was administered 6 weeks after the start of the school year, and the CS&S measures we used in the present study and their respective scoring criteria are outlined below: Participants were ﬁrst evaluated in their academic achieve- ment using CBAs, in their cognitive-linguistic abilities using Cognitive-LinguisticProtocolbyCapelliniet al.(2012)andintheir phonological abilities by using the collective phonological tasks referred to as ATEs (Andrade, 2010; Andrade et al., 2011, 2013). Participants were also screened for ADHD symptoms. (i) Reading – ﬂuency and accuracy in reading aloud a list of simple mono- syllabic, disyllabic, and trisyllable words, extracted from the textbooks used by the school – draw the meaning of a written word (silent reading) (ii) Writing – letter knowledge: write all the known letters of the alphabet (print or cursive letters); make a circle with red pencil around the vowels and blue pencil around the consonants; write the letter with which begins the name of a picture – syllable knowledge: write the correct syllables to complete the word – word knowledge: using the mobile alphabet form as many words as you know and then write them on the paper – spelling: write the dictated words – spelling: write the dictated words – oral language comprehension: draw what you understood of the story you just listened to. Frontiers in Psychology \| Educational Psychology MATERIALS AND METHODS PARTICIPANTS Furthermore, one important recent study (Fuchs et al., 2012) have used a cut point referenced to the class- room by selecting the six lower scores of each class from 42 ﬁrst-grade classrooms in 16 schools what corresponds approxi- mately to 25th to 30th percentile, for the ﬁrst stage of a two-stage screening process. (i) Reading Visual short term memory (individually administered) Visual short term memory (individually administered) This task was based on the Benton Visual Retention Test (BVRT; Sivan, 1992). In the BVRT’s original version, one or more simple geometric ﬁgures are drawn from memory after a brief exposure (typically 5 s for adults and 10 s for children), and two types of scores are computed: the number of correct representations and the number of errors. The visual short-term memory task from CS&S protocol is different from BVRT in that, instead of drawing from memory, children receive the same ﬁgures they just have After matching a word presented aurally by the examiner with three words depicted as pictures, children are asked to mark with “X” the picture whose name starts with the same sound as the spoken word. After three practice items, the test comprises 10 items leading to a maximum score of 10. Writing tasks (collectively administered) (i) Writing words: Number of words written with correct spelling from a list of 30 presented aurally (untimed and collectively administered in the classroom). Maximum score of 30. January 2015 \| Volume 5 \| Article 1581 \| 4 Frontiers in Psychology \| Educational Psychology Collective identiﬁcation of dyslexia Andrade et al. (ii) Writing pseudowords: Number of pseudowords written with correct spelling from a list of 10 presented aurally. Maximum score of 10. Task was untimed. seen, on cards in scrambled order, and have to organize them in the same order and position. (ii) Writing pseudowords: Number of pseudowords written with correct spelling from a list of 10 presented aurally. Maximum score of 10. Task was untimed. (i) Figure order: Ordering of solid abstract ﬁgures, i.e., after see- ing two to ﬁve-ﬁgure sequences over a 10 s period, student was asked to reassemble the ordered pattern of ﬁgures in the same order and rotation. Score is given by the number of sequences in which the ﬁgures were correctly ordered, with a maximum score of 8. ATE I-alliteration Childrenarepresentedwiththreewords,depictedasthreepictures, and are asked to mark with “X” the two pictures whose name start with the same sound. After three practice items, the test comprises 10 items leading to a maximum score of 10. Shapes copying (collectively administered) Correctly copied four archetypal forms: a circle, a square, a dia- mond, and a complex abstract ﬁgure. Students were able to see shapes while copying was performed. Students were only allowed to erase work on the last shape. Figures were scored through comparison with a standardized table of different visual represen- tations of the shapes and measured with a diagramed seven-point scale. Maximum score for the task was 7. Alternative tools for educators (iii) Verbal number sequence backward: Number of correctly repeated two to ﬁve-number sequences backward (two tri- als for each sequence) that were spoken by the examiner. Maximum score of 8. Consisted of phonological judgments by matching ﬁgures and ﬁgures to spoken words, developed by Andrade (2010) and Andrade et al. (2011) speciﬁcally with the goal of providing an effective non-literacy based tool for the collective assessment of the phonological abilities. Phonological tasks (individually administered) While rotation error score allows only the identiﬁcation of those who likely have impairments in visuospatial working memory, rotation hit provides information about each indi- vidual’s performance on this ability and in relation to the whole sample. (iii) Syllable segmentation: To repeat the word spoken by the examiner while tapping to each syllable. Correct syllable breakdown of 12 words leads to a maximum possible score of 12. (iv) Auditory word discrimination: To say whether a pair of words, spoken by the examiner and eventually differing in a single phoneme, are the same or different. Maximum possible of score of 19 for 19 trials. Rapid naming (individually administered) (i) Word sequence: Number of correctly repeated two to ﬁve- word sequences (two unique trials for each sequence with the exception of the ﬁrst example) spoken by the exam- iner in an interstimulus interval of 1 s. Familiar two or three-syllable words were presented. Score was given by the number of sequences correctly accomplished, with a maximum score of 7. (i) Rapid naming (Figure) of a list of four objects (house, ball, elephant, clock) displayed pictorially in a particular order. A different ordering was presented in each trial, with 10 total trials. Total time was recorded in seconds. (ii) Rapid naming (Number) of numbers one through nine as listed visually in a random order. Screening performed before task to conﬁrm that the child knew numbers one through nine. Time was recorded in seconds. (ii) Non-word repetition: Number of correctly repeated non- words from a list of 23 presented aurally by the examiner. Maximum score of 23 for non-words spoken correctly. Phonological tasks (individually administered) (i) Alliteration: Matching ﬁrst sounds in spoken words. Children are presented with three words spoken by the examiner and then are asked which of three spoken words start with the same sound. After three practice items, the test comprises 10 items leading to a maximum score of 10. (i) Alliteration: Matching ﬁrst sounds in spoken words. Children are presented with three words spoken by the examiner and then are asked which of three spoken words start with the same sound. After three practice items, the test comprises 10 items leading to a maximum score of 10. (ii) Figure rotation error: Number of errors made in rotation of the shapes in ﬁgure order task. 28 ﬁgures were presented throughout the sequences,leading to a maximum of 28 errors. (ii) Rhyme detection: Matching last sounds in spoken words. Chil- dren are presented with three words spoken by the examiner and then are asked which of three spoken words end with the same sound. After three practice items, the test comprises 20 items leading to a maximum score of 20. (ii) Rhyme detection: Matching last sounds in spoken words. Chil- dren are presented with three words spoken by the examiner and then are asked which of three spoken words end with the same sound. After three practice items, the test comprises 20 items leading to a maximum score of 20. (iii) Figure rotation hit: Number of hits made in rotation of the shapes in ﬁgure order task. 28 ﬁgures were presented throughout the sequences, leading to a maximum of 28 hits. Note: Rotation hit is a way to measure the number of cor- rect rotations proposed by Andrade et al. (in preparation) as a “positive measure” which provides information about the visuospatial working memory capacity of all subjects. While rotation error score allows only the identiﬁcation of those who likely have impairments in visuospatial working memory, rotation hit provides information about each indi- vidual’s performance on this ability and in relation to the whole sample. (iii) Figure rotation hit: Number of hits made in rotation of the shapes in ﬁgure order task. 28 ﬁgures were presented throughout the sequences, leading to a maximum of 28 hits. Note: Rotation hit is a way to measure the number of cor- rect rotations proposed by Andrade et al. (in preparation) as a “positive measure” which provides information about the visuospatial working memory capacity of all subjects. www.frontiersin.org PROCEDURE The initial sample of 69 children was screened for reading and writing difﬁculties using CBAs 2 weeks after the start of the school year as the ﬁrst stage, universal screening, of a two-stage screening process. One month later, we administered the CS&S protocol and the ATE tasks and screened the entire group for ADHD symptom as well, as a part of the second stage. ATE tasks and the collective subtests of the C&S protocol were administered to all participants concurrently in the classroom, followed by individual administra- tion of the linguistic and cognitive tests over the course of 6 weeks. Particularly, the accuracy with which CS&S writing tasks and ATE tasks predicted end of ﬁfth grade level poor literacy status was investigated. Analysisof individualdataacrossthedifferentdomainswasalso conducted in order to derive individual proﬁles of performance and, thus, to determine in which domains a given at-risk student did and not did show abnormal performance. It was adopted the 25th percentile as a ﬁrst criterion for deviance in the screening processes (Table 4). Poor literacy at the end of ﬁfth grade was des- ignated by performances 1 SD ≤M on Literacy, a composite score including all reading and writing measures of the CS&S protocol (Table 5). Finally, logistic regression analyses were performed to explore the accuracy (sensitivity, speciﬁcity, and accuracy) with which collective measures, namely, the writing tasks of the CS&S protocolandtheATEtasks,aswellastheircombinations,predicted reading at the end of ﬁfth grade (Table 6). All statistical analysis were conducted by using the SPSS software package, version 20.0 (SPSS Inc., Chicago, IL, USA) for Windows. All study participation took place during school hours, so class- room administration was implemented for time efﬁciency on tests that did not require one-on-one monitoring. Testing began at the beginning of the academic calendar year. After excluding children because exclusionary factor and after returning the consent form (approved by the “Ethics Committee from the Faculty of Science and Philosophy of São Paulo State University “Júlio de Mesquita Filho” – Faculdade de Filosoﬁa e Ciências/Universidade Estadual Paulista, Marília, São Paulo, Brazil, under the protocol 0630/2009) we were left with 45 partic- ipants. Data collection occurred over the course of 3 years at three separate time intervals (T), namely, at the beginning of the aca- demic calendar year of the second (T1) and third (T2) grades and at the end of ﬁfth grade (T3) grades. ATE IV-rhyme After matching a word presented aurally by the examiner with three words depicted as pictures, children are asked to mark with “X” the picture whose name ends with the same sound as the spoken word. After three practice items, the test comprises 10 items leading to a maximum score of 10. STATISTICAL ANALYSIS A PCA factor using a varimax rotation was performed on the mea- sures of C&S protocol and ATE tasks as well (Table 1). Although our sample size is considered small when it comes to PCA, thus requiring caution in interpretation, the generation of high dimen- sional data with small sample sizes [i.e., High Dimension, Low Sample Size] is fast becoming a common place occurrence in many areas of modern science, and a growing body of literature has shown that some rules of thumb in the factor analytic literature, such as recommendations on absolute N (ranging from 50 to 200) and the number of cases per variable or N/p ratio (ranging from 3 to 20) are inconsistent. Actually, when communalities are high and the number of factors is small, factor analysis can be reliable for sample sizes well below 50 (Mundfrom et al., 2005; Henson and Roberts, 2006; de Winter et al., 2009). Anyway, we reiterate that this is a pilot study with a small sample size so that all the results must be viewed as indicative and should be replicated with a much larger sample. ATE III-rhyme Childrenarepresentedwiththreewords,depictedasthreepictures, and are asked to mark with “X” the two pictures whose name end January 2015 \| Volume 5 \| Article 1581 \| 5 www.frontiersin.org www.frontiersin.org Collective identiﬁcation of dyslexia Andrade et al. with the same sound. After three practice items, the test comprises 10 items leading to a maximum score of 10. with the same sound. After three practice items, the test comprises 10 items leading to a maximum score of 10. ADHD symptoms Classiﬁcation of children as at-risk for ADHD (AR-ADHD) was based on pedagogical team (including teachers and the pedagog- ical coordinator) endorsements on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV; American Psychiatric Association, 2000) ADHD symptom checklist. DSM- IV checklist is an 18-item scale in which inattentive and impulsive behaviors are rated on a 4-point Likert scale (0 = not at all, 1 = just a little, 2 = pretty much, and 3 = very much). Children with ≥6 “pretty much”or“very much”ratings in either of the two domains (inattention or hyperactivity/impulsivity) were classiﬁed as “at- risk” for ADHD (AR-ADHD). Endorsements on ADHD checklist by the pedagogical team were further conﬁrmed by interviews with parents. Composite scores based on summed raw scores of writing (writing words + pseudowords), reading (reading ﬂuency + read- ing accuracy + reading non-words), phonological (alliteration + rhyme detection + syllable segmentation),VWM (word sequence, non-words repetition, verbal number sequence backward), RN (RN of ﬁgures and digits) and, ﬁnally, ATE tasks (ATE I + ATE II + ATE III + ATE IV) were created (Table 2) in order to perform t-tests aimed at examining differences between academic low- achievers and non-low achievers in cognitive-linguistic abilities from the CS&S protocol and ATE tasks as well (Table 3). PROCEDURE However, we will restrict our analysis only in the T1 and T3 periods. In addition to the adminis- tration of the CS&S protocol screening, educators’ endorsements on DSM-IV ADHD symptom checklist were also carried out at each of the three time intervals and by different teachers, once there were different teachers for each grade level. Frontiers in Psychology \| Educational Psychology RESULTS The results of the ﬁrst-stage universal screening based on CBMs identiﬁed 13 at-risk students. In the second stage of our two-stage screening process, some CS&S and ATE measures showed “ceil- ing effect” [more than 15% of respondents achieving the highest possible score scores (McHorney and Tarlov, 1995)], and a few signiﬁcant outliers were found in the data. However, the method of PCA is relatively robust to these effects because assumes mea- surement without error, does not provide standard error and does not require distributional assumptions such as normality and homoscedasticity (Quinn and Keough, 2002; Jolliffe, 2005; Schmitt, 2011). Ceiling effects were observed in 10 variables: auditory word discrimination (AWD; 75%), syllable segmentation January 2015 \| Volume 5 \| Article 1581 \| 6 Frontiers in Psychology \| Educational Psychology Collective identiﬁcation of dyslexia Andrade et al. Table 1 \| Principal components analysis for CS&S measures and ATE tasks after varimax rotation and retention of factor loadings ≥0.35. CS&S measures and ATE tasks Factors and variance explained (%) 1 2 3 Literacy (22%) Phonology (19%) Visual (12%) Writing words 0.818 0.379 Rapid naming (Figure) –0.791 Reading accuracy 0.787 0.419 Rapid naming (Number) –0.768 Writing pseudowords 0.765 0.370 Reading ﬂuency 0.752 0.408 Alphabet task 0.621 Word sequence 0.454 ATE III-rhyme 0.416 0.687 Non-word reading 0.413 0.677 Auditory word discrimination 0.665 Alliteration 0.645 ATE I-alliteration 0.634 0.459 Rhyme detection 0.412 0.629 ATE IV-rhyme 0.604 Verbal number sequence backward 0.452 ATE II-alliteration 0.358 Syllable segmentation Shapes copying Figure rotation hit 0.903 Figure rotation error –0.761 Figure order 0.639 Non-word repetition 0.490 Table 1 \| Principal components analysis for CS&S measures and ATE tasks after varimax rotation and retention of factor loadings ≥0.35. Table 1 \| Principal components analysis for CS&S measures and ATE tasks after varimax rotation and retention of factor loadings ≥0.35. (69%), reading non-words (64%),ATE III (55%), alphabet (51%), alliteration and non-words repetition (29%), reading accuracy (20%), ATE I and ATE IV (18%). As for the PCA, the further analyses based on composite scores, which do not show ceiling effects, should not be affected by these effects. component matrix which emphasizes differences in correlations (loadings) of the variables with each factor (maximizing high correlations and minimizing low ones) and was performed on these three components to give further support to the assump- tion that factors are indeed relatively independent from each other (Dancey and Reidy,2007, pp. 469–472). www.frontiersin.org RESULTS Table 2 \| Descriptive characteristics of performance on CS&S measures and on ATE tasks on the basis of composite scores (n = 45). Measure Mean ± SD Percentiles P 25 P 20 P 15 P 10 P 5 Reading 105.09 ± 28.689 94.00 90.20 81.80 63.80 27.20 Writing 30.09 ± 8.339 27.00 24.40 21.90 18.40 8.90 Literacy 135.18 ± 36.187 120.50 112.00 106.70 88.60 36.10 Phonological awareness 36.24 ± 4.488 33.50 33.00 32.00 30.00 25.00 Auditory word discrimination 18.62 ± 0.984 18.50 18.00 18.00 18.00 17.30 Verbal working memory 29.00 ± 3.090 26.50 26.00 26.00 25.00 24.30 Shapes copying 4.84 ± 2.132 3.00 2.20 2.00 1.60 1.00 Figure order 5.62 ± 1.134 5.00 5.00 4.00 4.00 4.00 Figure rotation errora,b 2.40 ± 3.129 3.00 4.80 6.10 7.00 8.70 Figures rotation hit 14.38 ± 6.065 9.00 8.20 7.00 6.00 3.60 Naming speedb 83.98 ± 18.165 90.50 94.80 99.10 105.80 126.30 ATE 32.16 ± 4.931 29.00 27.00 26.90 25.50 22.00 aOriginal non-composite scores; bValues represent percentiles 75, 80, 85, 90, and 95, respectively. cs of performance on CS&S measures and on ATE tasks on the basis of composite scores (n = 45). Table 3 \| Descriptive characteristics (mean ± standard deviation) of performance on composite scores of CS&S measures (except auditory word discrimination and visual tasks) and ATE tasks in children identiﬁed as academic low-achievers compared to those considered typical learners. Table 3 \| Descriptive characteristics (mean ± standard deviation) of performance on composite scores of CS&S discrimination and visual tasks) and ATE tasks in children identiﬁed as academic low-achievers compared to th cs (mean ± standard deviation) of performance on composite scores of CS&S measures (except auditory word and ATE tasks in children identiﬁed as academic low-achievers compared to those considered typical learners. Table 3 \| Descriptive characteristics (mean ± standard deviation) of performance on composite scores of CS&S measures (except auditory word discrimination and visual tasks) and ATE tasks in children identiﬁed as academic low-achievers compared to those considered typical learners. RESULTS Varimax rotation revealed the presence of a simple and clear structure of three compo- nents (Table 1), each showing a number of variables with speciﬁc and salient loadings. The few variables that loaded on two fac- tors were allocated to the factor with which it had the highest loading. (69%), reading non-words (64%),ATE III (55%), alphabet (51%), alliteration and non-words repetition (29%), reading accuracy (20%), ATE I and ATE IV (18%). As for the PCA, the further analyses based on composite scores, which do not show ceiling effects, should not be affected by these effects. Taken together, the presence of many coefﬁcients ≥0.30 revealed by a correlation matrix containing all measures under investigation, jointly with the value of 0.675 of the Kaiser- Meyer-Olkin measure of sample adequacy (KMO value) and the statistically signiﬁcant result of the Bartlett test of spheric- ity (p = 0.000), these results indicate that the data are suitable for factor analysis. A PCA suppressing coefﬁcients smaller than 0.35, conducted for all CS&S variables and all four ATE tasks (totalizing 23 measures), revealed the presence of six components with an eigenvalue exceeding 1.0 which accounted for 75.84% of the data variability. However, Cattell’s scree test, used as a factor retention test, recommended extracting only three components which accounted for 53.1% of the variability. By looking at the commonalities among variables that more strongly correlate with one of the three factors it allows one to know what cognitive dimension each factor represents and, hence, to choose an appro- priate name for each factor. Varimax is a method of rotating the Loadings on each of the three factors are outlined in Table 1. The ﬁrst component, which can be assumed to represent the lit- eracy dimension, accounted for 22% of data variance and had its highest loadings (between 0.70 and 0.80) on writing words (0.818), RN – ﬁgure (–0.791), reading accuracy (0.787), RN – number (–0.768), writing pseudowords (0.765), reading ﬂuency (0.752), whereas its lowest loadings are found for alphabet task (0.621), and word sequence (0.454). For the second component, which represented the phonology dimension and accounted for 19% of data variance, the highest loadings (between 0.60 and 0.70) are found for ATE III (0.687), non-word reading (0.677), January 2015 \| Volume 5 \| Article 1581 \| 7 www.frontiersin.org Collective identiﬁcation of dyslexia Andrade et al. RESULTS Table 4 \| Academic low-achievers composite scores at the beginning of the second grade on reading, writing, phonological awareness (PAs), verbal working memory (VWM), rapid naming (RN), and the alternative tool for educator’s (ATEs), and single scores of alphabet, auditory word discrimination (AWD), shapes copying (SHAPE), ﬁgure ordering (FIGOR), ﬁgure rotation error (ROTE) and ﬁgure rotation hit (ROTH) subtests (ascores ≤P 25 or > P 75 for ROT and RN; bscores ≤P 10 or > P 90 for ROT and RAN; cscores ≤P 5 or >P 95 for ROTE and NS). Low-achievers at the beginning of the second grade (A = ADHD symptoms) 04 05 06 10 18 19 22 24 27 30 32 41 43 Task (P 25) A A A A A Alphabet (24) 22a 26 19b 26 21b 21b 24a 26 25 24a 13 26 25 Reading (94) 95 80a 37b 53b 95 90a 71a 100 23c 82a 20c 91a 84a Writing (27) 16b 27a 11b 20a 21a 27a 28 24a 8c 22a 2c 33 23a Literacy (121) 111a 107a 48b 73b 116a 117a 99a 124 31c 104a 22c 124 107a PA (34) 32a 30a 36 25c 33a 25c 37 34a 25c 30a 33a 36 32a AWD (18.50) 18a 19 19 13c 19 19 19 18a 19 18a 18a 18a 17c VWM (27) 31 28 27a 26a 28 25b 26a 28 27a 22c 24c 30 25b SHAPE (3) 1c 7 7 7 4 2a 3a 7 3a 2a 1c 7 1c FIGOR (5) 7 6 5a 4b 5a 4b 5a 6 5a 4b 5a 6 4b ROTE (3) 1 1 7b 4a 9c 7b 0 0 0 7b 2 1 1 ROTH (9) 22 17 7a 6b 5b 3c 14 18 14 3c 12 17 9a RN (90.50) 80 100a 132c 88 88 94a 79 91a 152c 110b 113b 71 71 ATE (29) 31 27a 35 22c 34 26a 27a 29a 19c 27a 30 32 24b Table 5 \| Academic low-achievers’ composite scores at the end of the ﬁfth grade on reading, writing, and literacy (apoor literacy status, i.e., scores 1 SD ≤M; bscores ≤P 25; cscores ≤P 5) and the true positives (TPs), true negatives (TNs), false positives (FPs), and false negatives (FNs) produced by CS&S writing tasks (W) and ATE tasks, separately and combined (W/ATE), administered at the beginning of the second grade as the second stage screening. RESULTS Low-achievers at the end of the ﬁfth grade (A = ADHD symptoms) 04 05 06 10 18 19 22 24 27 30 32 41 43 Task (P 25) A A A A A Reading (129) 134 119b 103c 121a 134 122a 124a 143 99c 134 113b 121a 161 Writing (32) 33 31a 31a 28b 24c 31a 29b 24c 26c 22c 28c 36 34c Literacy (160) 167 150a 134a 149a 158b 153a 153a 177 125a 166 141a 157b 161 W FP TP TP TP FP TP FN FP TP FP TP TN FP ATE TN TP FN TP TN TP TP FP TP FP FN TN FP W/ATE FP TP TP TP FP TP TP FP TP FP TP TN FP Low-achievers at the beginning of the second grade (A = ADHD symptoms) 04 05 06 10 18 19 22 24 27 30 32 41 43 Task (P 25) A A A A A Alphabet (24) 22a 26 19b 26 21b 21b 24a 26 25 24a 13 26 25 Reading (94) 95 80a 37b 53b 95 90a 71a 100 23c 82a 20c 91a 84a Writing (27) 16b 27a 11b 20a 21a 27a 28 24a 8c 22a 2c 33 23a Literacy (121) 111a 107a 48b 73b 116a 117a 99a 124 31c 104a 22c 124 107a PA (34) 32a 30a 36 25c 33a 25c 37 34a 25c 30a 33a 36 32a AWD (18.50) 18a 19 19 13c 19 19 19 18a 19 18a 18a 18a 17c VWM (27) 31 28 27a 26a 28 25b 26a 28 27a 22c 24c 30 25b SHAPE (3) 1c 7 7 7 4 2a 3a 7 3a 2a 1c 7 1c FIGOR (5) 7 6 5a 4b 5a 4b 5a 6 5a 4b 5a 6 4b ROTE (3) 1 1 7b 4a 9c 7b 0 0 0 7b 2 1 1 ROTH (9) 22 17 7a 6b 5b 3c 14 18 14 3c 12 17 9a RN (90.50) 80 100a 132c 88 88 94a 79 91a 152c 110b 113b 71 71 ATE (29) 31 27a 35 22c 34 26a 27a 29a 19c 27a 30 32 24b Table 5 \| Academic low-achievers’ composite scores at the end of the ﬁfth grade on reading, writing, and literacy (apoor literacy status, i.e., scores 1 SD ≤M; bscores ≤P 25; cscores ≤P 5) and the true positives (TPs), true negatives (TNs), false positives (FPs), and false negatives (FNs) produced by CS&S writing tasks (W) and ATE tasks, separately and combined (W/ATE), administered at the beginning of the second grade as the second stage screening. www.frontiersin.org RESULTS Mean ± SD Task Non-low academic achievers (n = 32) Academic low-achievers (n = 13) t-test p-value Alphabet 25.09 ± 1.907 22.92 ± 3.774 1.974 0.680 Reading 119.00 ± 13.036 70.85 ± 28.151 5.915 0.000* Writing 34.13 ± 3.260 20.15 ± 8.783 5.581 0.000* Literacy 153.13 ± 14.788 91.00 ± 35.541 6.092 0.000* Phonological awareness 38.22 ± 2.791 31.38 ± 4.214 6.391 0.000* Verbal working memory 29.94 ± 2.850 26.69 ± 2.428 3.602 0.001** Auditory word discrimination 18.88 ± 0.336 18.00 ± 1.633 1.916 0.079 Shapes copying 5.19 ± 1.839 4.00 ± 2.614 1.494 0.153 Figure order 5.84 ± 1.139 5.08 ± 0.954 2.138 0.038* Figure rotation error 2.13 ± 3.077 3.08 ± 3.278 –0.923 0.361 Figure rotation hit 15.63 ± 5.615 11.31 ± 6.250 2.263 0.029* Rapid naming 78.44 ± 11.733 97.62 ± 23.894 –2.762 0.015* ATE 33.88 ± 3.966 27.92 ± 4.609 4.355 0.000*** Asterisks indicate signiﬁcance (p < 0.05, p < 0.01, **p < 0.001). facilitate analysis and interpretation of data, as well as to provide educators with a simple way to interpret children’s performance in a school context. Then, we grouped children’s responses in the composite scores (see Statistical Analysis in the Materials and Methods) of Reading, Writing, PA, VWM and, ﬁnally, ATE tasks that, jointly with AWD and visual measures presented in original single scores, are outlined in Table 2 which contains mean scores, standard deviation and percentiles. AWD (0.665), alliteration (0.645), ATE I (0.634), rhyme detection (0.629), ATE IV (0.604), and the two lowest loadings are for verbal number sequence backward (0.452) and ATE II (0.358). Finally, the third component, which represented the visual dimension and accounted for 12% of data variance, had its highest loadings for ﬁgure rotation hit (0.903), ﬁgure rotation error (–0.761) and ﬁgure order (0.639), and the lowest loading is for non-word repetition (0.490). According to these results we could reduce the number of vari- ables by using composite scores obtained from the sum of raw scores on conceptually related cognitive measures with the aim to After performing ranking transformation, Spearman correla- tion analysis (CA) was conducted in all measures under investiga- tion and showed that ATE tasks correlated with several composite January 2015 \| Volume 5 \| Article 1581 \| 8 Frontiers in Psychology \| Educational Psychology Collective identiﬁcation of dyslexia Andrade et al. RESULTS Low-achievers at the beginning of the second grade (A = ADHD symptoms) Table 5 \| Academic low-achievers’ composite scores at the end of the ﬁfth grade on reading, writing, and literacy (apoor literacy status, i.e., scores 1 SD ≤M; bscores ≤P 25; cscores ≤P 5) and the true positives (TPs), true negatives (TNs), false positives (FPs), and false negatives (FNs) produced by CS&S writing tasks (W) and ATE tasks, separately and combined (W/ATE), administered at the beginning of the second grade as the second stage screening. scores from CS&S measures related to literacy and phonologi- cal abilities. From the highest to the lowest, correlations of ATE tasks with CS&S measures are: literacy (r = 0.595, p = 0.000), reading (r = 0.589, p = 0.000), VWM (r = 0.572, p = 0.000), PA (r = 0.548, p = 0.000), writing (r = 0.505, p = 0.000), RN (r = 0.447, p = 0.002), ﬁgures order (r = 0.423, p = 0.004), ﬁgure rotation hit (r = 0.368, p = 0.013). These correlations give further support to our hypothesis that ATE tasks are suitable for measuring phonological abilities collectively in classrooms. scores from CS&S measures related to literacy and phonologi- cal abilities. From the highest to the lowest, correlations of ATE tasks with CS&S measures are: literacy (r = 0.595, p = 0.000), reading (r = 0.589, p = 0.000), VWM (r = 0.572, p = 0.000), PA (r = 0.548, p = 0.000), writing (r = 0.505, p = 0.000), RN (r = 0.447, p = 0.002), ﬁgures order (r = 0.423, p = 0.004), ﬁgure rotation hit (r = 0.368, p = 0.013). These correlations give further support to our hypothesis that ATE tasks are suitable for measuring phonological abilities collectively in classrooms. n = 32). Since our sample size (n = 45) ﬁts well with the central limit theorem, according to which sample sizes equal or greater than 30 are sufﬁciently large for concerns about unequal variance and non-normality (Sokal and Rohlf, 1987, p. 107), we conducted a t-test to compare performances on CS&S measures and ATE tasks of children designated as academic low-achievers by CBA measures and those children considered normal readers (Table 3). We used Levene’s test for inequality to verify whether variances between groups are equal and to decide what t-value to choose and, then, its signiﬁcance. RESULTS PA (t = 6.391, p < 0.001), Literacy abilities (t = 6.092), Reading (t = 5.915, p < 0.001), Writing (t = 5.581, p < 0.001) and ATE tasks (t = 4.355, p < 0.001), in this order, which were substantially greater than t-values for VWM (t = 3.602, p < 0.01), Naming Speed (t = –2.762, p < 0.05), Figure Rotation Hit (t = 2.263, p < 0.05), Figure Order (t = 2.138, p < 0.05). p g p An analysis of individual performance across the composite and individual measures of the CS&S protocol and the compos- ite scores of ATE tasks, as described in Tables 2 and 3, was also conducted on the academic low-achievers (Table 4). This allows both the identiﬁcation of at-risk students in the second stage and to derive individual proﬁles of performance (i.e., to deter- mine in which domains a given at-risk student did and not did show abnormal performance) for which it was adopted norm- referenced cutoff points to determine deviance. As shown in Table 4 the 25th percentile was the main criterion for deviance, but scores at or below 20th, 15th, 10th, and 5th percentiles were also included in the analysis. Table 4 shows that CBAs, collec- tively administered by teachers in the classrooms as part of the ﬁrst stage, was efﬁcient in identifying children at risk for dyslexia. CBA measures have identiﬁed 13 children as struggling learners, from which 10 were found to perform below 25th percentile on reading tasks and 11 on writing tasks from the CS&S protocol. Performances on the Literacy composite score were found to be handicapped in 11 of the 13 academic low-achievers. From the non-literacy based tasks of the CS&S protocol, PA (10 of 13) and FIGOR (9 of 13) were the most frequently impaired in the aca- demic low-achievers, followed by VWM (8 of 13), AWD, SHAPE and NS (7 of 13), ROTH (6 of 13), and ROTE (5 of 13). ATE tasks were handicapped in 8 of the 13 academic low-achievers thus comparably to VWM, the third most frequently impaired CS&S non-literacy based task. Children with ADHD symptoms, identiﬁed by the pedagogical team endorsements on the DSM- IV ADHD symptom checklist, is also shown in Table 4. Five of the 11 academic low-achievers also were identiﬁed as at risk for ADHD. RESULTS Since participant/variable ratio recommended in books for regression analysis range from 15 to 40 participants per variable (Dancey and Reidy, 2007), we considered that our sample size ﬁts well within Table 6 \| Prediction of PL status (Sens = sensitivity; Spec = speciﬁcity; Accur = accuracy) at the end of ﬁfth grade according to logistic regression models 1, 2, and 3 based on one-stage screening with collective tools. sensitivity; Spec = speciﬁcity; Accur = accuracy) at the end of ﬁfth grade according to logistic one-stage screening with collective tools. Table 6 \| Prediction of PL status (Sens = sensitivity; Spec = speciﬁcity; Accur = accuracy) at the end of ﬁfth grade according to logistic regression models 1, 2, and 3 based on one-stage screening with collective tools. From now on we will focus our analyzes on the accuracy with which our collective screening tools, namely, CS&S writing tasks and ATE tasks, predict poor literacy status at the end of ﬁfth grade (literacy scores 1 SD ≤M) as shown in Tables 5 and 6. Since participant/variable ratio recommended in books for regression analysis range from 15 to 40 participants per variable (Dancey and Reidy, 2007), we considered that our sample size ﬁts well within these requirements once we have only two variables under focus, i.e., composite scores of the collectively administered writing and ATE tasks. We conducted regression analysis with Writing and ATE composite scores, both separately and together, as predic- tors. Together Writing and ATE tasks accounted for nearly half the variation of literacy abilities at the end of ﬁfth grade (adjusted R2 = 0.486), F(2,38) = 19.886, p < 0.001. Writing has a larger absolute standardized coefﬁcient (0.651) compared to ATE tasks (0.118),thus reﬂecting its higher contribution to the model. Alone, Writing accounted for 48.8% of the variance [F(1,39) = 39.107, p < 0.001] in literacy abilities at the end of ﬁfth grade, against 16.3% of the variance explained by ATE tasks [F(1,39) = 8.777, p < 0.01]. RESULTS Model 1, combining both CS&S writ- ing and ATE tasks, and Model 2, based only on writing tasks, both resulted in a sensitivity of 100% and speciﬁcities of 89 5 Table 6 \| Prediction of PL status (Sens = sensitivity; Spec = speciﬁcity; Accur = accuracy) at the end of ﬁfth grade according to logistic regression models 1, 2, and 3 based on one-stage screening with collective tools. Models (1–4) B SE Wald p TN FN TP FP Sens Spec Accur Model 1 Writing –0.225 0.094 5.746 0.017 34 0 7 4 100 89.5 91.1 ATE –0.297 0.167 3.178 0.075 Constant 12.957 6.040 4.602 0.32 Model 2 Writing –0.227 0.079 8.292 0.004 32 0 7 6 100 84.2 86.7 Constant 4.373 2.078 4.427 0.035 Model 3 ATE –0.287 0.106 7,381 0.007 26 1 6 12 85.7 68.4 71.1 Constant 6,947 3,057 5,165 0.023 Table 6 \| Prediction of PL status (Sens = sensitivity; Spec = speciﬁcity; Accur = accuracy) at the end of ﬁfth grade according to logistic regression models 1, 2, and 3 based on one-stage screening with collective tools. Models (1–4) B SE Wald p TN FN TP FP Sens Spec Accur Model 1 Writing –0.225 0.094 5.746 0.017 34 0 7 4 100 89.5 91.1 ATE –0.297 0.167 3.178 0.075 Constant 12.957 6.040 4.602 0.32 Model 2 Writing –0.227 0.079 8.292 0.004 32 0 7 6 100 84.2 86.7 Constant 4.373 2.078 4.427 0.035 Model 3 ATE –0.287 0.106 7,381 0.007 26 1 6 12 85.7 68.4 71.1 Constant 6,947 3,057 5,165 0.023 PA (t = 6.391, p < 0.001), Literacy abilities (t = 6.092), Reading (t = 5.915, p < 0.001), Writing (t = 5.581, p < 0.001) and ATE tasks (t = 4.355, p < 0.001), in this order, which were substantially greater than t-values for VWM (t = 3.602, p < 0.01), Naming Speed (t = –2.762, p < 0.05), Figure Rotation Hit (t = 2.263, p < 0.05), Figure Order (t = 2.138, p < 0.05). An analysis of individual performance across the composite From now on we will focus our analyzes on the accuracy with which our collective screening tools, namely, CS&S writing tasks and ATE tasks, predict poor literacy status at the end of ﬁfth grade (literacy scores 1 SD ≤M) as shown in Tables 5 and 6. RESULTS No signiﬁcant difference in age was found between groups. As is outlined in Table 3, academic non- low achievers demonstrated signiﬁcantly better performance than academic low-achievers on a number of C&S measures and in ATE tasks as well. Note that the greater t-values are observed for n = 32). Since our sample size (n = 45) ﬁts well with the central limit theorem, according to which sample sizes equal or greater than 30 are sufﬁciently large for concerns about unequal variance and non-normality (Sokal and Rohlf, 1987, p. 107), we conducted a t-test to compare performances on CS&S measures and ATE tasks of children designated as academic low-achievers by CBA measures and those children considered normal readers (Table 3). We used Levene’s test for inequality to verify whether variances between groups are equal and to decide what t-value to choose and, then, its signiﬁcance. No signiﬁcant difference in age was found between groups. As is outlined in Table 3, academic non- low achievers demonstrated signiﬁcantly better performance than academic low-achievers on a number of C&S measures and in ATE tasks as well. Note that the greater t-values are observed for Table3 providesthe mean scores and standard deviation related to the measures described in the Table 2 for those children identi- ﬁed academic low-achievers (n = 13) by the ﬁrst stage of our two- stage screening procedure as well as for the remaining children not considered academic low-achievers (academic non-low achievers, January 2015 \| Volume 5 \| Article 1581 \| 9 Collective identiﬁcation of dyslexia Andrade et al. Table 6 \| Prediction of PL status (Sens = sensitivity; Spec = speciﬁcity; Accur = accuracy) at the end of ﬁfth grade according to logistic regression models 1, 2, and 3 based on one-stage screening with collective tools. RESULTS Models (1–4) B SE Wald p TN FN TP FP Sens Spec Accur Model 1 Writing –0.225 0.094 5.746 0.017 34 0 7 4 100 89.5 91.1 ATE –0.297 0.167 3.178 0.075 Constant 12.957 6.040 4.602 0.32 Model 2 Writing –0.227 0.079 8.292 0.004 32 0 7 6 100 84.2 86.7 Constant 4.373 2.078 4.427 0.035 Model 3 ATE –0.287 0.106 7,381 0.007 26 1 6 12 85.7 68.4 71.1 Constant 6,947 3,057 5,165 0.023 PA (t = 6.391, p < 0.001), Literacy abilities (t = 6.092), Reading (t = 5.915, p < 0.001), Writing (t = 5.581, p < 0.001) and ATE tasks (t = 4.355, p < 0.001), in this order, which were substantially greater than t-values for VWM (t = 3.602, p < 0.01), Naming Speed (t = –2.762, p < 0.05), Figure Rotation Hit (t = 2.263, p < 0.05), Figure Order (t = 2.138, p < 0.05). An analysis of individual performance across the composite and individual measures of the CS&S protocol and the compos- ite scores of ATE tasks, as described in Tables 2 and 3, was also conducted on the academic low-achievers (Table 4). This allows both the identiﬁcation of at-risk students in the second stage and to derive individual proﬁles of performance (i.e., to deter- mine in which domains a given at-risk student did and not did show abnormal performance) for which it was adopted norm- referenced cutoff points to determine deviance. As shown in Table 4 the 25th percentile was the main criterion for deviance, but scores at or below 20th, 15th, 10th, and 5th percentiles were also included in the analysis. Table 4 shows that CBAs, collec- tively administered by teachers in the classrooms as part of the ﬁrst stage, was efﬁcient in identifying children at risk for dyslexia. CBA measures have identiﬁed 13 children as struggling learners, from which 10 were found to perform below 25th percentile on reading tasks and 11 on writing tasks from the CS&S protocol. Performances on the Literacy composite score were found to be handicapped in 11 of the 13 academic low-achievers. From the non-literacy based tasks of the CS&S protocol, PA (10 of 13) and FIGOR (9 of 13) were the most frequently impaired in the aca- demic low-achievers, followed by VWM (8 of 13), AWD, SHAPE and NS (7 of 13), ROTH (6 of 13), and ROTE (5 of 13). RESULTS ATE tasks were handicapped in 8 of the 13 academic low-achievers thus comparably to VWM, the third most frequently impaired CS&S non-literacy based task. Children with ADHD symptoms, identiﬁed by the pedagogical team endorsements on the DSM- IV ADHD symptom checklist is also shown in Table 4 Five of From now on we will focus our analyzes on the accuracy with which our collective screening tools, namely, CS&S writing tasks and ATE tasks, predict poor literacy status at the end of ﬁfth grade (literacy scores 1 SD ≤M) as shown in Tables 5 and 6. Since participant/variable ratio recommended in books for regression analysis range from 15 to 40 participants per variable (Dancey and Reidy, 2007), we considered that our sample size ﬁts well within these requirements once we have only two variables under focus, i.e., composite scores of the collectively administered writing and ATE tasks. We conducted regression analysis with Writing and ATE composite scores, both separately and together, as predic- tors. Together Writing and ATE tasks accounted for nearly half the variation of literacy abilities at the end of ﬁfth grade (adjusted R2 = 0.486), F(2,38) = 19.886, p < 0.001. Writing has a larger absolute standardized coefﬁcient (0.651) compared to ATE tasks (0.118),thus reﬂecting its higher contribution to the model. Alone, Writing accounted for 48.8% of the variance [F(1,39) = 39.107, p < 0.001] in literacy abilities at the end of ﬁfth grade, against 16.3% of the variance explained by ATE tasks [F(1,39) = 8.777, p < 0.01]. We performed logistic regression to predict normal reader and poor literacy status at the end of ﬁfth grade (PL, i.e., chil- dren with performances ≤1 SD below the mean on literacy tasks). To maximize TPs and limit FPs we explored the util- ity of several classiﬁcation cutoffs in addition to the standard 0.5 classiﬁcation cutoff of the SPSS according to the suggestion of Neter et al. (1996). By adjusting the cutoff value at either 0.2 or 0.1, the accuracy of the three logistic regression mod- els based on one-stage screening in the entire group (n = 45) using Writing and ATE collective screening tools, either separately or combined, as predictors of PR at the end of ﬁfth grade, are described in the Table 6. DISCUSSION literacy abilities and 10 were handicapped in phonological abilities, from which eight were also handicapped in ATE tasks, this last the third most frequently impaired from all non-literacy based task. The most common way to implement RTI in the schools is the reliance on a single-stage universal screen to determine children at risk for learning disabilities and to provide them with Tier 2 interventions. Despite the advantages of being efﬁcient, requir- ing only a brief, one-time assessment which provides objective information about the academic status of students, a single-stage universal screening often overidentify at-risk status (Fuchs et al., 2012; Gilbert et al., 2012). There is evidence that use of a second stage screening reduces the number of FPs and, hence, the number of students in need of Tier 2 intervention by two thirds, reducing the total cost of approximately $260,000, considering a universe of 200 at-risk children, to approximately $96,500 (Fuchs et al., 2012). However, in a two-stage screening process both ﬁrst and second stages are based on individual assessments what implies in addi- tional time, personnel and ﬁnancial resources, even more if we take into account the expense of tutoring interventions in Tiers 2 and 3. Taken together, even in a RTI implemented on the basis of a two-stage screening process these aspects make the implemen- tation of RTI in Brazil a challenging endeavor if we consider the public funding limitations. q y p y Moreover, individual proﬁles of linguistic-cognitive deﬁcits in academic low-achievers are consistent with literature in that although a phonological processing deﬁcit is the main endophenotype associated with literacy acquisition difﬁculties it may not be sufﬁcient to fully explain dyslexia (Peterson and Pen- nington, 2012). Firstly, we found that attention deﬁcits are present in ﬁve of the 11 children identiﬁed as at risk by the second-stage screening (Table 4) who were later part of the seven poor literates at the end of ﬁfth grade (Table 5). Additionally, two of these poor literates had normal phonological processing, while both were severely impaired in naming speed and one of them was identiﬁed as at risk for ADHD. Therefore, their literacy difﬁculties could be explained only on the basis of naming deﬁcits, consistently with the double-deﬁcit hypothesis of dyslexia (Wolf and Bowers, 1999; Boada et al., 2012). DISCUSSION Individual analysis also revealed that all chil- dren identiﬁed as at risk for ADHD were impaired on RN and four were clearly impaired in VWM and in all three visuospatial measures (ﬁgure order, ﬁgure rotation error, and ﬁgure rotation hit). These ﬁndings are strikingly consistent with the notion that inattention symptoms are associated with weaknesses in naming speed and working memory (Shanahan et al., 2006; Lui and Tan- nock, 2007; Marzocchi et al., 2008; Arnett et al., 2012), and also consistent with evidence that tasks involving visuospatial manipu- lations are specially sensitive to inattentive behavior (Martinussen et al., 2005; Willcutt et al., 2005; Castellanos et al., 2006). One of the ways to reduce the costs of both one-stage and two-stage screening procedures is the use of screening tools that are able to be administered collectively (Andrade et al., 2013). The mainpurposeof thispilotstudywastoevaluatetheaccuracyof two collective screening tools in identifying children at risk for dyslexia in the beginning of the ﬁrst grade and to predict poor reading status at the end of ﬁfth grade. Writing tasks of the collective version of the CS&S protocol and a non-literacy based tool for the collective assessment of the phonological abilities based on phonological judgments by matching ﬁgures and ﬁgures to spoken words (ATE tasks), were tested as collective screening tools in the context of both a one-stage and a two-stage screening processes. , ; , ; , ) Taken together, these results support our proposal that the collective administration of the ATE tasks can substitute the indi- vidually administered phonological processing tasks from the CS&S protocol. A third important ﬁnding is regarding the col- lectively administered writing tasks of the CS&S protocol, namely, words and pseudowords, which represented the highest and the ﬁfth highest loadings on the literacy factor, respectively, thus suggesting that this task also has the potential to be used as a literacy-based screening tool. In fact, Snowling and Hulme (2012) argue that although few causal theories have addressed the com- plex relations between reading and writing development it is clear that most cognitive mechanisms underlying these abilities are shared, so that reading difﬁculties will be associated with writing difﬁculties most of the time. RESULTS It is worthy of noting that four of the ﬁve children with clear visuospatial difﬁculties (performing at or below 25th per- centile in all three visual measures) were identiﬁed as at risk for ADHD. We performed logistic regression to predict normal reader and poor literacy status at the end of ﬁfth grade (PL, i.e., chil- dren with performances ≤1 SD below the mean on literacy tasks). To maximize TPs and limit FPs we explored the util- ity of several classiﬁcation cutoffs in addition to the standard 0.5 classiﬁcation cutoff of the SPSS according to the suggestion of Neter et al. (1996). By adjusting the cutoff value at either 0.2 or 0.1, the accuracy of the three logistic regression mod- els based on one-stage screening in the entire group (n = 45) using Writing and ATE collective screening tools, either separately or combined, as predictors of PR at the end of ﬁfth grade, are described in the Table 6. Model 1, combining both CS&S writ- ing and ATE tasks, and Model 2, based only on writing tasks, both resulted in a sensitivity of 100% and speciﬁcities of 89.5 and 84.2%, respectively. Model 3, based only on the ATE tasks resulted in a sensitivity of 85.7% and speciﬁcity of 68.4%. The Hosmer–Lemeshow test showed small and non-signiﬁcant Chi- squared values indicating goodness of ﬁt for the logistic regression models. January 2015 \| Volume 5 \| Article 1581 \| 10 Frontiers in Psychology \| Educational Psychology Collective identiﬁcation of dyslexia Andrade et al. www.frontiersin.org REFERENCES American Psychiatric Association. (2000). Diagnostic and Statistical Manual of Mental Disorders, 4th Edn, Text Revision. Washington, DC: Author. Andrade, O. V. C. A. (2010). Instrumentalização Pedagógica Para Avaliação de Cri- Andrade, O. V. C. A. (2010). Instrumentalização Pedagógica Para Avaliação de Cri- anças com Risco de Dyslexia. Master Degree thesis, Faculdade de Filosoﬁa e Ciências, Universidade Estadual Paulista, Brazil. Available at: http://www.marilia. unesp.br/Home/Pos-Graduacao/Educacao/Dissertacoes/andrade_ovca_me_mar. pdf anças com Risco de Dyslexia. Master Degree thesis, Faculdade de Filosoﬁa e Ciências, Universidade Estadual Paulista, Brazil. Available at: http://www.marilia. unesp.br/Home/Pos-Graduacao/Educacao/Dissertacoes/andrade_ovca_me_mar. pdf Andrade, O. V., Andrade, P. E., and Capellini, S. A. (2013). Identiﬁcação precoce do risco para transtornos da atenção e da leitura em sala de aula. Psicol. Teoria Pesqui. 29, 167–176. doi: 10.1590/S0102-37722013000200006 There are several study limitations and admonitions regarding the interpretations of our study. The ﬁrst and most important is the small sample size which imposes serious limitations regarding the generalizability of our results. Therefore, our results should be interpreted in the context of a pilot study which took care of controlling homogeneity with respect to age, socioeconomic status and pedagogical approaches, so that our results should be consis- tent with studies on larger samples in order to be considered as a valid pilot study worthy of being conducted in larger, representa- tive samples. Secondly, once we do not have detailed information regarding the general non-verbal cognitive abilities of these chil- dren it remains unclear whether differences between groups are due to differences in general cognitive abilities, e.g., IQ or even overall processing speed. However, only students with no exclu- sionary factors participated in the study. We also have examined separatelythosetaskstraditionallyassociatedwithexecutivecogni- tive functions and noted that they were not signiﬁcantly correlated with reading abilities neither showed high loadings on literacy fac- tor. These tasks are Verbal Number Sequence Backward, which measures the ability of mental double-tracking of the memory and the reversing operations (Gathercole et al., 2004; Lezak et al., 2004), Figure Order, Rotation Error (and Figure Rotation Hit) which measure visual short-term memory and visuospatial abili- ties, respectively, thus tapping visual memory functions different from those assessed by verbal-auditory measures (Snow, 1998; Rosselli et al., 2001; Kyttälä, 2008) and tapping attention as well (Marzocchi et al., 2008). Therefore contributions of these abilities to the differences between groups and the relationships between variables should be minimal. REFERENCES The third limitation is regarding to the measures used to deﬁne poor reading status at the end of ﬁfth grade, which did not include a subtest of reading comprehension. To minimize this limitation we also took into account the ﬁfth Andrade, O. V. C. A., Prado, P. S. T., and Capellini, S. A. (2011). Desenvolvimento de ferramentas pedagógicas para identiﬁcação de escolares de risco para a dislexia. Rev. Psicopedag. 28, 14–28. Arnett, A. B., Pennington, B. F., Willcutt, E., Dmitrieva, J., Byrne, B., Samuelsson, S., et al. (2012). A cross-lagged model of the development of ADHD inattention symptoms and rapid naming speed. J. Abnorm. Child Psychol. 40, 1313–1326. doi: 10.1007/s10802-012-9644-5 Barth, A. E., Stuebing, K. K., Anthony, J. L., Denton, C. A., Mathes, P. G., Fletcher, J. M., et al. (2008). Agreement among response to intervention cri- teria for identifying responder status. Learn. Individ. Differ. 18, 296–307. doi: 10.1016/j.lindif.2008.04.004 Boada, R., Willcutt, E. 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DISCUSSION These authors further argue that once spelling demands explicit knowledge of the orthographic structure of words its development depends in large part on reading experience which, in short, “gives information in how phonology maps to orthography”; in this sense, spelling is protracted in rela- tion to reading which normally begins to signiﬁcantly inﬂuence spelling around the second year of reading instruction (Snowling and Hulme, 2012, p. 596). This rationale is consistent with the fact that spelling and writing problems are often more severe and more persistent than reading problems in people with dyslexia and our ﬁndings ﬁt well within this framework. By conducting a PCA analysis with varimax rotation on the CS&S measures we found the presence of a simple and clear structure of three components,namely,a literacy dimension repre- sented by measures of reading and writing abilities, a phonological dimension mainly represented by tasks involving phonological processing and, ﬁnally, a third dimension representing visu- ospatial processing. Overall, the results above indicate that the tasks proposed by CS&S protocol are, indeed, representing the cognitive-linguistic or conceptual dimensions which they are intended to measure. More relevant to our investigation is the ﬁnding that three of the collectively administered ATE tasks presented high loadings on the phonology factor (or phonological dimension) and corre- lated with several composite scores from CS&S measures related to literacy and phonological abilities. More speciﬁcally, ATE III showed the highest of all loadings on phonology factor, followed by ATE I being the ﬁfth highest loading and by ATE IV being the seventh highest loading. Secondly, an analysis of individual per- formance across the composite and individual measures of the CS&S protocol and the composite scores of ATE tasks, allowed both the identiﬁcation of at-risk students and to derive their indi- vidual proﬁles of performance. From the 13 children identiﬁed as struggling learners by CBA measures administered collectively by teacher in the classrooms, 11 were found to be handicapped in Logistic regression showed that both collective screening tools under investigation, namely CS&S writing subtests and ATE tasks, used as one-stage universal screening tools (in isolation or combination) were statistically signiﬁcant predictors of literacy abilities at the end of ﬁfth grade. In combination, Writing and January 2015 \| Volume 5 \| Article 1581 \| 11 Collective identiﬁcation of dyslexia Andrade et al. DISCUSSION ATE tasks (Model 1) accurately classiﬁed 91.1% of poor readers at the end of ﬁfth grade, yielding sensitivity of 100% and speciﬁcity of 89.5%. Writing alone (Model 2) classiﬁed 86.7% of poor read- ers with 100% of sensitivity and 84.2% of speciﬁcity, whereas ATE tasks accuracy (Model 3) was 71.1% with sensitivity of 85.7% and speciﬁcity of 68.4%. grade CBA measures administered by teachers and the interviews with the pedagogical team to conﬁrm the academic status of those children identiﬁed as poor readers. The fourth limitation is with respect to the cutoff point to designate low-achievement (1 SD ≤M) which could be considered too lenient. However, this cut point was used in relation to the entire sample, which also included academic low-achievers, so that it deﬁned a more stringent cutoff that if it were used in relation to only those students with normal literacy abilities. Of relevance is the ﬁnding that a ﬁrst-stage universal screen- ing based on CBA measures collectively administered by teachers in classrooms showed to be of great utility identifying 13 aca- demic low-achievers, corresponding to 29% of the whole sample. From the 13 children identiﬁed as academic low-achievers by CBA measures 11 were identiﬁed as at risk for dyslexia in the second-stage universal screening. Moreover, all seven students identiﬁed as poor readers at the end of the ﬁfth grade were aca- demic low-achievers screened by CBA measures in the beginning of the second grade. Although teachers’ judgments of student literacy abilities are too subjective and show signiﬁcant variabil- ity (Madelaine and Wheldall, 2005) these results are consistent with the notion that well clearly deﬁned pedagogical assessments administered by teachers in classrooms have the potential to be used as a ﬁrst-stage universal screening. CBA measures has reduced the number of children referred to Tier 2 interventions by 15.4%, and could be used as ﬁrst-stage universal screening fol- lowed by ATE tasks and Writing tasks as second-stage screening in a two-stage screening procedure. Overall our results provide empirical support for the notion that further studies on developing and testing of collective screen- ing tools for the identiﬁcation of children at risk for learning disabilities should be encouraged. ACKNOWLEDGMENTS We thank Colégio Criativo and all children and families who participated in this study. Frontiers in Psychology \| Educational Psychology REFERENCES de Winter, J. 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Contrasting deﬁcits on executive functions between ADHD and reading disabled children. J. Child Psychol. Psychiatry 49, 543–552. doi: 10.1111/j.1469-7610.2007.01859.x Citation: Andrade OVCA, Andrade PE and Capellini SA (2015) Collective screen- ing tools for early identiﬁcation of dyslexia. Front. Psychol. 5:1581. doi: 10.3389/fpsyg.2014.01581 McHorney, C. A., and Tarlov, A. R. (1995). Individual-patient monitoring in clinical practice: are available health status surveys adequate? Qual. Life Res. 4, 293–307. doi: 10.1007/BF01593882 This article was submitted to Educational Psychology, a section of the journal Frontiers in Psychology. in Psychology. Copyright © 2015 Andrade, Andrade and Capellini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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https://openalex.org/W2096779532	https://www.e3s-conferences.org/10.1051/e3sconf/20130125006/pdf	English	null	New low cost sorbents for Cr(VI) – batch and column experiments	E3S web of conferences	2,013	cc-by	1,652	H. Šillerová1 and M. Komárek1 H. Šillerová1 and M. Komárek1 1 Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 21, Prague 6, Czech Republic, sillerovah@fzp.czu.cz 1 Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 21, Prague 6, Czech Republic, sillerovah@fzp.czu.cz Abstract. The use of agricultural byproducts and industrial biowaste materials has been shown to be an attractive technique for removing Cr(VI) from contaminated waste waters. In this study, used brewers draff, peat moss, sawdust, grape stalks and husks were investigated as novel biosorbents for Cr(VI). The material was tested in two different modifications. The material was dried, cut and sieved and part of it was subjected to acid (2 M H2SO4) and alkali (0.5 M NaOH) pre-treatments to remove starch, proteins and sugars. Fourier transform infrared rays analysis on solid phase (FTIR-ATR) was used to determine the main functional groups that might control the metal uptake. Batch experiments were performed at different pH values (3, 4.5, 6) and at various initial concentration of Cr(VI) (25‒250 mg L-1). Two equilibrium empirical models, Langmuir and Freundlich, were used to describe Cr(VI) adsorption. In order to identify possible reduction processes, ion exchange separation on the AG1-X8 resin was used to separate the anionic Cr(VI) and the reduced cationic Cr(III) from the aqueous phase after biosorption. As expected, Cr(VI) removal was pH-dependent and fitted well both the Langmuir and Freundlich isotherm models. The ion exchange separation showed that Cr(VI) reduction had occurred in the solution during biosorption. The efficiency of draff as a biosorbent was comparable (or even higher) to highly organic materials (e.g., composted peat) showing its potential application for Cr(VI) decontamination. Key words: chromium, biosorption process, organic substrates, batch and packed bed column experiments Introduction This study is based on investigation of adsorption properties of five potential low-cost biosorbents for Cr(VI).The adsorption process under different conditions (various concentrations of Cr(VI) and various pH) will be studied using batch and column experiments. Also the ability of the sorbents to reduce Cr(VI) to Cr(III) will be investigated. Chromium can be find mainly in two forms in the environment, hexavalent and trivalent. Cr(VI) is highly soluble and mobile and it is a potential carcinogen (Kortenkamp et al., 1996) It is not typically naturally occurring in the environment but can be found in high concentrations in some regions of the Czech Republic (ultramafic rocks). Chromium as well as other metal elements is used widely in metallurgical and chemical industry. The majority of Cr is processed in stainless steel manufacturing followed by electroplating, lather and wood preservation, pigments production. Industrial wastewaters are the main pollution sources of Cr in natural waters and soils. Besides conventional processes used to remove Cr(VI) (precipitation as chromium hydroxide, ion exchange removal or adsorption). Recently, alternative sorbents have been investigated by many authors in order to find effective low cost technology for the treatment of metal-contaminated water (Fiol et al., 2008; Levankumar et al., 2009; Park et al., 2008). E3S Web of Conferences DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 2013 , 201 / 3012500 1 conf e3s 25006 (2013) 6 E3S Web of Conferences DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 2013 , 201 / 3012500 1 conf e3s 25006 (2013) 6 E3S Web of Conferences DOI: 10.1051/ , 201 / 3012500 1 conf e3s 25006 (2013) 6 C ⃝Owned by the authors, published by EDP Sciences, 2013 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2 0 , which . permits unrestricted use, distributi and reproduction in any medium, provided the original work is properly cited. on, Article available at http://www.e3s-conferences.org or http://dx.doi.org/10.1051/e3sconf/20130125006 New low cost sorbents for Cr(VI) – batch and column experimen H. Šillerová1 and M. Komárek1 Materials and Methods Biosorbents used in this study (brewers draff, peat moss, grape stalks and husks and digestate from biogass plant) were dried in an oven at 80°C for 24 hours and cut. Half of the material was subjected to pretreatment - an acid hydrolysis by 2 M H2SO4 (at 100°C for 1 hour) and an alkali treatment by 0.5 M NaOH (stirring for 24 hours at the room temperature). After each treatment the material was washed with deionized water several times until the pH reached 7 ± 0.5. Both types of the material were used for the batch and the column experiments. Fourier transform infrared rays analysis (FTIR) was used to determine the main functional groups. The This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2 0 , which . permits unrestricted use, distributi and reproduction in any medium, provided the original work is properly cited. on, E3S Web of Conferences specific surface area was measured using nitrogen as gas absorbent and the BET model. evaluated from the Langmuir isotherm is 350 mg.g-1), followed by grape stalks (331 mg.g-1), peat moss (237mg.g-1), brewers draff (139 mg.g-1) and sawdust (63 mg.g-1). FTIR-ATR analyses showed that the composition of the materials has changed by the acid and alkali pretreatment. Maximal sorption capacities of all the materials were enhanced: grape husks (378 mg.g-1), grape stalks (336 mg.g-1), peat moss (298 mg.g-1), brewers draff (261 mg.g-1) and sawdust (99 mg.g-1). By this treatment, starch, proteins and sugars are removed from the structure of biomaterial accessing thus the functional groups, such as carboxyl and hydroxyl, and increasing the specific surface area (Dupont and Guillon, 2003). Batch experiments were performed at plastic tubes by shaking a mass of 0.4 g of each material with 40 mL of chromium solution at different pH values (3, 4.5, 6) and different initial concentration of Cr(VI) (25-250 mg.L-1). Column experiments were carried out in plastic column (BioRad, USA). Initial Cr(VI) concentration was between 25 and 300mg.L-1 and the initial pH values were 3, 4.5 and 6. Effect of different flow rates (5, 10 and 15 mL.min-1) was investigated. Cr concentration was determined by ICP-OES. Cr(VI) and Cr(III) exist in anionic and cationic forms, respectively. That is why the separation of both forms can be realized by exchange resin. Separation of Cr(VI) and Cr(III) by resin AG1-X8 (Bio-Rad) was performed in this study. Materials and Methods Ion exchange columns were filled with 10 mL of the resin and initialized by 10 ml of 6 M HCl and 40 mL deionized water subsequently. The water sample was passed through the column. Cr(VI) was absorbed onto the resin and after that converted to Cr(III) with H2O2 and eluted by 6 M HCl (Ellis et al., 2002). The concentration of both Cr(III) and Cr(VI) in the filtrates was analyzed by ICP-OES (Agilent Technologies, USA). Chromium sorption proved to be pH-dependent. The sorption efficiency was the highest for all the materials at initial solution pH 3. At low pH values the sorption sites bound largely H+ and these can be easily exchanged by toxic metal ions. It was generally observed that increasing the initial Cr(VI) concentration led to adsorption decreases. In the range 0 – 100 mg.L-1 the adsorption percentage was between 25 and 90% for most of the materials (except sawdust); in solution with concentrations between 100 – 200 mg.l-1, from 15 to 50 % of the Cr has been removed and in the range 200 – 300 mg.L-1 only 0 to 30 % of Cr was removed. After the sorption process, Cr(III) was detected in the solution. This proves the presence of reduction reactions during the sorption process. Cr(VI) is partly reduced to Cr(III) by the sorbents. To ascertain the sorption mechanism and the oxidation state of Cr on the biomaterial surface more experiments are necessary. To characterize the oxidation state of Cr bound X-ray adsorption spectrometry might be used as a next step (Park et al., 2008). Acknowledgements The authors thank the financial supports providing from Technology Agency of the Czech Republic (TA01021055). Park D, Yun YS. XAS and XPS studies on chromium- binding groups of biomaterial during Cr(VI) biosorption. J. Colloid Interf. Sci. 2008; 317: 54- 61. Conclusion Ellis SA, Johnson MT, Bullen DT. Chromium Isotopes anf the Fate of Hexavalent Chromium in the Environment. Science 2002; 295: 2060-2062. All of the used materials, except sawdust, show good efficiency in removal of chromium from aqueous solution. The efficiency of the newly tested material, brewers draff, was comparable (or even higher) to highly organic materials (e.g., composted peat) showing its potential application for Cr(VI) decontamination. Chromium sorption is pH-dependent and the maximum sorption capacity is found at initial pH 3. Part of Cr(VI) is reduced to Cr(III) during the sorption process (5 - 15 % of the chromium at equilibrium concentration conditions). More experiments are necessary to ascertain the oxidation state of the chromium bound on the biomaterial. Dupont L, Guillon E. Removal of Hexavalent Chromium with a Lignocellulosic Substrate Extracted from Wheat Bran. Environ. Sci. Technol. 2003; 37: 4235-4241. Fiol et al. Chromium sorption and Cr(VI) reduction to Cr(III) by grape stalks and yohimbe bark. Bioresource technology. 2008; 99: 5030-5036. Kortenkamp et al. A Role for molecular oxygen in the formation of DNA damage during the reduction of the carcinogen chromium (VI) by glutathione. Arch.Biochem. Biophy. 1996; 329: 199-207. Levankumar et al. Batch adsorption and kinetics of chromium (VI) removal from aqueous solution by Ocimum americanum L. seed pods. Results and Discussion Langmuir and Freundlich isotherms were used to describe the sorption process and both fitted well the batch experimental data. As seen in Fig. 1, all the studied materials are able to adsorb Cr to some extent. Comparing non-pretreated materials, the most efficient one was grape husks (the maximal sorption capacity Fig. 1. Biosorption efficiency of non-pretreated brewers draff, peat moss, sawdust, grape stalks and husks on chromium solution at different pH. Fig. 1. Biosorption efficiency of non-pretreated brewers draff, peat moss, sawdust, grape stalks and husks on chromium solution at different pH. 25006-p.2 ICHMET 2012 References 25006-p.3
https://openalex.org/W3212363500	https://ph.pollub.pl/index.php/bia/article/download/817/2499	English	null	Forgotten shelters of Kłodzko Land. On architecture inspired by the local building tradition	Budownictwo i Architektura	2,021	cc-by	6,687	Budownictwo i Architektura 20(3) 2021, 119-133 DOI: 10.35784/bud-arch.817 Received: 04.12.2019; Revised: 02.01.2020; Accepted: 20.10.2021; Available online: 29.10.2021 Orginal Article © 2021 Budownictwo i Architektura This is an open-access article distributed under the terms of the CC-BY-SA 4.0 Budownictwo i Architektura 20(3) 2021, 119-133 DOI: 10.35784/bud-arch.817 Received: 04.12.2019; Revised: 02.01.2020; Accepted: 20.10.2021; Available online: 29.10.2021 Orginal Article © 2021 Budownictwo i Architektura This is an open-access article distributed under the terms of the CC-BY-SA 4.0 Budownictwo i Architektura 20(3) 2021, 119-133 DOI: 10.35784/bud-arch.817 Received: 04.12.2019; Revised: 02.01.2020; Accepted: 20.10.2021; Available online: 29.10.2021 Orginal Article © 2021 Budownictwo i Architektura This is an open-access article distributed under the terms of the CC-BY-SA 4.0 Orginal Article © 2021 Budownictwo i Architektura This is an open-access article distributed under the terms of the CC-BY-SA 4.0 Forgotten shelters of Kłodzko Land. On architecture inspired by the local building tradition Jacek Suchodolski Chair of Architecture and Visual Arts; Faculty of Architecture; Wrocław University of Science and Technology; ul. B. Prusa 53/55, Wrocław, Poland; jacek.suchodolski@pwr.edu.pl 0000-0002-4767-6169 Abstract: The article addresses the problem of the decline in the number of tourist facilities with outstanding features of regional architecture, built before 1945 in Kłodzko Land. Their destruction is an irreparable loss for the cultural heritage of the described region. Usually designed in the spirit of the local building tradition – born out of centuries of expe- rience of the people living here – hostels, inns and taverns were a characteristic, regional distinguishing feature of the landscape of Kłodzko Land. Meanwhile, the new architecture of objects related to tourism, built after the end of the Second World War, most often does not refer to the characteristic forms and structures of local buildings. The article stresses the need to put an end to the often deliberate practice of destroying old pre-war buildings and to draw the attention of architects working today to the importance of continuing local, regional forms in the emerging tourist facilities. Keywords: architecture, shelters, Kłodzko Land Keywords: architecture, shelters, Kłodzko Land 1. Introduction Kłodzko Land, partially covering the areas of the Central and Eastern Sudetes, is certainly one of the most diverse and interesting regions in the Sudetes in terms of tourist attractive- ness. The reasons for that are its history, a significant number of architectural monuments, including wooden buildings in the regional style, and the landscape diversified by the ranges of the Stołowe, Orlické, Bystrzyckie, Bardzkie, Bialskie and Golden Mountains, as well as the Śnieżnik Mountains, the Kłodzko Valley and the Upper Nysa Rift. What nature created here in the form of an undoubtedly attractive landscape has been used by man for centuries, i.a. in tourism: in developing new maps, setting out hiking trails, buildings shelters, inns and viewpoints. The second half of the nineteenth century brought a lot of investments in the Sude- tes, related to the expansion of railway routes and roadways accompanying the mining and textile industry emerging here, as well as glassworks, porcelain factories and other types of 120 Jacek Suchodolski production. This had a major impact on the development of tourism, which also became one of the profitable branches of the economy. A more important and clearly visible element of tourism development, which also constitutes a form of its architectural expression, were the emerging shelters and other tourist facilities, more and more of which were built on mountain slopes and in valleys in the Sudetes, and thus in Kłodzko Land, providing travellers with accommodation and rest. Unfortunately, the period of development of these lands and the expansion of tourist facilities in the Sudetes was interrupted in 1945, after the borders were changed. Lower Sile- sia became a part of Poland, and settlers from various parts of the former Commonwealth of Poland came to replace the current German population; however, they were not usually familiar with the specificity of farming in the mountains. To make matters worse, Poland found itself in the zone of Soviet influence, which meant that a command-and-distribution economy was established, which is a denial of logical and established principles in economics. Moreover, uncertainty prevailed among settlers whether the decision to annex the Western Territories to Poland would last or not. It was often used by the authorities as leverage in the event of social unrest. All these factors did not favour stabilisation, especially in the initial period after the end of World War II. 2. Aim of the study The aim of the study is to restore and preserve the memory about those tourist shel- ters which had an interesting regional form and constituted an important part of the cultural landscape of Kłodzko Land before 1945. These buildings should become an inspiration for contemporary architects in order to restore the cultural continuity of this region and to create new architecture, enriched with traditional elements. The current article presents and publicises this problem in an attempt to halt the process of degradation, which consists in decapitalis- ing and then demolishing the objects from before 1945. It should be the task of institutions established for this purpose, mainly the Provincial Heritage Monuments Protection Office in Wrocław, because these objects are often valuable examples of old, unique work of local builders. The above-mentioned examples are only a fragment of what was irretrievably lost in Kłodzko Land after the end of the war and in later years. This problem concerns the entire range of the Sudetes on the Polish side of these mountains. 1. Introduction As a consequence, many tourist and leisure facilities were devastated and liquidated, including those with significant value of the architectural and regional features they represented. 3. Tourist and sport resorts in Kłodzko Land Over the past centuries, three main centres of tourism and winter sports were formed in Kłodzko Land, in which most of the buildings with recreational and tourist functions were concentrated, including shelters, mountain inns and taverns. These include the Śnieżnik Moun- tains, whose name is derived from the highest mountain in the former Kłodzko County –1425 m above sea level – along with summer resorts and famous winter resorts such as Międzygórze and Sienna with the ski resort in Czarna Góra, and Jodłów, Potoczek or Goworów [18], situ- ated slightly away from Śnieżnik. The following one is Masyw Orlicy – 1084 m, located in the Orlické Mountains – with Zieleniec, Kozia Hala, Graniczna and Podgórze, which are now parts of Duszniki-Zdrój [17]. The third mountain massif, whose first ascent, in 1790, is one of the earliest known, [13], [19], is Szczeliniec Wielki (919 m) located in the Stołowe Mountains, with the adjacent Karłów, Karłówko and Pasterka. 121 Forgotten shelters of Kłodzko Land… In addition to the main resorts, there were also smaller ones, in which tourist and recrea- tional architecture was constructed, including well-known buildings [15]. Among others, towns on the Dzika Orlica River should be mentioned here: Lasówka, Piaskowice, Mostowice, Rudawa, Poniatów, Niemiów and Lesica. Sudeten huts with an interesting regional form [17], [24], [25] were also located in Spalona, Wójtowice, Młoty, Biała Woda in the Bystrzyckie Mountains or the Puchaczowska Pass separating the Śnieżnik Mountains from the Krowiarki Mountains. Before 1945, in the above-mentioned resorts and individual towns, as well as in particularly attractive places, such as passes or summits in the mountains, mountain shelters, hotels and mountain inns formed a well-organised system of to urist and recreational facilities, which suffered significant degradation after the Second World War. Places that used to be densely populated became deserted; where buildings once stood, hills covered with grass and trees are now seen – these can be easily identified and stimulate the viewer’s imagination even stronger. Out of 56 shelters in Kłodzko Land (Fig. 1) which existed before 1945, as many as 43 are no longer in operation and only thirteen have a tourist function. After the war, only three new facilities of this type were built [4], [13], [16], [25]. 4. Regional architecture as a source of inspiration for the form and architectural details of shelters and inns in Kłodzko Land As in the neighbouring ranges of the Sudetes on the Silesian and Czech sides, character- istic features of the local wooden architecture also developed over the centuries in Kłodzko Land. The fact that the Kłodzko County used to belong to the Czech Crown for many centuries is also apparent in the form of its wooden architecture, largely resembling its Czech versions [1], [9], [13], [23]. The most common object of regional origin on both sides of the border is a one-storey building with a log structure of wooden walls and a steep gable roof with a slope of 45-55 degrees. At first roofs were covered with straw or reed thatch, then with wood shingles, less often with phyllite slate. Even before the First World War, in some buildings sheet metal was nailed to shingle roofing through intermediary ridge beams as an external covering, which increased fire resistance, but changed the character of the whole [9], [23] (Fig. 2). 122 Jacek Suchodolski Fig. 1. Location of shelters in Kłodzko Land (points: green – new shelters, built after 1945; black – non-ex- istent shelters, shelters that perform a different function; red – shelters in operation today, built after 1945; lines: red – Polish and Czechia state border; rose – poviat (district) border). Author’s study Fig. 1. Location of shelters in Kłodzko Land (points: green – new shelters, built after 1945; black – non-ex- istent shelters, shelters that perform a different function; red – shelters in operation today, built after 1945; lines: red – Polish and Czechia state border; rose – poviat (district) border). Author’s study Fig. 2. The village of Młoty in the Bystrzyckie Mountains. A rural residential building with log walls, a gabled roof covered with sheet metal, with a slope of 45 degrees. Photo by Jacek Suchodolski 2005 Fig. 2. The village of Młoty in the Bystrzyckie Mountains. A rural residential building with log walls, a gabled roof covered with sheet metal, with a slope of 45 degrees. Photo by Jacek Suchodolski 2005 123 Forgotten shelters of Kłodzko Land… Among the varieties of regional architecture, there are also single-storey wooden houses with an attic storeroom, i.e. a single-storey extension in the form of a dormer (extending beyond the perimeter of the building), supported on posts. 4. Regional architecture as a source of inspiration for the form and architectural details of shelters and inns in Kłodzko Land Known in Polish as wyżka, pięterko or ryzalit wejściowy, it was originally used for storing hay, and over time it became an additional living space for holidaymakers visiting these areas [23] (Fig. 3). Buildings in the region had gable roofs or roofs with a mansard structure. Buildings with log walls and mansard roofs are also a distinctive group among wooden architecture. Larger in volume, they often had the above-mentioned attic storerooms and served as taverns, shelters and inns. These roofs were mostly covered with diamond-shaped eternit or sheet metal tiles (Fig. 4). Fig. 3. Pstrążna – an open-air museum of wooden buildings. The so-called Zajazd (Inn) moved from Szalejów Dolny (probably built in the first half of the nineteenth century). The modest form of a single-storey house with log walls is diversified by an attic storeroom, i.e. an extension protruding beyond the eaves, supported on posts, in which there were rooms for tourists. Photo by Jacek Suchodolski 2015 Fig. 3. Pstrążna – an open-air museum of wooden buildings. The so-called Zajazd (Inn) moved from Szalejów Dolny (probably built in the first half of the nineteenth century). The modest form of a single-storey house with log walls is diversified by an attic storeroom, i.e. an extension protruding beyond the eaves, supported on posts, in which there were rooms for tourists. Photo by Jacek Suchodolski 2015 Fig. 4. Orlica shelter in Zieleniec, built in 1878. The building with log walls, covered from the outside with vertically-laid boards and with a mansard roof with dormers, also has an attic storeroom in the form of an extension, supported by the masonry walls of the ground floor. There was a second tavern near Orlica (Gasthaus zur Mense), of a similar shape and structure, but it was pulled down in the 1960s. Photo by Jacek Suchodolski 2009 Fig. 4. Orlica shelter in Zieleniec, built in 1878. The building with log walls, covered from the outside with vertically-laid boards and with a mansard roof with dormers, also has an attic storeroom in the form of an extension, supported by the masonry walls of the ground floor. There was a second tavern near Orlica (Gasthaus zur Mense), of a similar shape and structure, but it was pulled down in the 1960s. Photo by Jacek Suchodolski 2009 Fig. 4. Orlica shelter in Zieleniec, built in 1878. 4. Regional architecture as a source of inspiration for the form and architectural details of shelters and inns in Kłodzko Land The building with log walls, covered from the outside with vertically-laid boards and with a mansard roof with dormers, also has an attic storeroom in the form of an extension, supported by the masonry walls of the ground floor. There was a second tavern near Orlica (Gasthaus zur Mense), of a similar shape and structure, but it was pulled down in the 1960s. Photo by Jacek Suchodolski 2009 Fig. 4. 124 Jacek Suchodolski An important distinguishing feature of the regional architecture is also the architectural detail which gives it a distinct character and a kind of charm. The most characteristic elements include the arrangement of boards on the side and gable walls, the size and form of window openings and the shape of their trims, door lintels, types of structural joints in the walls and the most common types of roofing. It should be emphasised here that the main feature of regional houses were their proportions – the height of the walls of the ground floor in relation to the height of the roof (ranging from 1:2 or 1:2.5 to 1:3). Buildings intended for tourist purposes, put up in the mountains in the described region, were characterised by form and detail mostly based on regional motifs, and houses that were designed in isolation from local tradition were rare [9], [23]. 5. Non-existent tourist facilities with regional features The places where the largest number of tourism-related facilities, i.e. inns and shelters, ceased to exist after 1945, certainly include Zieleniec (Grunwald) in the Orlické Mountains, which is currently a part of Duszniki-Zdrój. Initially a poor mountain village, founded in 1719, over time it became one of the better known winter resorts with excellent conditions for skiing, as well as for hiking and cycling in the summer, thanks to extensive routes on the Polish and Czech side of the border [14], [25], [26]. Even before the First World War, when it was inhabited by almost 900 people, it boasted many well-known shelters and inns located on the slopes of Šerlich and Orlica, forming small complexes of buildings, scattered over the mountainous terrain. The most famous ones, now non-existent, were Hindenburgbaude, Hohe Mense – Baude, Gasthaus zur Mense (later called Ritterbaude) and Berghof Rübartsch (formerly called Reinerzer “Ski – Hütte”), or Mensehorst am Goldenen Stolen. Also in the central part of Zieleniec there was an inn that gained the rank of a shelter – Grunwaldbaude, whose name was changed in the 1930s to Adlerbaude. In 1948, Hindenburgbaude, built in 1928, ceased to exist. The author of this successful project was Konrad Goebel, a member of GGV and the architect of the city of Kłodzko. GGV was also the investor. Located at an altitude of 975 m above sea level, at the foot of Orlica, it was characterised by a compact form, inspired by regional architecture. Wood was the basic building material here, both for construction and finishing – apart from the basement walls and fragments of the ground floor made of stone. The skilful combination of the texture of wooden walls with stone elements in the entrance area emphasised the regional character of the building. The huge, steep, gable roof with pediments, covered with shingles, certainly stood out in the body of the shelter. The plan for the functions of the shelter also deserved praise, especially its ground floor with rooms intended for guests, where the designer managed to create interiors with an individual character. Decorated in a regional style with wooden carvings, they gave a sense of solidity and security. There was a fuel dispenser next to the shelter, as it could be accessed by car. Unfortunately, this building burned down in November 1948, probably as a result of a boiler room failure [14], [22] (Figs. 5-7). 5. Non-existent tourist facilities with regional features 125 Forgotten shelters of Kłodzko Land… Fig. 5. Zieleniec (Grunwald). Hindenburgbaude shelter. North-east façade. The interwar period. Source: Jacek Suchodolski’s collection Fig. 5. Zieleniec (Grunwald). Hindenburgbaude shelter. North-east façade. The interwar period. Source: Jacek Suchodolski’s collection Fig. 6. Zieleniec. Hindenburgbaude shelter. North-west façade. The interwar period. Source: the collection of the Museum of Kłodzko Land in Kłodzko Zieleniec. Hindenburgbaude shelter. North-west façade. The interwar period. Source: the collection of the Museum of Kłodzko Land in Kłodzko Fig. 7. Zieleniec. Hindenburgbaude shelter. The interior of the dining room. The interwar period. Source: the collection of the Museum of Kłodzko Land in Kłodzko Fig. 7. Zieleniec. Hindenburgbaude shelter. The interior of the dining room. The interwar period. Source: the collection of the Museum of Kłodzko Land in Kłodzko 126 Jacek Suchodolski This shelter, as it should be emphasised once again, was one of the most beautiful in the Sudetes, alongside Samotnia (Kleine Teichbaude) on Mały Staw in the Karkonosze, and Lucyna (Max Müllerbaude) in the Owl Mountains, which makes its loss even more severe. The second shelter – Hohe Mense-Baude (1084 m) – was built gradually, thanks to the efforts of its owner Heinrich Rübartsch, just below the summit of Orlica, at the Austrian-Prussian border. It was characterised by a simple form, and it consisted of two buildings with log and masonry construction, covered with steep gable roofs, with a wooden observation tower next to it (Fig. 8). They provided rest and accommodation for 10 people in bedrooms and 25 places in a shared room. The main advantage of this place, it seems, was the surrounding nature as well as peace and quiet, which was so valued – especially at the end of his life – by Heinrich Rübartsch, the precursor of skiing in Zieleniec, who contributed to the development and significance of this place in the Sudetes as a tourist resort. When he left the world of the living in 1930, he left behind an equally valuable thing: a memory of a person who never refused to help anyone, sometimes in extreme mountain conditions [24], [26]. Fig. 8. Zieleniec. Hohe Mensee-Baude shelter. The interwar period. Source: www.ansichtskarten.de Fig. 8. Zieleniec. Hohe Mensee-Baude shelter. The interwar period. Source: www.ansichtskarten.d The first shelter building was constructed in 1883, the second in the first years of the twentieth century. The shelter was demolished in the 1940s. 5. Non-existent tourist facilities with regional features The next building, Gasthaus zur Mense (872 m above sea level), is also associated with Rübartsch, because this was where he began his work as an innkeeper. Located opposite the main entrance to the church, the Gasthaus building was one of the oldest in Zieleniec (apart from the wooden church demolished at the beginning of the twentieth century), and its form referred to the regional tradition of wooden architecture of Kłodzko Land. It offered 35 beds in 15 rooms. Gasthaus changed its name to Ritterbaude after it was taken over by a new owner. This one-storey building, covered with a mansard roof with a pediment and an attic storeroom, was one of many objects of this type found on the Kłodzko-Orlické border, as well as in other parts of the region. To a large extent, it resembled the Orlica shelter from 1878 – which still stands next to it – in terms of form and detail, but it was probably older. Rübartsch eventually left Gasthaus zur Mense in 1903, when he moved permanently to his Hohe Mense-Baude shelter at the top of Orlica. After the war, the 127 Forgotten shelters of Kłodzko Land… inn building fulfilled auxiliary functions for some time as Orlica III. Gasthaus zur Mense was demolished in the 1960s [24]. Another shelter – Berghof Rübartsch (975 m above sea level) – situated on steeply sloping ground, was a relatively small facility, but had visible regional influences in its architecture. It provided 25 beds. The original name of this Orlické hut on the slope of Šerlich Mountain was Reinerzer “Ski Hütte”. After 1945, the building ceased to exist 11 Fig. 9. Mensehorst am Goldenen Stollen shelter: (A) - the first building from 1927, (B) the shelter after its expansion (after 1936) Source: Andrzej Wziątek’s collection A B B A Fig. 9. Mensehorst am Goldenen Stollen shelter: (A) – the first building from 1927, (B) the shelter after its expansion (after 1936). Source: Andrzej Wziątek’s collection B B B Fig. 9. Mensehorst am Goldenen Stollen shelter: (A) - the first building from 1927, (B) the shelter after i i ( f 1936) S A d W k’ ll Fig. 9. Mensehorst am Goldenen Stollen shelter: (A) – the first building from 1927, (B) the shelter after its expansion (after 1936). 5. Non-existent tourist facilities with regional features Source: Andrzej Wziątek’s collection Mensehorst am goldenen Stollen youth hostel, located about two kilometres from Ziele- niec, by the Orlicka Road, was built in 1927 as a small one-storey building with a steep gable roof (Fig. 8 A). It had 23 rooms with 40 beds. In 1936, a second, also one-storey building (Fig. 8 B) was erected on the plot of the existing facility, with architecture reminiscent with its form of border guard buildings in many Sudeten towns along the border (e.g. in Szklarska Poręba, on the Okraj Pass, in Jarkowice, Pasterka, Zieleniec, Lasówka, Rudawa, Lesica, Międzylesie). In the 1960s, Mensehorst am Goldenen Stollen was demolished. Mensehorst am goldenen Stollen youth hostel, located about two kilometres from Ziele- niec, by the Orlicka Road, was built in 1927 as a small one-storey building with a steep gable roof (Fig. 8A). It had 23 rooms with 40 beds. In 1936, a second, also one-storey building (Fig. 8B) was erected on the plot of the existing facility, with architecture reminiscent with its form of border guard buildings in many Sudeten towns along the border (e.g. in Szklarska Poręba, on the Okraj Pass, in Jarkowice, Pasterka, Zieleniec, Lasówka, Rudawa, Lesica, Międzylesie). In the 1960s, Mensehorst am Goldenen Stollen was demolished. Fig. 10. Zieleniec. In the foreground, on the right, the Grunwaldbaude shelter, later known as Adlerbaude. A characteristic element in the body of the shelter was the observation porch. The interwar period. Source: Andrzej Wziątek’s collection Fig. 10. Zieleniec. In the foreground, on the right, the Grunwaldbaude shelter, later known as Adlerbaude. A characteristic element in the body of the shelter was the observation porch. The interwar period. Source: Andrzej Wziątek’s collection 128 Jacek Suchodolski Fig. 11. Lasówka (Kaiserswalde). Three-field postcard, the interwar period. Bottom right corner: Max Mader Gasthaus. This object, which had probably been built in the mid-nineteenth century, ceased to exist in 1954. Source: www.klodzkikatek.pl Fig. 11. Lasówka (Kaiserswalde). Three-field postcard, the interwar period. Bottom right corner: Max Mader Gasthaus. This object, which had probably been built in the mid-nineteenth century, ceased to exist in 1954. Source: www.klodzkikatek.pl The last shelter in Zieleniec from this group, located in the central part of the town, which no longer exists today, was Grunwaldbaude (900 m above sea level), called Adlerbaude since 1934. It had 16 beds. Initially, it was an inn, which then became a shelter (Fig. 10). 5. Non-existent tourist facilities with regional features A porch was added in the 1930s, enlarging this object and giving it a tourist character. The architecture of this Sudeten hut, though it did include some regional elements in its architec- tural details, did not stand out as anything special. However, the volume of this object was adapted to the surrounding buildings and did not cause visible disharmony, which, unfortu- nately, is now often seen in the expanding Zieleniec. After the war, in the 1950s, Adlerbaude was renamed Janosikowa Hala, which was then taken over by the Wrocław University of Physical Education and demolished. All the above-mentioned shelters – perhaps the last one the least – had a common quality: namely, their architecture was strongly saturated with local and regional features, which, together with their location in the mountainous terrain (which made them harmoniously fit in with the surrounding landscape), made the panorama of Zieleniec uncluttered and clear. In the neighbouring Lasówka (Kaiserswalde), known before the war for a crystal glassworks and a glass cutting plant, Max Mader Gasthaus (Fig. 11) shelter/inn – a one-storey, wooden building situated next to the church – was demolished in the 1950s. Its form and detail clearly attested to its regional origins, which were evident in such features as a steep gable roof covered with shingles, log walls of the ground floor, covered with vertically-laid boards, and the gable wall covered with shingles. Small window openings with trims represented the modest architectural details of Mader’s Gasthaus. As befits the regional Sudeten structures, the ratio of the height of the ground floor walls to the roof height was 1:2. [25] (Fig. 11). Forgotten shelters of Kłodzko Land… 129 Fig. 12. Lasówka (Kaiserswalde), Erlitztalbaude shelter, the interwar period. Source: Jacek Suchodolski’s collection Fig. 12. Lasówka (Kaiserswalde), Erlitztalbaude shelter, the interwar period. Source: Jacek Suchodolski’s collection Fig. 13. Lasówka (Kaiserswalde). Former Erlitztalbaude shelter, now a residential building. The metal sheet on the roof was nailed to the original shingle covering through ridge beams. Photo by Jacek Suchodolski 2005 Fig. 13. Lasówka (Kaiserswalde). Former Erlitztalbaude shelter, now a residential building. The metal sheet on the roof was nailed to the original shingle covering through ridge beams. Photo by Jacek Suchodolski 2005 The second shelter, Erlitztalbaude (Fig. 11, 12), located near the Mostowy Stream, which also has a classic, regional form, has survived to our times, becoming a residential building after 1945. 5. Non-existent tourist facilities with regional features After the war, when Pośna waterfalls ceased to exist, which was caused by the creation of the artificial lake in Radków, the shelter fell into ruin. As in other mountain ranges in the tourist county of the Sudetes, there were also buildings in the Table Mountains that until 1945 served as shelters, such as Rabenkoppenbaude (721 m above sea level) in Dańczów, characterised by a classic regional form. After the war they became private residential buildings [25]. In the village of Biała Woda (Weisswasser), located near the Puchaczówka Pass, by the road connecting Bystrzyca Kłodzka, Lądek and Stronie Śląskie, a youth hostel Jugendherberge Weisswas- ser was built with over 100 beds; its architecture was modelled on regional motifs. After 1945, the shelter ceased to exist. A little above the pass, on the northern slope of Czarna Góra, by the road mentioned above, in the first half of the nineteenth century there was an inn called Puhu Wirtshaus, transformed at the beginning of the twentieth century into the Puhu-Baude shelter (899 m above sea level). (Fig. 14). include log walls of the ground floor, covered with vertically-laid boards, a steep gable roof and small window openings with trims. The proportions of the ground floor walls to the roof height are 1:2. The places from which several inns and shelters also disappeared after 1945 and to which the tourist industry has not returned since were the Waldhufen- dorf-type village of Lesica (Freiwalde) on the Jelonik stream and the nearby settlement Czerwony Strumień (Rothflössel), located on the stream of the same name, which is, as the former, a tributary of the Dzika Orlica. The hamlet of Lesica, Hirschenhäuser, was characterised by particular landscape values, and it had two inns in the second half of the nineteenth century. One of them was transformed into Hirschenhaus shelter (650 m above sea level) with a wooden structure. Above the ground floor with a log wall there was a high, steep roof covered with shingles, which also protected the gable wall against atmospheric influences. The building was representative of one of the varieties of regional construction, often found in Kłodzko Land. After 1945, the shelter ceased to exist. Regional features were also visible in the case of the facility located in Czerwony Strumień, namely Gruppenbaude shelter (which was an SS resort during the war), whose ruins are located at the foot of Bochniak Mountain1. 1 Its ruins are marked on the tourist map: Góry Bystrzyckie, Góry Orlickie, scale 1:35 000, Wydawnictwo Compas, Cracow, catalogue number: 1473/2019 [7]. 5. Non-existent tourist facilities with regional features Initially, it housed the Stumpfgasthaus inn, and in the 1930s it became a shelter. A characteristic element in the body of the building is the raised roof slope (Frakdach), intended to provide larger cubic capacity in the attic. This was often used in facilities intended for tourist and leisure purposes. This building – like the previous one – represents one of the varieties of the local regional architecture, whose main features 130 Jacek Suchodolski include log walls of the ground floor, covered with vertically-laid boards, a steep gable roof and small window openings with trims. The proportions of the ground floor walls to the roof height are 1:2. The places from which several inns and shelters also disappeared after 1945 and to which the tourist industry has not returned since were the Waldhufen- dorf-type village of Lesica (Freiwalde) on the Jelonik stream and the nearby settlement Czerwony Strumień (Rothflössel), located on the stream of the same name, which is, as the former, a tributary of the Dzika Orlica. The hamlet of Lesica, Hirschenhäuser, was characterised by particular landscape values, and it had two inns in the second half of the nineteenth century. One of them was transformed into Hirschenhaus shelter (650 m above sea level) with a wooden structure. Above the ground floor with a log wall there was a high, steep roof covered with shingles, which also protected the gable wall against atmospheric influences. The building was representative of one of the varieties of regional construction, often found in Kłodzko Land. After 1945, the shelter ceased to exist. Regional features were also visible in the case of the facility located in Czerwony Strumień, namely Gruppenbaude shelter (which was an SS resort during the war), whose ruins are located at the foot of Bochniak Mountain1. Among the well-known shelters with regional archi- tectural features which have not survived to our times, Baude Carl Rast (680 m above sea level) can be mentioned, located near the also non-existent today waterfalls Pośny in the Table Mountains, in Karłówek (Klein Karlsberg), which disappeared after the war. The inn functioning here was transformed into a shelter in the mid-nineteenth century, and then expanded in the first years of the twentieth century. In two wooden buildings, one of which had an attic storeroom, there were 14 beds. 5. Non-existent tourist facilities with regional features Among the well-known shelters with regional archi- tectural features which have not survived to our times, Baude Carl Rast (680 m above sea level) can be mentioned, located near the also non-existent today waterfalls Pośny in the Table Mountains, in Karłówek (Klein Karlsberg), which disappeared after the war. The inn functioning here was transformed into a shelter in the mid-nineteenth century, and then expanded in the first years of the twentieth century. In two wooden buildings, one of which had an attic storeroom, there were 14 beds. After the war, when Pośna waterfalls ceased to exist, which was caused by the creation of the artificial lake in Radków, the shelter fell into ruin. As in other mountain ranges in the tourist county of the Sudetes, there were also buildings in the Table Mountains that until 1945 served as shelters, such as Rabenkoppenbaude (721 m above sea level) in Dańczów, characterised by a classic regional form. After the war they became private residential buildings [25]. In the village of Biała Woda (Weisswasser), located near the Puchaczówka Pass, by the road connecting Bystrzyca Kłodzka, Lądek and Stronie Śląskie, a youth hostel Jugendherberge Weisswas- ser was built with over 100 beds; its architecture was modelled on regional motifs. After 1945, the shelter ceased to exist. A little above the pass, on the northern slope of Czarna Góra, by the road mentioned above, in the first half of the nineteenth century there was an inn called Puhu Wirtshaus, transformed at the beginning of the twentieth century into the Puhu-Baude shelter (899 m above sea level). (Fig. 14). Forgotten shelters of Kłodzko Land… 131 Fig. 14. The Puchaczówka Pass (899 m above sea level) separating the Śnieżnik Mountains from the Krowiarki Mountains. The Puhu-Baude shelter, the interwar period. Drawing by Jacek Suchodolski [17] Fig. 14. The Puchaczówka Pass (899 m above sea level) separating the Śnieżnik Mountains from the Krowiarki Mountains. The Puhu-Baude shelter, the interwar period. Drawing by Jacek Suchodolski [17] Along with the change of the inn into a shelter, it was expanded and its architecture was given a regional form. It had 17 beds in 14 rooms and a shared room. The wooden-brick build- ing gained a compact body with a steep, gable roof with a long dormer. 2 In the School of Wood Carving in Cieplice near Jelenia Góra (Holzschnitzschule Bad Warmbrunn), founded in 1902, which educated both sculptors-artists and craftsmen, a characteristic style was born, inspired by the local folk tradition as well as that of south Tyrol. The school was animated by Cirillo Dell’Antonio (a teacher and later the principal of the school in Cieplice in 1920-1940). The works of the graduates of the school could be found throughout the Sudetes, often in the form of stylised images of groups of people that decorated information boards, signposts or the interiors of shelters [20]. 5. Non-existent tourist facilities with regional features The gable walls were covered with boards arranged in a herringbone pattern, and the ground floor windows were fitted with shutters, which clearly referred to regional motifs. The interior of the shelter was in a rustic style, which was emphasised by the beam structure of the wooden ceiling. Similarly, tables and chairs were made of wood, and it was all complemented by a large tiled stove. White areas between the ceiling beams were covered with floral motifs. Porcelain plates decorated with local motifs hung on the walls, and under the ceiling there was a wooden candelabrum depicting a group of musicians, reminiscent in its form of the works of woodcarvers from the Cieplice school of crafts2. This shelter, which was very popular before the war, ceased to exist after 1945. Near Puhu-Baude, another small shelter was built in the interwar years – Schwarze- Berg-Häusel (900 m above sea level), located on the slope of Czarna Góra. An alpine garden was founded around it, the remains of which are still visible today. The building was crowned with a characteristic steep gable roof, giving it a somewhat “fairy-tale” character. Like the neighbouring shelter, Domek Czarnogórski was pulled down after 1945 132 Jacek Suchodolski 6. Conclusion The examples of non-existent shelters in Kłodzko Land described here reflect only a small scale of the problem which was and still is the devastation of the cultural environment after 1945 in these areas as well as in other parts of the Sudetes. Unfortunately, it is especially visible on the Silesian, i.e. Polish side of these mountains. The listed non-existent objects, distinguished by their regional architecture, were an inseparable component of the mountain landscape, currently very incomplete and often further damaged by the foreign form and an inadequate scale of the buildings being erected today, which does not contribute to restoring the cultural continuity of the region. Therefore, it is indispensable to provide professional and systematic conservation care and legal protection over the still existing wooden buildings of regional character, including tourist and recreational facilities, as well as compliance with the provisions contained in detailed land use plans. This has become exceptionally important in recent years, as the volume and form of buildings under construction that serve tourist func- tions are a contradiction of tradition and often common sense (examples are the Gołębiewski Hotel in Karpacz or the Infinity apartment building in Zieleniec, which is under construction, intended for around 1,000 people). Similar facilities are planned and if these intentions are not stopped by public opinion and competent authorities, the degradation process will deepen. According to the author, in order to restore former splendour and uniqueness to the cultural landscape of Kłodzko Land, one should consider recreating some former shelters and inns in their original locations. This would apply to objects with special aesthetic and regional values of their architecture, i.e. some of those referred to in the article. As it has already been stated, new facilities should incorporate solutions inspired by the local building tradition in their form and structure. Examples of such an approach are residential buildings and those with a tourist function, designed in Alpine areas in Germany, Austria, Switzerland or Italy. Thanks to the use of mainly wood and stone and modest architectural details, the buildings fit in well with the surrounding landscape, at the same time being a contemporary, creative complement to it and its continuation, i.e. they meet the assumptions that should also be implemented in the Kłodzko Region and other parts of the Sudetes in terms of shaping architecture. References [1] Bocheński S., Trocka-Leszczyńska E., Wiatrzyk S., “Architektura regionalna Ziemi Kłodzkiej – tradycja, stan istniejący, możliwość kontynuacji”, Raporty Instytutu Architektury Politechniki Wrocławskiej (PRE series), no. 384, (1986). [2] Brygier W., Dudziak T., Ziemia Kłodzka. Pruszków: Oficyna Wydawnicza Rewasz, 2010. [3] Chmielewski W.J., Regionalizm współczesnej architektury reakcją na procesy globalizacji. Cracow: Dział Poligrafii Politechniki Krakowskiej, 2017. [4] Dziedzic M., “Schroniska Kłodzkiego Towarzystwa Górskiego”, Sudety, no. 8, (2009), p. 37. [5] Dziedzic M., Kłodzkie Towarzystwo Górskie -1945, Wrocław: Quaestio, 2013. [6] Ferien und Wochenend in der Grafschaft Glatz, Herausgegeben vom Hauptvorstand des G.G.V.. Glatz: Kłodzkie Towarzystwo Górskie, 1932. [7] Góry Bystrzyckie, Góry Orlickie. Mapa turystyczna skala 1:35000. Catalogue numer: 1473/2019, Cracow: Wydawnictwo Compas, 2019. [8] Kosiński W., Setkowicz B., “Śnieżnik – organizacja przestrzenna rekreacji”, Turystyka w Sude- tach, Prace Karkonoskiego Towarzystwa Naukowego, no. 10, part 2, Jelenia Góra: Wydawnictwo Turystyczne „PLAN”, 1977. [9] Löwe L., Schlesische Holzbauten. Düsseldorf: Werner-Verlag, 1969. 133 Forgotten shelters of Kłodzko Land… [10] Marx J., Vom Schneeberg zur Hohen Eule. Leimen/Heidelberg: Marx Verlag, 1975. [11] Mazurski K.R., Góry Bystrzyckie i Orlickie, Sudety. Wrocław 1993. [12] Mazurski K.R., “Schronisko PTTK Orlica”, Na Szlaku, no. 10(136), (2000), p. 19. [13] Mazurski R.K., Historia turystyki sudeckiej. Cracow: Oficyna Wydawnicza Wierchy, 2012. 14] Michna W., Zieleniec i okolice. Okruchy historii. 1st ed., Paczków: Wiesław Michna, 201 [15] Otto A., Glatzer Wanderbuch. Ein Erinerungsbuch an die Grafschaft Glatz / Schlesien. 3rd ed., Leimen/Heidelberg: Marx Verlag, 1981. [16] Potocki J., Rozwój zagospodarowania turystycznego Sudetów od połowy XIX wieku do drugiej wojny światowej. Jelenia Góra: Wydawnictwo Turystyczne „PLAN”, 2004. [17] Słownik Geografii Turystycznej Sudetów. Góry Bystrzyckie i Orlickie. Vol. 14, ed. M. Staffa, Warsaw: Wydawnictwo PTTK Kraj, 1992. [18] Słownik Geografii Turystycznej Sudetów, T. XIV Góry Bystrzyckie i Orlickie. M. Staff (ed.), Warsaw: Wydawnictwo PTTK “Kraj, – Krakow 1992. [19] Słownik Geografii Turystycznej Sudetów. Masyw Śnieżnika, Góry Bialskie. M. Staff (ed.), vol. 16, Warsaw: Wydawnictwo PTTK Kraj, 1993. [20] Bździach K., ed., Wspaniały krajobraz – Artyści i kolonie artystyczne w Karkonoszach w XX wieku. Berlin: Gesellschaft für Interregionalen Kulturaustausch; Jelenia Góra: Muzeum Okręgowe, 1999, pp. 223-237. [21] Przerwa T., Między lękiem a zachwytem. Sporty zimowe w śląskich Sudetach i ich znaczenie dla regionu (do 1945 r.). Wrocław: Atut, 2012. [22] Sroka P.,“Hindenburgbaude w płomieniach”, Sudety, no. 113, (2010), p. 29. [23] Suchodolski J., Regionalizm w kształtowaniu formy architektury współczesnej na obszarze Sudetów. Habilitation thesis, Wrocław: Oficyna Wydawnicza PWR, 1996. References [24] Suchodolski J., „Architektura regionalna schronisk i zajazdów w Masywie Orlicy”, Konferencja Kłodzko-Orlicka, Duszniki Zdrój 21-22.04.2005, Biuletyn pokonferencyjny, Nowa Ruda, pp. 47-62.i [25] Suchodolski J., Architektura schronisk górskich w Sudetach. Wrocław: Oficyna Wydawnicza PWR, 2005. [26] Tomczak M., “Orlica – szczyt zapomniany”, Pielgrzymy´88. Informator krajoznawczy XXXIII Ogólnopolskiego Rajdu Sudeckiego, Wrocław: SKPS, 1988, pp. 77-89.
https://openalex.org/W4361003748	https://www.scielo.br/j/mr/a/dJWG6wmhFWXqxcJ6FcJNjjg/?lang=en&format=pdf	English	null	Wear Behavior of Brass Based Composite Reinforced with SiC and Produced by Stir Casting Process	Materials research	2,023	cc-by	4,931	DOI: https://doi.org/10.1590/1980-5373-MR-2022-0196 Materials Research. 2023; 26: e20220196 Received: April 13, 2022; Revised: February 16, 2023; Accepted: March 01, 2023 The current investigation presents the wear-worn surface analysis of a silicon carbide-reinforced brass-based composite synthesized by stir casting. Wear behavior of the brass composite pin was analyzed by disc tribometer. Wear characterization studies and confirmation of elemental composition are investigated through scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) respectively. The worn surface of the synthesized brass composite was analyzed using atomic force microscopy (AFM). The aim of the investigation is to examine the surface morphology of the worn specimen. Based on the input constraints, the wear rate ranges from 0.0135 to 0.0893 mm3/min. The applied load is the predominant factor in the wear rate (83.75%). Sliding velocity has a minor effect on wear rate (1.06%). The improved surface roughness of 15.27 nm was produced on the worn surface. The novelty of the research work is to study the various surface parameters of the worn surface, such as roughness average, root mean square roughness, maximum height of the roughness, skewness, and kurtosis. These parameters were analyzed at different wear-worn surfaces of the synthesized brass composite. The wear-worn surface was deeply investigated and incorporated with SEM and AFM analysis. Keywords: Brass composite, Silicon carbide, Wear characterization, Wear worn surface, Atomic force microscopy. e-mail: mohanmechmz@gmail.com E. Mohana , G. Anbuchezhiyanb, R. Pugazhenthic, F. Peter Prakashd E. Mohana* , G. Anbuchezhiyanb, R. Pugazhenthic, F. Peter Prakashd aShanmuganathan Engineering College, Department of Mechanical Engineering, Pudukkottai, India. bSaveetha Institute of Medical and Technical Sciences, Saveetha School of Engineering, Department of Mechanical Engineering, 60125, Chennai, India. cVels Institute of Science, Technology & Advanced Studies, Department of Mechanical Engineering, Chennai, India. dMount Zion College of Engineering and Technology, Department of Mechanical Engineering, Pudukkottai, India. 1. Introduction Wear and strength play an essential role in materials and composites. Brass is stronger and stiffer than copper. It has excellent corrosion resistance, malleability, and formability characteristics. Brass and its alloys are used in gears, valves, bearings, couplings, and electrical and electronic components. It has a tensile strength of 39 Kg/mm2, Vickers hardness of 105, and a density of 84 kg/mm3 1. The brass alloy has an exceptional abrasive wear rate and frictional behavior. An abrasive wear rate of 0.0003 mm3/ Nm was attained at 50 N of load for brass2. Friction coefficient and wear behavior experiment was conducted on the brass alloy. It was observed that sliding velocity played an essential role in wear rate3. In the wear behavior of copper and brass alloys, the coefficient of friction decreases as the applied load increases. SiC reinforcement particles were used to enhance the tribological and material properties4. The wear rate mainly depends on the weight percentage of the reinforcement particles and the material properties. It was concluded that the wear layer was found on the surface of the duplex brass5. Wear and strength are the essential material properties of the composite. The volume fraction of reinforcement and its effect on wear rate were analyzed under different temperature conditions6. The wear experimentation was conducted on a titanium carbide-based copper composite. The influential factor and its role in wear rate were analyzed7. Reduction in grain size and the accumulation of reinforcement increased the material’s properties and wear resistance in copper composite8. The wear behavior of the copper composite was analyzed under different input constraints. The disc rotational speed produced a major effect on wear rate9. Wear rate and coefficient of friction were evaluated for different compositions of copper composite. The wear surface of the copper composite under different load conditions was analyzed by scanning electron microscope images10. Wear volume and wear rate were decreased by an applied load of 20N. The coefficient of friction was decreased by the increase of reinforced particles in the brass alloy. Delamination of wear particles and debris was observed in the wear test on brass11. Mechanical and wear behaviors were investigated in the sandblasting and annealing processes of a brass alloy. The grain size of the reinforced particle had the greatest influence on the wear rate in brass alloy12. The purpose of the Mohan et al. 2.3. Characterization of the brass composite The characterization of the synthesized brass composite is shown in Figure 2. From the figure, it was observed that the SiC particles are entrenched on the brass composite and uniformly spotted over the surface due to the least amount of the reinforced particles, as was clearly shown in Figure 2. 2.1. Raw material The wear characteristics were analyzed by a pin-on-disc tribometer. The pin was made of brass composite with a 10 mm diameter and 20 mm height. The ASTM G-99 standard was used to follow the wear test. Initial roughness of the pin is 11.2 nm. It was measured by surface roughness tester. Various sizes of emery paper were used to finish the pin surfaces, which were cleaned carefully with an acetone solution. EN-31 steel was used as the disc material. As per the ASTM E8 M11 standard, the specimens were prepared. The total length of the specimen is 50 mm, with front and back end diameters of 10 mm. The neck diameter is 6 mm, and the length is 30 mm was fabricated for the tensile test. A Brinell hardness tester with a ball diameter of 10 mm and a load of 500 kgf was used. The test results noted that the composite has better material properties, such as a hardness of 510 BHN, a density of 8.6 g/cc, and a tensile strength of 475 MPa. AFM (OEM model, Korea) deeply analyzed the worn surface of the brass composite. The scanning resolution is 0.2 nm in the XY direction and 0.05 nm in the Z direction. AFM is the best tool to provide information about surface topography parameters. The scan size of 100 mm by 100 mm was used in the atomic microscope to study the roughness parameters. Imaging software was involved in calculating the roughness parameters. The chemical composition ensured by EDS is shown in Figure 3. EDS (Carl Zeiss, Jena, Germany) with sensor active areas of 10mm2 to 100mm2 and a resolution of 123–133 eV was used. The primary composition of the composite is copper and zinc. Copper and zinc are the main compositions of pure brass. It is lead-free brass, and it is considered for experimental investigation. It is more malleable than other materials, and it has exceptional material properties. It consists of 66% copper and 34% zinc. The brass billets are purchased from NEXTGEN Metals in Mumbai, India. The total weight of the raw material is around 4 kg, and the diameter is 1 inch. Between copper and zinc, silicon carbide acts as reinforcement. It has outstanding hardness and mechanical strength at elevated temperatures, higher thermal conductivity, and corrosion and oxidation resistance. 1. Introduction 2 Materials Research present article was to investigate the worn surface on brass composites produced by the stir casting process. The alloying composition and characterization of the composite were confirmed by EDS and SEM. The wear-worn surface analysis on the brass composite was conducted by AFM analysis. 2.2. Sample preparation A silicon carbide-reinforced brass-based composite was synthesized by the stir casting technique. It is an exceptional and popular technique for developing reinforced composite materials. The reinforcement phase and uniform mixture of molten metal play an essential role in the stir-casting process. A muffle furnace preheats the SiC particles at 1500 °C for 50 minutes. A graphite crucible furnace develops the melts from brass (69 wt.% of copper and 29.5 wt.% of zinc) with silicon carbide particles (1.5 wt.%). The stirrer was applied to the molten metal at 600 rpm. Silicon carbide particles with a size of 20 mm were chosen. The preheated silicon carbide particles were placed into the molten metal and stirred at 700 rpm for 20 minutes. Figure 1 clearly depicts the shape morphology of SiC particles. The molten metal was poured into the cast iron mould. After the solidification process, the metal part was removed and machined to 50 mm in length and 10 mm in diameter. 3. Result and Discussion Experimental results for frictional wear are shown in Table 1. The sliding distance and track diameter for the entire test are Figure 1. SEM image of SiC particles. Figure 1. SEM image of SiC particles. Figure 2. SEM image of the brass composite. Figure 2. SEM image of the brass composite. Figure 2. SEM image of the brass composite. Figure 1. SEM image of SiC particles. Wear Behavior of Brass Based Composite Reinforced with SiC and Produced by Stir Casting Process 3 Wear Behavior of Brass Based Composite Reinforced with SiC and Produced by Stir Casting Process Figure 4a-4b describe the wear rate of the brass composite samples concerning velocity and disc speed. Figure 4a demonstrates the wear rate as a function of velocity for the different loads, such as 15N, 20N, and 25N. This figure confirms that the low wear rate was perceived (0.0135 mm3/ min). The maximum wear rate was attained at a load of 20 N and a sliding velocity of 6 m/s. It was observed that the samples were quickly worn and had a wear loss of 0.0893 mm3 due to severe abrasive action13,14. Figure 4b illustrates the composite’s wear rate as related to disc speed and load condition. The figure clearly stated that the wear rate gradually increased with an increase in disc rotational speed. More rubbing action was observed due to the temperature that developed between the contact surfaces of the pin and disc15. Owing to high disc speeds, sudden increases in temperature cause a decrease in the specimen’s strength and wear16. It was interesting to note that the wear rate gradually increased from 400 rpm to 1200 rpm. It was concluded that the 34% wear rate had increased from 400 rpm to 1200 rpm. The minimum wear rate of 0.0587 mm3/min was attained at maximum load conditions. The variance analysis of frictional wear is shown in Table 2. The wear rate of the brass composite depended on the different input constraints. Rubbing action was increased with an increase in load. The load developed the most elevated effect on kept constant (18 m and 100 mm, respectively). Different control factors and their levels were used to evaluate the wear rate. The various input constraints and their levels are 15–25 N of applied load, 400–1200 rpm of disc rotational speed, and 3–9 m/s of velocity. Equation 1 was used to evaluate the wear rate. / Wear rate Volume of wear loss applied load x sliding distance = (1) Standard deviation of the wear rate = 0.0237 3. Result and Discussion Standard deviation of the wear rate is 0.0237 / Wear rate Volume of wear loss applied load x sliding distance = (1) Figure 3. EDS image of brass composite. Figure 3. EDS image of brass composite. Figure 3. EDS image of brass composite. Figure 3. EDS image of brass composite. Figure 4. Wear rate of the samples a) function of velocity b) function of disc speed. Figure 4. Wear rate of the samples a) function of velocity b) function of disc speed. Figure 4. Wear rate of the samples a) function of velocity b) function of disc speed. Table 1. Experimental results for wear rate of brass composite. S.No Load (N) Disc speed (rpm) Velocity (m/s) Wear rate (mm3/min) 1 15 400 3 0.0135 2 15 800 6 0.0231 3 15 1200 9 0.0322 4 20 400 6 0.0486 5 20 800 9 0.0534 6 20 1200 3 0.0667 7 25 400 9 0.0587 8 25 800 3 0.0782 9 25 1200 6 0.0893 Standard deviation of the wear rate = 0.0237 e 1. Experimental results for wear rate of brass composite. Mohan et al. 4 Materials Research Table 2. Variance analysis for wear rate. Basis DF SS MS F P % Load 2 0.004229 0.002115 214.57 0.005 83.57 Disc speed 2 0.000757 0.000379 38.42 0.025 14.96 Velocity 2 0.000054 0.000027 2.73 0.268 01.06 Error 2 0.000020 0.000010 ---- ---- 00.41 Total 8 0.005060 --- ---- ---- 100 Table 2. Variance analysis for wear rate. clearly examined how the surface was damaged owing to the speed, load, and velocity throughout the wear test. It was principally focused on studying and clearly assessing the surface topography of the material. In the current study, AFM measurement is critical in associating surface characteristics such as roughness average, root mean square roughness, maximum height of the roughness, maximum roughness peak height, skewness, and kurtosis with the load value, sliding velocity, and disc speed in the wear test analysis. From Table 1, It was clearly stated that at the maximum load, disc speed, and velocity condition, a greater wear rate of 0.0893 mm3/min was recorded. It was likewise duplicated in the AFM measurement of the specimen 5, which has a maximum roughness peak height of 188.5 nm, a roughness average value of 43.12 nm, and a maximum root mean square value of 56.76 nm. 3. Result and Discussion At the same time, a minimum wear rate of 0.01355 mm3/min was clearly apparent on Specimen 3 at the conclusion of the wear test. The average roughness maximum root mean square roughness value, which has also been connected with the AFM measurement, has a lower value when compared to the other specimens in the surface Investigation. The worn surface of the brass composite has different shapes of cliffs and valleys. Average roughness, root mean square roughness, the maximum height of the roughness, roughness valley depth, peak height, skewness, and kurtosis were neatly delivered in Table 3. Figures 6-10 show 3D atomic force microscope images of the specimen under various wear and load conditions. The line profile, which depicts the texture, waviness, and surface roughness of the material, is also shown near the 3D AFM images. Table 3 shows that specimen 5 has the highest average roughness value of 43.12 nm, while specimen 3 has the lowest average roughness value of 15.27 nm. The root mean square value of 56.76 nm is also the highest in specimen 5 and the lowest in specimen 3. Kurtosis is another important parameter to use to characterize the surface of the specimen; if the value is less than 3, the surface seems to be flat; if it is greater than 3, the surface has a strong spike-like peak profile. The greatest value of kurtosis was clearly stated to resemble a spike profile on the surface22. In this study, specimen 3 with a wear load condition of 25 N produced a higher waviness profile, as seen in Figure 8. The root mean square value and maximum height of the roughness for the specimen 5 are 56.76 nm and 376.9 nm, respectively, which create a rougher surface23. Similar to the investigation24, negative skewness indicates less spiky surfaces, as is obviously indicated in Figure 6, and a higher skewness value evidently indicates hill-like surface morphology, as is clearly shown wear rate among all the input constraints. It has produced an 83.75% effect on wear rate. The bonding strength between the surface layers was unstable and increased with the loss of materials17. The attained R squared, adjusted R squared, and predicted R squared are all greater than 90%. The disc speed and velocity contributions to wear rate are 14.96% and 1.06%, respectively. 3.1. SEM analysis The worn surface of the brass composite was analyzed by scanning electron microscopy with varying loading conditions. SEM images of the specimen under different wear and load conditions were neatly demonstrated in Figure 5a-5e. From the figure, it has been confirmed that silicon carbide particles are scattered over the specimen. The wear debris spread over the surface due to the localized heat of the pin and abrasive wear. The amount of heat generation depends on the frictional force between the tool and the workpiece18. It was also demonstrated in a wear study by silicon particles being separated across the surface due to increased friction between the two surfaces19,20. The wear rate has increased without the addition of SiC particles in a brass alloy. The addition of SiC particles manipulates properties such as density and hardness in the brass21. Figure 5a shows the wear-worn surface at 25 N of load, 400 rpm of disc speed, 9 m/s of velocity, and a wear rate of 0.0587 mm3/min. Wear debris was found on the surface. Figure 5b shows the wear-worn surface at 20 N of load, 800 rpm of disc speed, 9 m/s of velocity, and a wear rate of 0.0534 mm3/min. Wear tracks and delamination of the wear layer were observed on the surface. Figure 5c shows the wear-worn surface at 15N of load, 400 rpm of disc speed, 3 m/s of velocity, and a wear rate of 0.01355 mm3/min. The pin profile on the surface produced the wear track lines. Figure 5d established the wear-worn surface at 20 N load, 400 rpm disc speed, 6 m/s of velocity, and a wear rate of 0.0486 mm3/min. The penetration of the wear track lines and localized positions of silicon particles were observed on the surface. Figure 5e shows the wear-worn surface at 25 N of load, 1200 rpm of disc speed, 6 m/s of velocity, and a wear rate of 0.0893 mm3/min. The impact of the pin produced wear penetration, and resistance against load was observed. Figure 5e revealed the wear-worn surface at 25 N of load, 1200 rpm of disc speed, 6 m/s of velocity, and a wear rate of 0.0893 mm3/min. The impact of the pin produced wear penetration, and resistance against load was observed. 3.2. AFM analysis AFM analysis is the best approach to visualize the surface characteristics of the worn-out specimen. The atomic force microscopic image of the specimen was significantly employed to identify the surface characteristics, and it 5 Wear Behavior of Brass Based Composite Reinforced with SiC and Produced by Stir Casting Process Figure 5. SEM images of different worn surfaces (a) 25N of Load, 400 rpm of disc speed, 9m/s of velocity and wear rate of 0.0587 mm3/min(b) 20N of Load, 800 rpm of disc speed, 9 m/s of velocity and wear rate of 0.0534 mm3/min (c)15N of Load, 400 rpm of disc speed, 3 m/s of velocity and wear rate of 0.01355 mm3/min (d) 20N of Load, 400 rpm of disc speed, 6 m/s of velocity and wear rate of 0.0486 mm3/min (e) 25N of Load, 1200 rpm of disc speed, 6 m/s of velocity and wear rate of 0.0893 mm3/min. Figure 5. SEM images of different worn surfaces (a) 25N of Load, 400 rpm of disc speed, 9m/s of velocity and wear rate of 0.0587 mm3/min(b) 20N of Load, 800 rpm of disc speed, 9 m/s of velocity and wear rate of 0.0534 mm3/min (c)15N of Load, 400 rpm of disc speed, 3 m/s of velocity and wear rate of 0.01355 mm3/min (d) 20N of Load, 400 rpm of disc speed, 6 m/s of velocity and wear rate of 0.0486 mm3/min (e) 25N of Load, 1200 rpm of disc speed, 6 m/s of velocity and wear rate of 0.0893 mm3/min. Figure 5. SEM images of different worn surfaces (a) 25N of Load, 400 rpm of disc speed, 9m/s of velocity and wear rate of 0.0587 mm3/min(b) 20N of Load, 800 rpm of disc speed, 9 m/s of velocity and wear rate of 0.0534 mm3/min (c)15N of Load, 400 rpm of disc speed, 3 m/s of velocity and wear rate of 0.01355 mm3/min (d) 20N of Load, 400 rpm of disc speed, 6 m/s of velocity and wear rate of 0.0486 mm3/min (e) 25N of Load, 1200 rpm of disc speed, 6 m/s of velocity and wear rate of 0.0893 mm3/min. Table 3. Surface texture characteristics for the wear specimens. Table 3. Surface texture characteristics for the wear specimens. Sl.No Parameters Values in nm (Nano meters) Std. 3.2. AFM analysis AFM images for the specimen 9 (25N of Load, 1200 rpm of disc speed, 6 m/sec of velocity and wear rate of 0.0893 mm3/min). ges for the specimen 9 (25N of Load, 1200 rpm of disc speed, 6 m/sec of velocity and wear rate of 0.0893 mm3/min). igure 10. AFM images for the specimen 9 (25N of Load, 1200 rpm of disc speed, 6 m/sec of velocity and wear rate of 0. • A silicon carbide-reinforced brass-based composite was fabricated by the stir casting method. The chemical composition was validated by an EDS image. roughness of the surface to the root mean square value is around 1.31 for confirming engineering surfaces, and it follows the Gaussian normal distribution. The kurtosis value for all 5 specimens is above 3, which clearly represents how surface roughness affects wear rate26-28. The average roughness measuring method was effectively used by AFM, and it was very useful to investigate the specimen before it went to the manufacturing process. Average roughness and the maximum height of the roughness are the two important parameters to characterize the surface topography of the specimen. In this research, specimen 3 has a minimum average roughness value of 15.27 nm, and specimen 4 has a minimum Rt value of 178 nm, as found in 3D AFM. Similar results were attained in nanoparticles based aluminium composite29,30. • A silicon carbide-reinforced brass-based composite was fabricated by the stir casting method. The chemical composition was validated by an EDS image. • A silicon carbide-reinforced brass-based composite was fabricated by the stir casting method. The chemical composition was validated by an EDS image. • Synthesized brass composite has better mechanical properties, such as a hardness of 510BHN, 8.6 g/cc of density, and 475 MPa of tensile strength. • The wear characteristics were analyzed with a pin- on-disc tribometer. The wear rate was evaluated by different control factors, such as 15–25 N of applied load, 400–1200 rpm of disc rotational speed, and 3–9 m/s of velocity. • Based on the input constraints and their level, the wear rate varied from 0.0135 to 0.0893 mm3/minf • Load has produced 83.75% of its effect on wear rate. The contributions of disc speed and velocity to wear rate are 14.96% and 1.06%, respectively. • Atomic force microscopy was used to analyze the wear surface of the brass composite under different loads, disc speeds, and velocities. 3.2. AFM analysis Deviation Specimen -1 Specimen -2 Specimen -3 Specimen -4 Specimen -5 1 Roughness Average (Ra) 34.04 27.66 15.27 17.49 43.12 10.35 2 Root mean square roughness (Rq) 47.79 37.95 22.29 22.12 56.76 13.74 3 Maximum height of the roughness (Rt) 350.2 269.1 296.2 178 376.9 69.44 4 Maximum roughness Valley depth (Rv) 224.7 124.1 129.7 89.39 188.3 48.57 5 Maximum roughness Peak height (Rp) 125.5 145 166.5 88.61 188.5 34.32 6 Skewness (Rsk) -0.8543 0.3263 0.9655 0.02416 0.0761 0.586 7 Kurtosis (Rku) 6.422 4.46 13.39 3.389 3.589 3.727 Mohan et al. Materials Researc 6 Mohan et al. Materials Research Mohan et al. Figure 6. AFM images for the specimen1 (25N of Load, 400 rpm of disc speed, 9 m/sec of velocity and wear rate of 0.0587 mm3/min). Figure 6. AFM images for the specimen1 (25N of Load, 400 rpm of disc speed, 9 m/sec of velocity and wear rate of 0.0587 mm3/min). Figure 7. AFM images for the specimen 2 (20N of Load, 800 rpm of disc speed, 9 m/sec of velocity and wear rate of 0.0534 mm3/min). Figure 7. AFM images for the specimen 2 (20N of Load, 800 rpm of disc speed, 9 m/sec of velocity and wear rate of 0 ges for the specimen 2 (20N of Load, 800 rpm of disc speed, 9 m/sec of velocity and wear rate of 0.0534 mm3/min). Figure 8. AFM images for the specimen 3 (15N of Load, 400 rpm of disc speed, 3 m/sec of velocity and wear rate of 0.01355 mm3/min). Figure 8. AFM images for the specimen 3 (15N of Load, 400 rpm of disc speed, 3 m/sec of velocity and wear rate of 0.01355 mm3/min). Wear Behavior of Brass Based Composite Reinforced with SiC and Produced by Stir Casting Process Figure 9. AFM images for the specimen 4 (20N of Load, 400 rpm of disc speed, 6 m/sec of velocity and wear rate of 0.0486 mm3/min). Figure 9. AFM images for the specimen 4 (20N of Load, 400 rpm of disc speed, 6 m/sec of velocity and wear rate of 0.0486 mm3/min). Figure 9. AFM images for the specimen 4 (20N of Load, 400 rpm of disc speed, 6 m/sec of velocity and wear rate o Figure 10. AFM images for the specimen 9 (25N of Load, 1200 rpm of disc speed, 6 m/sec of velocity and wear rate of 0.0893 mm3/min). Figure 10. 5. References 1. Alkarkhi N, Naif M. Study on the parameter optimization in magnetic abrasive polishing for brass plate using Taguchi method. Iraqi J Mech Mater Eng. 2012;12:596-615. 2. Alotaibi JG, Yousif BF, Yusaf TF. Wear behaviour and mechanism of different metals sliding against stainless steel counterface. Proc Inst Mech Eng Part J J Engineer Tribology. 2014;228(6):692-704. 17. Tyagi R, Xiong D, Li J. Effect of load and sliding speed on friction and wear behavior of silver/h-BN containing Ni-base P/M composites. Wear. 2011;270(7-8):423-30. 18. Oliveira MD Jr, Costa HL, Silva W Jr, Mello JDB. 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https://openalex.org/W1538830058	https://pure.aber.ac.uk/portal/files/5947611/Duller_et_al_2015_JHE_85_111_125.pdf	English	null	New investigations at Kalambo Falls, Zambia: Luminescence chronology, site formation, and archaeological significance	Journal of Human Evolution/Journal of human evolution	2,015	cc-by	16,199	Aberystwyth University New investigations at Kalambo Falls, Zambia Duller, G. A. T.; Tooth, Stephen; Barham, Lawrence; Tsukamoto, Sumiko Citation for published version (APA): Duller, G. A. T., Tooth, S., Barham, L., & Tsukamoto, S. (2015). New investigations at Kalambo Falls, Zambia: Luminescence chronology, site formation, and archaeological significance. Journal of Human Evolution, 85, 111- 125. https://doi.org/10.1016/j.jhevol.2015.05.003 Citation for published version (APA): Duller, G. A. T., Tooth, S., Barham, L., & Tsukamoto, S. (2015). New investigations at Kalambo Falls, Zambia: Luminescence chronology, site formation, and archaeological significance. Journal of Human Evolution, 85, 111- 125. https://doi.org/10.1016/j.jhevol.2015.05.003 Citation for published version (APA): Duller, G. A. T., Tooth, S., Barham, L., & Tsukamoto, S. (2015). New investigations at Kalambo Falls, Zambia: Luminescence chronology, site formation, and archaeological significance. Journal of Human Evolution, 85, 111- 125. https://doi.org/10.1016/j.jhevol.2015.05.003 General rights General rights Copyright and moral rights for the publications made accessible in the Aberystwyth Research Portal (the Institutional Repository) are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of private study or research. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of p ch esearch. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal New investigations at Kalambo Falls, Zambia: Luminescence chronology, site formation, and archaeological signiﬁcance a Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth, SY23 3DB, UK b Department of Archaeology, Classics and Egyptology, University of Liverpool, L69 3GS, UK c a Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth, SY23 3DB, UK b Department of Archaeology, Classics and Egyptology, University of Liverpool, L69 3GS, UK c Leibniz Institute for Applied Geophysics, Geochronology and Isotope Hydrology, Stilleweg 2, Hannover D-30655, Germany a r t i c l e i n f o Article history: Received 29 August 2014 Accepted 1 May 2015 Available online 12 June 2015 Article history: Received 29 August 2014 Accepted 1 May 2015 Available online 12 June 2015 Keywords: Fluvial deposits Geochronology Meander South-central Africa Stone Age Fluvial deposits can provide excellent archives of early hominin activity but may be complex to interpret, especially without extensive geochronology. The Stone Age site of Kalambo Falls, northern Zambia, has yielded a rich artefact record from dominantly ﬂuvial deposits, but its signiﬁcance has been restricted by uncertainties over site formation processes and a limited chronology. Our new investigations in the centre of the Kalambo Basin have used luminescence to provide a chronology and have provided key insights into the geomorphological and sedimentological processes involved in site formation. Excava- tions reveal a complex assemblage of channel and ﬂoodplain deposits. Single grain quartz optically stimulated luminescence (OSL) measurements provide the most accurate age estimates for the youngest sediments, but in older deposits the OSL signal from some grains is saturated. A different luminescence signal from quartz, thermally transferred OSL (TT-OSL), can date these older deposits. OSL and TT-OSL results are combined to provide a chronology for the site. Ages indicate four phases of punctuated deposition by the dominantly laterally migrating and vertically aggrading Kalambo River (~500e300 ka, ~300e50 ka, ~50e30 ka, ~1.5e0.49 ka), followed by deep incision and renewed lateral migration at a lower topographic level. A conceptual model for site formation provides the basis for improved inter- pretation of the generation, preservation, and visibility of the Kalambo archaeological record. This model highlights the important role of intrinsic meander dynamics in site formation and does not necessarily require complex interpretations that invoke periodic blocking of the Kalambo River, as has previously been suggested. The oldest luminescence ages place the Mode 2/3 transition between ~500 and 300 ka, consistent with other African and Asian sites where a similar transition can be found. The study approach adopted here can potentially be applied to other ﬂuvial Stone Age sites throughout Africa and beyond. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Article history: Received 29 August 2014 Accepted 1 May 2015 Available online 12 June 2015 Keywords: Fluvial deposits Geochronology Meander South-central Africa Stone Age g Accepted 1 May 2015 Available online 12 June 2015 Keywords: Fluvial deposits Geochronology Meander South-central Africa Stone Age * Corresponding author. E-mail address: ggd@aber.ac.uk (G.A.T. Duller). Contents lists available at ScienceDirect Contents lists available at ScienceDirect Take down policy Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wor vestigate your claim. tel: +44 1970 62 2400 email: is@aber.ac.uk tel: +44 1970 62 2400 email: is@aber.ac.uk Download date: 24. Oct. 2024 Journal of Human Evolution 85 (2015) 111e125 nature, timing, and controls of river and ﬂoodplain activity. While it is recognised that ﬂuvial deposits can provide exc archives of early hominin activity (Mishra et al., 2007), * Corresponding author. E-mail address: ggd@aber.ac.uk (G.A.T. Duller). http://dx.doi.org/10.1016/j.jhevol.2015.05.003 0047-2484/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2015.05.003 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction play an important role in discussions about the timing and importance of the characteristic technological changes of this transition (Barham et al., 2009). The signiﬁcance of the Kalambo Falls record, however, has been diminished by uncertainties about the geomorphological and sedimentological processes involved in site generation and preservation (Sheppard and Kleindienst, 1996; Schick, 2001) and by a very limited chronology (Clark, 2001), thereby contributing to the marginalisation of the site in such discussions. All four of Clark's (1969, 2001) excavations (designated sites A, B, C, and D) are immediately adjacent to the Kalambo River, and while ﬂuvial processes are known to have played a key role in site formation (e.g., Schick, 2001), many uncertainties surround the nature, timing, and controls of river and ﬂoodplain activity. Kalambo Falls, located in northern Zambia on the Tanzanian border (Fig. 1a, b), was the site of detailed archaeological excava- tions between 1956 and 1966. Deposits in a basin upstream of the falls produced one of the most important artefact archives span- ning the late Acheulean (Mode 2) to early Middle Stone Age (Mode 3) transition in south-central Africa (Clark, 1969, 2001). Owing to the rich record of artefacts recovered from the site and the limited number of other stratiﬁed sequences covering the Mode 2 to Mode 3 transition in this part of Africa, Kalambo Falls could potentially While it is recognised that ﬂuvial deposits can provide excellent archives of early hominin activity (Mishra et al., 2007), most 112 G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 Figure 1. a) Digital Elevation Model of the southern part of the African continent (from NASA) showing the location of Kalambo Falls at the southern end of one arm of the East African Rift Systems (now partly occupied by Lake Tanganyika). b) Oblique aerial view of the Kalambo River meandering through the small Kalambo Basin (ﬂow direction from right to left) prior to its ~200 m long passage through a quartzite ridge (spillway gorge) and over Kalambo Falls. Image taken from Google Earth. c) Panoramic view of Site C North. The Kalambo River ﬂows from centre right to upper left and the meander bend shown in this photograph is indicated by an arrow in (b). 1. Introduction Note how the modern channel is incised up to 9 m below the surface of the basin ﬁll, with lateral migration and renewed ﬂoodplain formation now taking place at a lower topographic level. The original Site C of Clark (2001) is thought to have been located near to the foreground of this image but has been eroded by the ongoing meander bend migration. Excavations in 2006 took place at locations shown by arrows on the left of the image where recent erosion had provided near-vertical exposures. Figure 1. a) Digital Elevation Model of the southern part of the African continent (from NASA) showing the location of Kalambo Falls at the southern end of one arm of the East African Rift Systems (now partly occupied by Lake Tanganyika). b) Oblique aerial view of the Kalambo River meandering through the small Kalambo Basin (ﬂow direction from right to left) prior to its ~200 m long passage through a quartzite ridge (spillway gorge) and over Kalambo Falls. Image taken from Google Earth. c) Panoramic view of Site C North. The Kalambo River ﬂows from centre right to upper left and the meander bend shown in this photograph is indicated by an arrow in (b). Note how the modern channel is incised up to 9 m below the surface of the basin ﬁll, with lateral migration and renewed ﬂoodplain formation now taking place at a lower topographic level. The original Site C of Clark (2001) is thought to have been located near to the foreground of this image but has been eroded by the ongoing meander bend migration. Excavations in 2006 took place at locations shown by arrows on the left of the image where recent erosion had provided near-vertical exposures. acid racemization (e.g., Penkman et al., 2011) have been applied to ﬂuvial deposits, but in many cases the resolution of ages is limited. This paper combines luminescence dating of deposits in the central Kalambo Basin with new geomorphological and sedimen- tological insights to propose a conceptual model outlining the key ﬂuvial processes involved in site formation. It then discusses the implications of this model for the generation, preservation, and visibility of the archaeological record. Barham et al. 2015 provides details of the artefacts recovered during these excavations, as well as additional sedimentological and palaeoenvironmental information. 1. Introduction attention has focused on ﬂights of alluvial river terraces, as these represent former ﬂoodplains within which archaeological materials may be preserved and placed in a relative chronology (e.g., Bridgland, 2006; Gibbon et al., 2009). In the Kalambo Basin and some other important ﬂuvial archaeological sites worldwide, however, rivers have not undergone the episodic net incision necessary to generate alluvial terrace ﬂights. In these situations, basin or valley alluvial ﬁlls containing important archaeological material may be generated and preserved (e.g., Lewin and Macklin, 2003) but the sedimentary archives may be more complex to interpret than those characterising terrace ﬂights, and without deep incision and/or extensive gully formation, the archaeological record may remain largely or completely invisible (e.g., Brink et al., 2012; Tooth et al., 2013). 3.2. Laboratory procedures To determine an age using luminescence methods requires measurement of two parameters, the equivalent dose (De) and the dose rate (Dr; Duller, 2008a). The De is the radiation dose received by the sample since the event being dated and is measured by making luminescence measurements of the sample. De has the SI unit of Grays (Gy), where 1 Gy is equivalent to an energy of 1 J/kg being absorbed by the sample. When dating the deposition of sediments, the event being dated is the last exposure of the sedi- ment mineral grains to daylight, as this bleaching reduces the De to a very low level. The Dr is the rate at which the sample is exposed to ionizing radiation in the natural environment and has the units Gy per thousand years (Gy/ka). The age is calculated by dividing De by Dr (Duller, 2004, 2008a). Over the past two decades, measurement of the optically stimulated luminescence (OSL) signal from quartz has been applied widely to provide ages for deposits from a few years to in excess of 100 ka (Murray and Olley, 2002). The method has played an increasingly important role in providing an absolute chronology for Middle Stone Age archaeological sites around the world, including India (e.g., Petraglia et al., 2007), Arabia (e.g., Armitage et al., 2011), southern Africa (e.g., Jacobs et al., 2008), and northern Africa (e.g., Clark-Balzan et al., 2012). OSL has proved especially important in providing ages for sites that lie beyond the range of radiocarbon, and where tephra are not found. In short, the chronology of the Kalambo Falls site is poorly known. Clark (2001) recognised the potential contribution of luminescence dating to improve the chronology, but only with recent developments in the suite of luminescence techniques, especially as applied to ﬂuvial deposits containing quartz-rich sand, has there been an opportunity to fulﬁl this potential and thus re- assess the signiﬁcance of the Kalambo Falls archaeological record. 2. Previous chronological investigations at Kalambo Falls This wood has been the subject of two attempts to provide a numerical chronol- ogy. Lee et al. (1976) used the racemization of proline and hy- droxyproline in the wood to estimate a minimum age of 110 ka. McKinney (2001) applied uranium-series methods to wood sam- ples attributed to the Sangoan, Acheulean Upper level, and Acheulean Lower level, leading to isochron ages of 76 ± 10 ka, 182 ± 10 ka, and 182 ± 16 ka, respectively. However, uranium series methods are rarely applied to wood. The occurrence of U in fossil wood in sufﬁcient concentrations to make uranium-series methods possible implies that uranium migrated into the system after deposition, but it is difﬁcult to be conﬁdent that the system has been geochemically closed since this initial input of uranium, and hence the ages may be unreliable. by directly collecting sediment into black plastic bags under a light- tight plastic sheet. The ends of any sample tubes were packed with plastic to prevent movement of sediment inside the tube and then sealed to allow transport back to the laboratory. For each sample, with the exception of KB11, in situ measurement of the gamma dose rate was made using a portable gamma spectrometer (Ortec MicroNomad equipped with a 2 inch diameter NaI crystal). result of waterlogging of the deposits (Clark, 2001). This wood has been the subject of two attempts to provide a numerical chronol- ogy. Lee et al. (1976) used the racemization of proline and hy- droxyproline in the wood to estimate a minimum age of 110 ka. McKinney (2001) applied uranium-series methods to wood sam- ples attributed to the Sangoan, Acheulean Upper level, and Acheulean Lower level, leading to isochron ages of 76 ± 10 ka, 182 ± 10 ka, and 182 ± 16 ka, respectively. However, uranium series methods are rarely applied to wood. The occurrence of U in fossil wood in sufﬁcient concentrations to make uranium-series methods possible implies that uranium migrated into the system after deposition, but it is difﬁcult to be conﬁdent that the system has been geochemically closed since this initial input of uranium, and hence the ages may be unreliable. 3.2. Laboratory procedures 2. Previous chronological investigations at Kalambo Falls A number of different geochronological methods have previ- ously been used on material collected from Kalambo Falls, but with only limited success for the pre-Holocene. Samples of organic-rich clays and some scattered charcoal were collected for radiocarbon dating during Clark's excavations in the 1950s and 1960s. By applying enrichment techniques at the Groningen Radiocarbon Laboratory, ﬁnite ages up to 60 ka BP were obtained from Acheu- lean levels in site A (Sheppard and Kleindienst, 1996; Clark, 2001). Although enrichment of these samples provided radiocarbon measurements that were above instrumental background, the techniques are not able to overcome contamination arising during burial. Less than 0.1% of contamination by modern carbon would result in a sample that is hundreds of thousands of years old, giving an apparent radiocarbon age of 60 ka BP (Taylor, 1987), and the radiocarbon ages obtained for material from Kalambo are all thought to underestimate the true age of the site (Sheppard and Kleindienst, 1996; Clark, 2001). In addition to these challenges of interpretation and visibility at ﬂuvial archaeological sites, geochronological methods are needed to establish the timing of deposition of different sedimentary packages and to develop conceptual models of site formation (e.g., Tooth et al., 2013; Lyons et al., 2014). Radiocarbon provides a valuable method back to ~40 ka, but beyond this range it is commonly difﬁcult to provide a numerical chronology. However, over the last two decades developments in luminescence dating have provided an excellent chronological tool that is now widely applied (Rhodes, 2011). The optically stimulated luminescence (OSL) signal from quartz (Duller, 2004) has been demonstrated to yield accurate ages for alluvial river terrace and other ﬂuvial de- posits ranging in age from a few tens of years to in excess of a hundred thousand years (e.g., Rittenour, 2008; Macklin et al., 2010; Lyons et al., 2014), but for many older deposits the OSL signal may be saturated. Beyond the range of quartz OSL methods, cosmogenic burial dating (e.g., Gibbon et al., 2009; Erlanger et al., 2012), biostratigraphy (e.g., Schreve et al., 2002), electron spin resonance (Rosina et al., 2014), and relative dating methods such as amino An unusual feature of the Kalambo Falls site is the preservation in sites A and B of wood in the Sangoan and Acheulean levels as a G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 113 result of waterlogging of the deposits (Clark, 2001). 3.1. Field sampling Re-examination of the Kalambo Falls archaeological site was undertaken in 2006 (Barham et al., 2009) and involved four unit excavations at Site C North (S835031.0100; E3114033.3700), located on a 5e7 m tall bank section immediately north of the original Site C of Clark (1969). From south to north, the excavations were designated C, C3, C1, and C2 (Figs. 1c and 2). A total of 18 samples (KB1-KB17 and KB19) were collected from the four excavations for luminescence dating (Fig. 2). Sample lo- cations were selected to establish the timing of major breaks in the sedimentary succession and to bracket key archaeological horizons, especially those associated with Mode 2 and Mode 3 artefacts (Fig. 2). Care was taken to avoid sampling within 20 cm of major sedimentary boundaries in order to avoid complexities in the gamma dose rate. Wherever possible, layers containing abundant sand were targeted, and samples were collected by hammering 5 cm diameter opaque plastic tubes into freshly excavated faces or Expanding our knowledge of the Mode 2 to Mode 3 transition and the broader sequence of changes in the Kalambo Basin is dependent on being able to place recovered artefacts in an absolute chronological framework. Whilst quartz OSL has played a valuable role in constraining events within the MSA, it may be of limited value in dating older sites. The upper age limit possible using quartz OSL measurements varies from one sample to another (Duller, 2008a) but is commonly in the range of 100e150 ka. At certain sites, it is possible to use quartz OSL to calculate older ages because Figure 2. Simpliﬁed stratigraphy exposed in the excavations at Site C North in the central Kalambo Basin, indicating the major stratigraphic boundaries, the archaeological horizons, and luminescence sample locations. Detailed sedimentological logs for Units C, C1, and C2 are provided in Barham et al. (2015). The horizontal distance between units is indicated at the bottom of the diagram. Figure 2. Simpliﬁed stratigraphy exposed in the excavations at Site C North in the central Kalambo Basin, indicating the major stratigraphic boundaries, the archaeological horizons, and luminescence sample locations. Detailed sedimentological logs for Units C, C1, and C2 are provided in Barham et al. (2015). The horizontal distance between units is indicated at the bottom of the diagram. G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 114 Figure 3. 3.1. Field sampling Comparison of beta and gamma dose rates derived using different dosimetry methods for the samples dated in this study. the dose rate (Dr) is unusually low. For example at Kathu Pan, Porat et al. (2010) were able to obtain an age of 291 ± 45 ka because the dose rate was extremely low (0.47 ± 0.07 Gy/ka). However, even with the low dose rates found at this site, the lowermost sample (464 ± 47 ka) in the study was near the limit of OSL, and it was the correspondence with ESR measurements that increased the likeli- hood that this OSL age was reliable. To provide a chronology extending back to the ESA/MSA transition, new luminescence methods are required. The thermally transferred OSL (TT-OSL) signal from quartz (Duller and Wintle, 2012) is one potential signal that has been explored and is applied here in combination with quartz OSL. 3.2.1. Sample processing All sample processing and analysis was undertaken in Aberystwyth Luminescence Research Laboratory. Standard preparation procedures were followed to separate sand- sized grains of quartz for luminescence measurements. Organic material and carbonates were removed by immersing the samples in 10% H2O2 and then 10% HCl until no subsequent reaction was observed. After drying, the samples were sieved to obtain grains ranging from 180e212 mm or 180e250 mm in diameter depending upon the availability of material. Solutions of sodium polytungstate at densities of 2.62 and 2.70 g/cm3 were then used to remove the majority of feldspars and heavy minerals. Grains with a density between these two values were then subject to etching in 40% hydroﬂuoric (HF) acid for 45 min. This treatment served two purposes: ﬁrst, to dissolve any remaining feldspar grains, and second, to etch away the outer ~10 mm of the quartz grains and thus remove the alpha irradiated layer. After rinsing in concentrated HCl to ensure that no ﬂuorides were precipitated, the grains were then resieved to remove grains with a diameter less than 180 mm, with the aim of removing any remaining feldspar fragments that had survived the HF etching. 3.2.2. Dosimetry measurements Assessing the radiation dose rate to which samples were exposed during burial (Dr) is a crucial part of luminescence dating. For the samples from Kalambo Falls, three sets of measurements were made. 3.1. Field sampling At the time of collection, an Ortec MicroNomad portable gamma spectrometer was inserted into the sample holes left after collection of the luminescence samples. A gamma spectrum was collected, permitting both an assessment of the concentration of U, Th, and K and an assessment of the total gamma dose using the threshold method (Mercier and Falgueres, 2007). In the laboratory, a dried subsample of the material collected for each luminescence sample was milled to a ﬁne powder and homogenised. This powdered subsample was then measured using two emission counting methods, thick source alpha counting (TSAC) and beta counting (using a GM-25 beta counter). Figure 3. Comparison of beta and gamma dose rates derived using different dosimetry methods for the samples dated in this study. based and laboratory based measurements of dose rate for KB4. In the part of Unit C1 below where KB4 was collected, mud balls up to 10 cm in diameter were observed within the sandy matrix. The selection of sites for OSL sampling aimed to avoid such complex stratigraphy, but the tube used to sample KB4 inadvertently passed through one of these mud balls, and caused this discrepancy be- tween the dose rates measured in situ and in the laboratory (Fig. 3). The consistency of the data collected by these three methods was assessed by comparing the values calculated for the beta and gamma dose rates using different methods. The beta dose rate measured directly using the GM-25 beta counter was compared with the beta dose rate calculated using the estimates of U, Th, and K concentrations derived from the ﬁeld gamma spectrometry (Fig. 3a). All values fall close to the 1:1 line, with the exception of sample KB4 for which the beta dose rate measured in the labora- tory (2.64 ± 0.08 Gy/ka) is more than double that calculated from the ﬁeld gamma spectrometry measurements (1.21 ± 0.02 Gy/ka). A similar situation is observed when comparing gamma dose rates determined from ﬁeld measurements using the threshold method with the gamma dose rate calculated from laboratory TSAC and beta counting (Fig. 3b). All samples lie close to the 1:1 line, but the gamma dose rate for KB4 (not shown) determined in the ﬁeld is 1.26 ± 0.04 Gy/ka, while that from laboratory measurements is 3.39 ± 0.15 Gy/ka. Table 1 i content (%) Grain size (mm) Beta dose (Gy/ka) Gamma dose (Gy/ka) Cosmic dose (Gy/ka) Total dose (Gy/ka) been saturated, while lower average values are likely higher in the succession. Values varying from 20 ± 5% at the base to 10 ± 5% at the top of the units were used, with these values being based on esti- mates of the saturated water content made in the laboratory and measurements of the water content of the samples at the time of collection. The dose rate due to cosmic rays was calculated using the equations in Prescott and Hutton (1994) and assuming the current thickness of overburden. a residual remains, leading to the potential for luminescence ages to be overestimated (Rittenour, 2008). This incomplete bleaching is important for young samples, where the residual is large compared with the dose accumulated during burial. For older samples, this residual becomes less important (Jain et al., 2004). been saturated, while lower average values are likely higher in the succession. Values varying from 20 ± 5% at the base to 10 ± 5% at the top of the units were used, with these values being based on esti- mates of the saturated water content made in the laboratory and measurements of the water content of the samples at the time of collection. The dose rate due to cosmic rays was calculated using the equations in Prescott and Hutton (1994) and assuming the current thickness of overburden. Single grain OSL dating can be used to test explicitly whether a sample is affected by incomplete bleaching at deposition, and this method has been applied here. The single aliquot regenera- tive (SAR) dose method has been used to measure equivalent dose (De), with a preheat of 240 C for 10 s and a cutheat of 220 C for the test dose. Screening of the data was undertaken so that only those grains that had appropriate luminescence char- acteristics were used for dating. The acceptance criteria are those described in Duller (2012), based on those previously described by Jacobs et al. (2006). Fig. 4 shows the OSL decay curve from a single grain of quartz from sample KB15 that passes all these criteria, along with the SAR dose response curve generated for this grain. 3.2.3. Luminescence equipment All luminescence measurements were made on Risø automated luminescence readers (TL-DA-15; Bøtter-Jensen et al., 2003). Table 1 i Optical stimulation of single grains was achieved using a 532 nm Nd:YVO4 laser, focused so that it could be directed onto a single ~200 mm diameter grain of quartz. Single grain measurements were either made for 1 s duration using 90% of the laser power, or 4 s duration using the laser at 25% of full power. This latter setting was used for the majority of the measurements reported here in order to give greater detail in the measurement of the OSL decay curves. An instrumental uncertainty of 2% has been included in the analysis of all single grain data. Further details of this equipment and the advantages of single grain measurements are given in Bøtter-Jensen et al. (2003), Jacobs and Roberts (2007), and Duller (2008b). Duller (2012) undertook a series of dose recovery experiments on sample KB15 using these measurement parameters and demonstrated that excellent dose recovery can be achieved up to doses of approximately 95 Gy. Above this dose, saturation of the OSL signal leads to increasing difﬁculty in accurately recovering a laboratory administered dose. All 18 samples have had single grain measurements made upon them, although for some samples the natural OSL signal of many of the grains is saturated. A large pro- portion (24%) of the quartz grains from Kalambo Falls yield an OSL signal suitable for constructing a dose response curve, and between 800 and 2000 grains from each sample have been measured (Table 2). The single grain dose distributions observed for these samples fall into three categories: (1) those affected by incomplete bleaching; (2) those that can best be modelled assuming a single dose population; and (3) those affected by saturation of the OSL signal. These are discussed in turn below. Stimulation of the TT-OSL signals was achieved using blue light emitting diodes (470D20 nm). Luminescence emissions (both OSL and TT-OSL) were measured using EMI 9635QA photomultiplier tubes ﬁtted with 7.5 mm thickness of Hoya U-340 glass ﬁlters. The Risø readers were equipped with Sr/Y beta sources for laboratory irradiation, and these were calibrated against a secondary inter- national standard in Denmark. Table 1 i Table 1 Dosimetry data for luminescence samples collected at Kalambo Fallsa. Sample Depth below surface (m) Water content (%) Grain size (mm) Beta dose (Gy/ka) Gamma dose (Gy/ka) Cosmic dose (Gy/ka) Total dose (Gy/ka) Unit C KB19 1.75 10 ± 5 180e212 0.76 ± 0.05 0.59 ± 0.04 0.169 ± 0.009 1.516 ± 0.061 KB15 3.25 15 ± 5 180e250 0.55 ± 0.04 0.43 ± 0.03 0.141 ± 0.007 1.120 ± 0.045 KB16 4.30 15 ± 5 180e250 0.39 ± 0.03 0.39 ± 0.02 0.125 ± 0.006 0.905 ± 0.034 KB17 5.17 20 ± 5 180e250 0.30 ± 0.02 0.29 ± 0.02 0.114 ± 0.006 0.705 ± 0.026 Unit C1 KB3 3.10 10 ± 5 180e212 0.92 ± 0.06 0.78 ± 0.05 0.144 ± 0.007 1.843 ± 0.077 KB4 3.65 15 ± 5 180e212 1.95 ± 0.12 1.07 ± 0.06 0.135 ± 0.007 3.158 ± 0.136 KB5 4.25 15 ± 5 180e250 0.34 ± 0.02 0.46 ± 0.03 0.123 ± 0.006 0.918 ± 0.036 KB6 5.25 20 ± 5 180e250 0.49 ± 0.03 0.53 ± 0.03 0.113 ± 0.006 1.124 ± 0.043 Unit C2 KB14 1.45 10 ± 5 180e212 1.20 ± 0.08 1.15 ± 0.07 0.175 ± 0.009 2.518 ± 0.103 KB13 2.35 10 ± 5 180e212 1.39 ± 0.09 1.06 ± 0.07 0.157 ± 0.008 2.608 ± 0.111 KB12 2.88 15 ± 5 180e212 1.74 ± 0.11 1.52 ± 0.09 0.147 ± 0.008 3.407 ± 0.138 KB10 4.03 15 ± 5 180e212 1.16 ± 0.07 0.91 ± 0.05 0.129 ± 0.007 2.206 ± 0.089 KB9 4.73 20 ± 5 180e212 0.75 ± 0.05 0.56 ± 0.03 0.119 ± 0.006 1.430 ± 0.055 KB7 5.46 20 ± 5 180e212 0.78 ± 0.05 0.49 ± 0.03 0.110 ± 0.006 1.380 ± 0.055 Unit C3 KB1 1.25 10 ± 5 180e212 1.54 ± 0.10 1.03 ± 0.06 0.179 ± 0.009 2.756 ± 0.115 KB2 1.70 10 ± 5 180e212 0.64 ± 0.04 0.61 ± 0.04 0.170 ± 0.009 1.418 ± 0.056 KB8 3.05 15 ± 5 180e212 1.42 ± 0.09 1.16 ± 0.07 0.144 ± 0.007 2.727 ± 0.110 KB11 4.50 20 ± 5 180e250 0.41 ± 0.03 0.35 ± 0.03 0.122 ± 0.006 0.882 ± 0.041 a Beta and gamma dose rates listed have been corrected for the effect of water content and grain size. Table 1 Dosimetry data for luminescence samples collected at Kalambo Fallsa. 3.1. Field sampling There is a clear discrepancy between the ﬁeld The dose rates used for age calculation are shown in Table 1. The beta dose rate was calculated using the GM-25 beta counter data, and the gamma dose rate using in situ gamma spectrometry calculated using the threshold method. The advantage of these methods is that they directly measure the beta and gamma dose received by the sample, rather than making any assumptions about homogeneity in the dose rate over different spatial scales. For KB4, this makes the assumption that the dosimetry sample measured in the laboratory accurately represents the dosimetry of the part of the sample used to separate quartz for OSL measurements. The beta dose rate was corrected for grain size attenuation, and both the beta and gamma dose rates were corrected for water content. Water content for these samples varies depending upon their stratigraphic position. Geomorphological ﬁeld evidence sug- gests that samples near the base of the excavated units will have G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 115 Table 1 4.1. Single grain optically stimulated luminescence (OSL) measurements First, KB19, KB15, KB3, and KB9 have De values that are on average much higher than those shown in Fig. 5; use of the central age model (CAM) gives values of 58.4 ± 1.2 Gy, 44.1 ± 1.4 Gy, 61.5 ± 1.4 Gy, and 62.5 ± 1.6 Gy, respectively. Second, the dose distributions for the samples shown in Fig. 6 do not appear asymmetric as do KB1, KB2, KB8, and KB14. Finally, the overdispersion (OD) values for KB19, KB15, KB3, and KB9 are in the range 30e33%, while the younger samples have values between 74 and 110% (Table 2). The average characteristic saturation dose (D0) for KB15 measured by Duller (2012) was 47 Gy. The calculated CAM De values for these four samples are below the threshold of two times the value of D0 suggested by Wintle and Murray (2006) and supported by the dose recovery data of Duller (2012) and the measurements of Chapot et al. (2012). This observation, along with the lack of asymmetry in the dose distributions gives conﬁdence that the CAM De values given in Table 2 for these four samples are reliable esti- mates to use for age calculation. 4.1.3. Samples affected by saturation of the quartz OSL signal The remaining 10 samples (listed in the same order as they appear in the tables: KB16, KB17, KB4, KB5, KB6, KB13, KB12, KB10, KB7, and KB11) may be affected by saturation of the OSL signal. To illustrate this problem, Fig. 7 shows the dose response curve for a single grain of quartz from sample KB16. The natural signal measured from this grain is at, or above, the asymptotic value determined from laboratory regeneration measurements. Thus, it is not possible to determine a De for this grain, and it is saturated. Grains were deﬁned as saturated if the normalised natural OSL signal was above the asymptotic value for the dose response curve or if one of the error margins was above this value. The impact of saturation becomes increasingly apparent as older samples are analysed. Fig. 8 shows the percentage of grains that give a De value as a function of dose. Duller (2012) showed in laboratory measurements that as the given dose increased from 50 to 170 Gy the proportion of grains which gave a De decreased from almost 100% to approximately 50%. 4.1. Single grain optically stimulated luminescence (OSL) measurements For some of the samples analysed from Kalambo Falls, 25% of grains or less gave De values. The comparison shown in Fig. 8 is made difﬁcult because the dose received by samples in nature is unknown, and the large proportion of grains that are saturated suggests that the calculated De may be underestimated. Figure 4. a) OSL decay curve for a single grain of quartz from sample KB15. The decay curve has been ﬁtted with the sum of three exponentials, representing the fast, me- dium, and slow components of the quartz OSL signal. b) The dose response curve generated for this grain of quartz. The lowest dose point was measured three times; the ﬁrst two measurements were used to calculate the recycling ratio (0.99 ± 0.04) and the third measurement was made after exposing the grain to infrared stimulation so that an IR depletion ratio (1.02 ± 0.04) could be measured. The equivalent dose (De) is 35.9 ± 1.8 Gy. While the CAM De calculated for some samples is almost certainly affected by saturation (e.g., KB7, where only 14% of grains give a De), the impact on other samples is more difﬁcult to assess. For instance, samples KB13, KB12, and KB10 have CAM De values between 79.0 and 82.1 Gy, overdispersion values of 28e36%, and between 48 and 52% of grains give De values. These samples give CAM De values that are less than twice the value of D0, but it is unclear from this single grain data alone whether these CAM De values are reliable or whether they are minimum values. For this reason, TT-OSL measurements were undertaken to attempt to overcome saturation of the OSL signal. values obtained for these samples. In all cases, there is a wide range of De values. The distribution of De values observed in samples KB1 and KB2 at the top of the sequence is typical of what has been observed from other young ﬂuvial deposits (Rodnight et al., 2005). The majority of the grains give a De of ~1.3 Gy (KB1) and ~1.0 Gy (KB2) and then a small proportion of grains give much larger De values up to ~20 Gy, consistent with the idea that these grains were not exposed to sufﬁcient light at deposition to reset the OSL signal. Sample KB8 was collected 30 cm above the erosional base of the feature, and the De distribution for this sample (Fig. 4.1. Single grain optically stimulated luminescence (OSL) measurements 5c) suggests that a much smaller proportion of the grains were exposed to sufﬁcient daylight prior to deposition. For all three samples, the minimum age model (MAM; Galbraith et al., 1999) was applied to determine the appropriate estimate of equivalent dose (De) to use for age calculation. These De values are given in Table 2 and are shown by a grey bar on each of the radial plots in Fig. 5. Only one other sample has a similar distribution, and this is KB14 near the top of Unit C2 (Fig. 5d). The distribution is very similar to KB8 in that only a small proportion of grains appear to have had their OSL signal reset at the time of deposition. The MAM was applied to this sample as well (Table 2). values obtained for these samples. In all cases, there is a wide range of De values. The distribution of De values observed in samples KB1 and KB2 at the top of the sequence is typical of what has been observed from other young ﬂuvial deposits (Rodnight et al., 2005). The majority of the grains give a De of ~1.3 Gy (KB1) and ~1.0 Gy (KB2) and then a small proportion of grains give much larger De values up to ~20 Gy, consistent with the idea that these grains were not exposed to sufﬁcient light at deposition to reset the OSL signal. Sample KB8 was collected 30 cm above the erosional base of the feature, and the De distribution for this sample (Fig. 5c) suggests that a much smaller proportion of the grains were exposed to sufﬁcient daylight prior to deposition. For all three samples, the minimum age model (MAM; Galbraith et al., 1999) was applied to determine the appropriate estimate of equivalent dose (De) to use for age calculation. These De values are given in Table 2 and are shown by a grey bar on each of the radial plots in Fig. 5. Only one other sample has a similar distribution, and this is KB14 near the top of Unit C2 (Fig. 5d). The distribution is very similar to KB8 in that only a small proportion of grains appear to have had their OSL signal reset at the time of deposition. The MAM was applied to this sample as well (Table 2). 4.1. Single grain optically stimulated luminescence (OSL) measurements 4.1.1. Samples affected by incomplete bleaching Based on geomorphological observations (see below and Barham et al., 2015), Unit C3 was excavated in a relatively young channel cut- and-ﬁll feature, with samples KB1, KB2, and KB8 collected within the ﬁll (Fig. 2). Fig. 5 shows radial plots of the single grain De All 18 of the samples collected from Site C North were deposited by ﬂuvial processes associated with the Kalambo River. Previous work has demonstrated that in some ﬂuvial settings the OSL signal in quartz may not be completely reset to zero at deposition and that G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 116 Figure 4. a) OSL decay curve for a single grain of quartz from sample KB15. The decay curve has been ﬁtted with the sum of three exponentials, representing the fast, me- dium, and slow components of the quartz OSL signal. b) The dose response curve generated for this grain of quartz. The lowest dose point was measured three times; the ﬁrst two measurements were used to calculate the recycling ratio (0.99 ± 0.04) and the third measurement was made after exposing the grain to infrared stimulation so that an IR depletion ratio (1.02 ± 0.04) could be measured. The equivalent dose (De) is 35.9 ± 1.8 Gy. 4.1.2. Samples dominated by a single dose population Fig. 6 shows the single grain dose distributions for four samples (KB19, KB15, KB3, and KB9). These samples differ from those shown in Fig. 5 in three main respects. First, KB19, KB15, KB3, and KB9 have De values that are on average much higher than those shown in Fig. 5; use of the central age model (CAM) gives values of 58.4 ± 1.2 Gy, 44.1 ± 1.4 Gy, 61.5 ± 1.4 Gy, and 62.5 ± 1.6 Gy, respectively. Second, the dose distributions for the samples shown in Fig. 6 do not appear asymmetric as do KB1, KB2, KB8, and KB14. Finally, the overdispersion (OD) values for KB19, KB15, KB3, and KB9 are in the range 30e33%, while the younger samples have values between 74 and 110% (Table 2). 4.1.2. Samples dominated by a single dose population Fig. 6 shows the single grain dose distributions for four samples (KB19, KB15, KB3, and KB9). These samples differ from those shown in Fig. 5 in three main respects. Table 2 Si l Table 2 Single grain OSL equivalent dose data and ages for samples collected at Kalambo Fallsa. Sample Number of grains OD (%) Age model Equivalent dose (Gy) Dose rate (Gy/ka) Age (ka) Measured Pass all criteria Give De Unit C KB19 1200 407 306 (75%) 33 CAM 58.4 ± 1.2 1.516 ± 0.061 38.5 ± 1.7 KB15 800 128 110 (86%) 31 CAM 44.1 ± 1.4 1.120 ± 0.045 39.4 ± 2.0 KB16 1000 172 53 (31%) 54 CAM >123 ± 9.9 0.905 ± 0.034 >136 ± 12 KB17 1000 196 71 (36%) 52 CAM >117 ± 7.8 0.705 ± 0.026 >166 ± 13 Unit C1 KB3 1200 297 246 (83%) 32 CAM 61.5 ± 1.4 1.843 ± 0.077 33.4 ± 1.6 KB4 1000 197 39 (20%) 67 CAM >98 ± 11 3.158 ± 0.136 >31.0 ± 3.7 KB5 1000 202 60 (30%) 50 CAM >140 ± 10 0.918 ± 0.036 >152 ± 12.4 KB6 1000 275 86 (31%) 59 CAM >146 ± 10 1.124 ± 0.043 >130 ± 10.1 Unit C2 KB14 1900 597 597 (100%) 110 MAM 1.68 ± 0.05 2.518 ± 0.103 0.67 ± 0.03 KB13 2000 476 247 (52%) 28 CAM >80.8 ± 1.7 2.608 ± 0.111 31.0 ± 1.5 KB12 2000 490 234 (48%) 36 CAM >82.1 ± 2.2 3.407 ± 0.138 24.1 ± 1.2 KB10 1900 413 214 (52%) 28 CAM >79.0 ± 1.8 2.206 ± 0.089 35.8 ± 1.7 KB9 1000 205 169 (82%) 30 CAM 62.5 ± 1.6 1.430 ± 0.055 43.7 ± 2.0 KB7 1000 185 26 (14%) 61 CAM >121 ± 16 1.380 ± 0.055 >87.7 ± 12.1 Unit C3 KB1 1000 243 243 (100%) 74 MAM 1.34 ± 0.05 2.756 ± 0.115 0.49 ± 0.02 KB2 1100 283 283 (100%) 93 MAM 1.04 ± 0.02 1.418 ± 0.056 0.73 ± 0.03 KB8 1000 369 360 (98%) 76 MAM 3.86 ± 0.21 2.727 ± 0.110 1.42 ± 0.10 KB11 900 199 88 (44%) 90 CAM >98.9 ± 9.8 0.882 ± 0.041 >112 ± 12 a All ages are reported in thousands of years (ka) before the date of measurement (AD 2010). Where the MAM was used, an overdispersion value of 15% was assumed for the model. Table 2 Single grain OSL equivalent dose data and ages for samples collected at Kalambo Fallsa. Figure 5. 4.2. Thermally transferred optically stimulated luminescence (TT- OSL) measurements TT-OSL is a signal that has been explored in quartz in recent years (Duller and Wintle, 2012). It has been applied in a small number of archaeological settings to extend the age range over which luminescence dating methods can be applied (e.g., Kim et al., 2010; Arnold et al., 2014). The principal advantage of TT-OSL over the more commonly used OSL signal is the dose response charac- teristic (Fig. 9), which shows continued growth of the signal at G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 117 Table 2 Si l Dose response curve for the OSL signal from a single grain of quartz from sample KB16. The open square and the dashed horizontal line show the natural signal. The dose response curve determined from laboratory regeneration doses does not intersect with the natural signal, demonstrating that the natural OSL signal in this grain had reached saturation. Figure 8. The percentage of grains that pass all appropriate selection criteria (e.g., recycling, IR-OSL ratio, recuperation) which give an equivalent dose (De) value. At higher doses, a decreasing percentage of grains yield a De value because the OSL signal is saturated. Two datasets are shown. The ﬁrst is dose recovery data (white circles) for sample KB15 from Duller (2012) and the x-axis is the known dose administered in the laboratory. The second is for the suite of 18 samples reported in this paper and the x- axis is the estimate of equivalent dose based on OSL measurements. For this second dataset, samples shown in black are ﬁnite values, while for those in grey the De may be a minimum value because of saturation. Values of D0 and twice the value of D0 are taken from Duller (2012). Figure 7. Dose response curve for the OSL signal from a single grain of quartz from sample KB16. The open square and the dashed horizontal line show the natural signal. The dose response curve determined from laboratory regeneration doses does not intersect with the natural signal, demonstrating that the natural OSL signal in this grain had reached saturation. Figure 8. The percentage of grains that pass all appropriate selection criteria (e.g., recycling, IR-OSL ratio, recuperation) which give an equivalent dose (De) value. At higher doses, a decreasing percentage of grains yield a De value because the OSL signal is saturated. Two datasets are shown. The ﬁrst is dose recovery data (white circles) for sample KB15 from Duller (2012) and the x-axis is the known dose administered in the laboratory. The second is for the suite of 18 samples reported in this paper and the x- axis is the estimate of equivalent dose based on OSL measurements. For this second dataset, samples shown in black are ﬁnite values, while for those in grey the De may be a minimum value because of saturation. Values of D0 and twice the value of D0 are taken from Duller (2012). Figure 8. Table 2 Si l Radial plots of equivalent dose data for four samples where incomplete bleaching is signiﬁcant: a) KB1; b) KB2; c) KB8; and d) KB14. Figure 5. Radial plots of equivalent dose data for four samples where incomplete bleaching is signiﬁcant: a) KB1; b) KB2; c) KB8; and d) KB14. re 5. Radial plots of equivalent dose data for four samples where incomplete bleaching is signiﬁcant: a) KB1; b) KB2; c) KB8; and d) KB G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 118 ( ) Figure 6. Equivalent dose distributions for four samples dominated by a single population: a) KB19; b) KB15; c) KB3; and d) KB9. Figure 6. Equivalent dose distributions for four samples dominated by a single population: a) KB19; b) KB15; c) KB3; and d) KB9. much higher doses than is observed for the OSL signal. However, the TT-OSL signal is normally at least one hundred times weaker than the OSL signal (Duller and Wintle, 2012). Initial experiments on samples from Kalambo Falls attempted to measure TT-OSL from much higher doses than is observed for the OSL signal. However, the TT-OSL signal is normally at least one hundred times weaker than the OSL signal (Duller and Wintle, 2012). Initial experiments on samples from Kalambo Falls attempted to measure TT-OSL from single grains of quartz, but signal levels were too low. Thus, all measurements of the TT-OSL signal reported here were made on medium sized aliquots (consisting of ~ 500 grains of quartz) in order to obtain a sufﬁciently strong signal. Figure 8. The percentage of grains that pass all appropriate selection criteria (e.g., recycling, IR-OSL ratio, recuperation) which give an equivalent dose (De) value. At higher doses, a decreasing percentage of grains yield a De value because the OSL signal is saturated. Two datasets are shown. The ﬁrst is dose recovery data (white circles) for sample KB15 from Duller (2012) and the x-axis is the known dose administered in the laboratory. The second is for the suite of 18 samples reported in this paper and the x- axis is the estimate of equivalent dose based on OSL measurements. For this second dataset, samples shown in black are ﬁnite values, while for those in grey the De may be a minimum value because of saturation. Values of D0 and twice the value of D0 are taken from Duller (2012). Figure 7. Table 2 Si l (2008) measured 17 Gy in coastal dunes in South Africa and similar values are summarised from the literature in Duller and Wintle (2012)), but smaller than those found for ﬁne grained overbank deposits from the Yellow River in China (259 ± 108 and 282 ± 38 Gy; Hu et al., 2010). Although the values measured for KB1 and KB2 are large, the De values for the two samples are very reproducible (both have overdispersion values less than 10%) and are similar to each other (102 ± 10 Gy and 115 ± 6.4 Gy, respectively). The depositional environment for all of the OSL samples collected here is thought to be similar, and therefore it is assumed that this residual dose should be sub- tracted from the De values obtained for all samples from Site C North. Figure 9. Dose response curves for two luminescence signals from quartz grains of sample KB7. Single grain OSL measurements show the onset of saturation in the ﬁrst 150e200 Gy, while the TT-OSL signal (derived from a multiple grain aliquot) shows a linear growth in signal up to 1000 Gy. The single aliquot regenerative (SAR) dose protocol developed by Adamiec et al. (2010) was used for all TT-OSL measurements in this study. This protocol holds the aliquot at 350 C for 200 s at the end of each SAR cycle in order to remove any charge remaining in the TT-OSL defect and thus improves recycling. The effectiveness of this protocol was tested by undertaking a dose recovery experi- ment on sample KB1. Sixteen aliquots were used, with eight receiving no treatment and eight receiving a known laboratory beta dose of 182 Gy prior to any other treatment. All sixteen aliquots then had their De measured using the Adamiec et al. (2010) TT-OSL SAR protocol. After subtracting the residual dose in KB1 (discussed below), the ratio of the given to the recovered dose was 0.97 ± 0.09, demonstrating that it was possible to accurately measure the known laboratory radiation dose. For each sample, eight medium sized aliquots were measured using the SAR TT-OSL protocol. Standard rejection criteria were applied, namely a signal at least three times above the standard deviation of the background, a recycling ratio within 10% of unity, and recuperation less than 5% of the natural. Table 2 Si l The percentage of grains that pass all appropriate selection criteria (e.g., recycling, IR-OSL ratio, recuperation) which give an equivalent dose (De) value. At higher doses, a decreasing percentage of grains yield a De value because the OSL signal is saturated. Two datasets are shown. The ﬁrst is dose recovery data (white circles) for sample KB15 from Duller (2012) and the x-axis is the known dose administered in the laboratory. The second is for the suite of 18 samples reported in this paper and the x- axis is the estimate of equivalent dose based on OSL measurements. For this second dataset, samples shown in black are ﬁnite values, while for those in grey the De may be a minimum value because of saturation. Values of D0 and twice the value of D0 are taken from Duller (2012). Figure 7. Dose response curve for the OSL signal from a single grain of quartz from sample KB16. The open square and the dashed horizontal line show the natural signal. The dose response curve determined from laboratory regeneration doses does not intersect with the natural signal, demonstrating that the natural OSL signal in this grain had reached saturation. G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 119 Figure 9. Dose response curves for two luminescence signals from quartz grains of sample KB7. Single grain OSL measurements show the onset of saturation in the ﬁrst 150e200 Gy, while the TT-OSL signal (derived from a multiple grain aliquot) shows a linear growth in signal up to 1000 Gy. Falls (e.g., Fig. 5) show that not all grains have been exposed to daylight sufﬁciently to reset their OSL signal, and thus it would be expected that there would be a large residual TT-OSL signal at deposition. Eight aliquots of each of the two youngest samples (KB1 and KB2) from the channel cut-and-ﬁll feature in Unit C3 (Fig. 2) had their De measured using the TT-OSL SAR protocol. When the data from these two samples are combined, they give an average residual dose of 111 ± 5.6 Gy. This TT-OSL residual is larger than that typically measured in aeolian environments (e.g., Tsukamoto et al. Table 2 Si l The De values measured are listed in Table 3, and the De values after subtraction of the 111 ± 5.6 Gy residual are also shown. One means of checking the veracity of the TT-OSL values is by comparing the De values with those obtained using single grain quartz OSL (Fig. 10). For the group of samples shown in Fig. 6 that are dominated by a single population of grains and where satura- tion is not signiﬁcant, there is very good agreement between the OSL and TT-OSL De values, especially given the large residual dose that has to be subtracted. One of the drawbacks of the TT-OSL signal is that it is reset by daylight much more slowly than the quartz OSL signal (Jacobs et al., 2011; Duller and Wintle, 2012), and hence most previous applications of this method have been on aeolian samples. The single grain OSL data for the youngest samples from Kalambo Uncertainty about the reliability of the single grain quartz OSL data for samples KB13, KB12, and KB10 was highlighted previously. Table 3 Table 3 TT-OSL equivalent dose data and ages for samples collected at Kalambo Falls, and TT-OSL ages corrected for the impact of the lifetime of the charge, assuming a burial temperature of 17 ± 2 C. Sample Dose rate (Gy/ka) TT-OSL equivalent dose (Gy) Residual subtracted TT-OSL De (Gy) Uncorrected Age (ka) Lifetime corrected age (ka) Unit C KB19 1.516 ± 0.061 166 ± 5.2 54.3 ± 7.6 35.8 ± 5.2 36.4 ± 5.5 KB15 1.120 ± 0.045 152 ± 14 40.4 ± 15 36.1 ± 14 36.7 ± 15 KB16 0.905 ± 0.034 446 ± 18 335 ± 19 370 ± 25 455 ± 103 KB17 0.705 ± 0.026 339 ± 24 228 ± 25 323 ± 37 386 ± 94 Unit C1 KB3 1.843 ± 0.077 164 ± 8.2 52.7 ± 10 28.6 ± 5.5 29.0 ± 5.7 KB4 3.158 ± 0.136 580 ± 37 468 ± 37 148 ± 13 159 ± 17 KB5 0.918 ± 0.036 427 ± 27 316 ± 28 342 ± 33 416 ± 91 KB6 1.124 ± 0.043 473 ± 27 362 ± 28 322 ± 27 384 ± 72 Unit C2 KB14 2.518 ± 0.103 145 ± 4.3 33.8 ± 7.0 13.4 ± 2.9 13.5 ± 2.9 KB13 2.608 ± 0.111 232 ± 5.7 121 ± 8.0 46.3 ± 3.6 47.3 ± 3.8 KB12 3.407 ± 0.138 201 ± 9.5 89.7 ± 11 26.3 ± 3.4 26.6 ± 3.5 KB10 2.206 ± 0.089 175 ± 6.9 63.3 ± 8.9 28.7 ± 4.2 29.1 ± 4.3 KB9 1.430 ± 0.055 157 ± 5.2 45.4 ± 7.6 31.8 ± 5.5 32.3 ± 5.7 KB7 1.380 ± 0.055 511 ± 22 400 ± 23 290 ± 20 339 ± 49 Unit C3 KB1 2.756 ± 0.115 102 ± 10 9.3 ± 12 3.4 ± 4.2 n/a KB2 1.418 ± 0.056 115 ± 6.4 4.1 ± 8.5 2.9 ± 6.0 2.9 ± 6.0 KB8 2.727 ± 0.110 140 ± 11 28.4 ± 13 10.4 ± 4.7 10.5 ± 4.8 KB11 0.882 ± 0.041 480 ± 14 369 ± 15 418 ± 26 532 ± 133 G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 120 Figure 10. A comparison of equivalent doses calculated for all 18 samples from Kalambo Falls using the OSL signal from single grains of quartz and the TT-OSL signal from medium-sized aliquots. Table 3 The same data are shown in (a) and (b), but an expanded scale is used in (a) for clarity. the single grain OSL CAM De is around 100 Gy or higher (KB16, KB17, KB4, KB5, KB6, KB7, KB11), the TT-OSL suggests that these values are strongly affected by saturation of the OSL signal and signiﬁcantly underestimate the true burial dose. In these cases, TT-OSL provides the most reliable age estimates. 4.2.1. Correcting for the impact of the lifetime of the TT-OSL signal Adamiec et al. (2010) calculated the lifetime of charge in the trap responsible for the TT-OSL signal in quartz based upon laboratory experiments and obtained a value of 4.5 Ma at 10 C. Thiel et al. (2012) compared ages determined using TT-OSL with those determined using the post-IR IR signal from feldspars, and assuming that the post-IR IRSL ages were correct, they saw underestimation of the TT-OSL ages consistent with the kinetic parameters calculated by Adamiec et al. (2010). It therefore seems that TT-OSL ages require correction for the instability of charge in the trap responsible for the signal. Although this correction is small for samples less than 100 ka, the corrections are larger for samples from near the base of each of the excavation units. 4.2.1. Correcting for the impact of the lifetime of the TT-OSL signal Adamiec et al. (2010) calculated the lifetime of charge in the trap responsible for the TT-OSL signal in quartz based upon laboratory experiments and obtained a value of 4.5 Ma at 10 C. Thiel et al. (2012) compared ages determined using TT-OSL with those determined using the post-IR IR signal from feldspars, and assuming that the post-IR IRSL ages were correct, they saw underestimation of the TT-OSL ages consistent with the kinetic parameters calculated by Adamiec et al. (2010). It therefore seems that TT-OSL ages require correction for the instability of charge in the trap responsible for the signal. Although this correction is small for samples less than 100 ka, the corrections are larger for samples from near the base of each of the excavation units. p The lifetime of charge in the TT-OSL trap is temperature dependent. The mean annual temperature at Mbala (30 km from Kalambo Falls) based on instrumental records covering the period from 1967 to 1986 is 18.7 C (NOAA; ftp://ftp.atdd.noaa.gov/pub/ GCOS/WMO-Normals/TABLES/REG__I/ZA/67413.TXT). Table 3 Over the long time periods (>50 ka) that some of these samples have been buried, temperatures are likely to have been lower on average than present, but the magnitude of cooling in the tropics is poorly con- strained. The most relevant data for this part of Africa are the studies by Tierney et al. (2008) and Woltering et al. (2011) who presented TEX86 based temperature reconstructions for Lakes Tanganyika and Malawi over the last 60 and 74 ka, respectively. The two temperature constructions are consistent with each other and imply that although temperatures may have varied by as much as 6 C, the average temperature is likely to be only ~1e2 C cooler than the present. In order to correct the TT-OSL ages for charge instability, a mean burial temperature of 17 ± 2 C has been assumed. Using the kinetic parameters of Adamiec et al. (2010), the lifetime of charge in the TT-OSL defect at this temperature is 1.06 Ma. Ages in the ﬁnal column of Table 3 have been corrected to account for this thermal lifetime using the following equation (based on that given in Adamiec et al., 2010): Figure 10. A comparison of equivalent doses calculated for all 18 samples from Kalambo Falls using the OSL signal from single grains of quartz and the TT-OSL signal from medium-sized aliquots. The same data are shown in (a) and (b), but an expanded scale is used in (a) for clarity. Age ¼ t,ln 1 Agem t The TT-OSL De values (63.3e121 Gy) obtained for these samples are broadly similar to the single grain OSL values (79.0e82.1 Gy), but the two samples (KB4 and KB11) with the next largest single grain OSL De values (98 ± 11 and 99 ± 10 Gy, respectively) have TT-OSL De values (468 ± 37 and 369 ± 15 Gy) that imply that the quartz OSL is severely affected by saturation. In the case of KB4, the OSL signal of 80% of the grains is saturated, and this differentiates the sample from those where 50% or less of the grains are saturated. However, for KB11, 44% of the grains give De values, a proportion that is not very different from those samples where the TT-OSL and single grain OSL De values are consistent. Table 3 The TT-OSL De values (63.3e121 Gy) obtained for these samples are broadly similar to the single grain OSL values (79.0e82.1 Gy), but the two samples (KB4 and KB11) with the next largest single grain OSL De values (98 ± 11 and 99 ± 10 Gy, respectively) have TT-OSL De values (468 ± 37 and 369 ± 15 Gy) that imply that the quartz OSL is severely affected by saturation. In the case of KB4, the OSL signal of 80% of the grains is saturated, and this differentiates the sample from those where 50% or less of the grains are saturated. However, for KB11, 44% of the grains give De values, a proportion that is not very different from those samples where the TT-OSL and single grain OSL De values are consistent. where the measured age (Agem) is corrected for the impact of the lifetime (t). This approach assumes that growth of the TT-OSL signal is linear during the period being considered, and for the Kalambo samples this is an appropriate assumption (Fig. 9). The lifetime is calculated from the values of trap depth (E), escape frequency (s), and mean annual temperature (T) using the equation: It thus appears that it is difﬁcult to devise criteria by which to assess the reliability of De values obtained using the quartz OSL signal from samples as they approach saturation. For this suite of samples, the single grain OSL data appear reliable when the CAM De is less than about 70 Gy, and this is also the point at which more than 75% of grains that pass all the acceptance criteria yield De values (Table 2). There is then a group of samples where the single grain OSL CAM De is about 80 Gy and where about 50% of the grains give De values. These values are similar to the TT-OSL De values, and thus the OSL appears to be reliable. Given the challenge of measuring the TT-OSL and the need to subtract a substantial re- sidual signal, the OSL ages for samples KB13, KB12, and KB10 are considered more reliable than the TT-OSL ages. For samples where t ¼ 1 se E kT Uncertainties in the corrected ages were determined by incor- porating the error on the TT-OSL age and the uncertainty on the burial temperature (17 ± 2 C) using a Monte Carlo method. 4.3. Table 4 Table 4 Summary of the luminescence chronology for the four units excavated at Kalambo Falls. Sample Signal used Age Model Age (ka) Unit C KB19 OSL CAM 38.5 ± 1.7 KB15 OSL CAM 39.4 ± 2.0 KB16 TT-OSL CAM 455 ± 103 KB17 TT-OSL CAM 386 ± 94 Unit C1 KB3 OSL CAM 33.4 ± 1.6 KB4 TT-OSL CAM 159 ± 17 KB5 TT-OSL CAM 416 ± 91 KB6 TT-OSL CAM 384 ± 72 Unit C2 KB14 OSL MAM 0.67 ± 0.03 KB13 OSL CAM 31.0 ± 1.5 KB12 OSL CAM 24.1 ± 1.2 KB10 OSL CAM 35.8 ± 1.7 KB9 OSL CAM 43.7 ± 2.0 KB7 TT-OSL CAM 339 ± 49 Unit C3 KB1 OSL MAM 0.49 ± 0.02 KB2 OSL MAM 0.73 ± 0.03 KB8 OSL MAM 1.42 ± 0.10 KB11 TT-OSL CAM 532 ± 133 Summary of the luminescence chronology for the four units excavated at Kalambo Falls. Figure 12. Unit C3 excavation showing the location of the luminescence samples (red circles) and the ages obtained. Scale bar is shown for the front face of the excavation. package (package 1) is found in the basal parts of all four excava- tions, with ages ranging from ~500 to 300 ka (Unit C: KB16 and KB17; Unit C1: KB5 and KB6; Unit C2: KB7; Unit C3: KB11). The next youngest sedimentary package (package 2) is poorly preserved, being represented by only a single age in the ~300 to 50 ka range (Unit C1: KB4), and is discussed further below. The next youngest sedimentary package (package 3) is found in three of the units and indicates deposition from ~50 to 30 ka (Unit C: KB 19 and KB 15; Unit C1: KB3; Unit C2: KB13, KB10, and KB9). The youngest sedi- mentary package (package 4) records deposition from ~1.5 to 0.49 ka and is seen most clearly in Unit C3 (KB1, KB2, and KB8), but is also at the top of Unit C2 (KB14). been used, while TT-OSL is used for the older samples. The OSL signal is preferred for the younger samples because it bleaches more rapidly by exposure to daylight. The derived ages are in stratigraphic order, within uncertainties, except for sample KB12 in Unit C2 (Fig. 14), which is discussed later. Across the four unit excavations, there appears to be four sedi- mentary packages with distinct age ranges. The oldest sedimentary Figure 11. Table 3 Luminescence chronology of deposits at Kalambo Falls Table 4 summarises the luminescence chronology for the four unit excavations at Site C North, and the ages are shown in Figs. 11e14. For the younger samples, which were not affected by saturation, ages derived from single grain OSL measurements have G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 121 Figure 12. Unit C3 excavation showing the location of the luminescence samples (red circles) and the ages obtained. Scale bar is shown for the front face of the excavation. Table 4 These younger deposits represent sedimentary package 3. Stage 4 Sometime prior to ~1.5 ka, a small channeldpossibly a side branch (ﬂoodplain chute) channel of the main Kalambo Riv- erdincised partway through the ﬂuvial succession. Rapid reﬁlling of the incised channel with dominantly ﬁne-grained (silt, sand) sediment occurred between ~1.5 and 0.49 ka, with minor ﬁne- grained sedimentation also extending laterally beyond the chan- nel margins atop sedimentary package 3. These young channel ﬁll and overbank deposits represent sedimentary package 4. Figure 14. Unit C2 excavation showing the location of the luminescence samples (red circles) and the ages obtained. Scale bar is shown for the front face of the excavation. Stage 5 After ~0.49 ka, the main Kalambo River incised up to 9 m into the ﬂuvial succession, so that the base and lower banks of the channel were formed in the preserved sedimentary package 1 de- posits, and the upper banks were formed in the preserved parts of sedimentary packages 2, 3, and 4. made in situ and those made in the laboratory (Fig. 3), and the dose rate listed in Table 1 is much higher than for most other samples. The high dose rate observed in the laboratory may be an artefact of having subsampled a clay-rich unit. If only the in situ measure- ments are used for dosimetry, then the lifetime corrected TT-OSL age for KB4 is 249 ± 33 ka, but this still does not ﬁt within the age ranges for sedimentary packages 1 and 3, as described above. Stage 6 Following this deep incision, renewed lateral migration is currently occurring at a lower topographic level. Bank erosion, primarily on outer meander bends, is providing new exposures of sedimentary packages 1e4, while concomitant deposition is occurring primarily on inner bends, leading to formation of another package of young deposits in juxtaposition with these older, luminescence-dated, sedimentary packages. Sample KB12 (Fig. 14) gives an OSL age (24.1 ± 1.2 ka) that is not consistent with the age of KB13 (31.0 ± 1.5 ka) immediately above. Like KB4, KB12 has a very high dose rate (3.41 ± 0.14 Gy/ka) and it is possible that the discrepancy between this sample and those around it is due to local variations in the dose rate that have not been captured by our dosimetry measurements. Table 4 Unit C excavation showing the location of the luminescence samples (red circles) and the ages obtained. Sample KB19 was taken from a small excavation just to the upper left of the main excavation. Scale bar is shown for the front face of the excavation. Sedimentary package 2 is designated on the basis of the TT-OSL age of 159 ± 17 ka for sample KB4, as this appears to represent a period of deposition distinct from the underlying (package 1) and overlying (package 3) deposits. However, the dosimetry for KB4 was complex, with a large difference between the measurements Figure 13. Unit C1 excavation showing the location of the luminescence samples (red circles) and the ages obtained. Scale bar is shown for the front face of the excavation. Figure 11. Unit C excavation showing the location of the luminescence samples (red circles) and the ages obtained. Sample KB19 was taken from a small excavation just to the upper left of the main excavation. Scale bar is shown for the front face of the excavation. Figure 13. Unit C1 excavation showing the location of the luminescence samples (red circles) and the ages obtained. Scale bar is shown for the front face of the excavation. G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 122 deposits in the centre of the Kalambo Basin. These deposits represent sedimentary package 1. Figure 14. Unit C2 excavation showing the location of the luminescence samples (red circles) and the ages obtained. Scale bar is shown for the front face of the excavation. Stage 2 Between ~300 ka and 50 ka, the Kalambo River laterally migrated back and forth across the centre of the Kalambo Basin but again with a minor component of vertical aggradation. During lateral migration, channel bed erosion partially truncated the top part of sedimentary package 1 (generally the ﬁner-grained de- posits), but younger, relatively coarse-grained channel and ﬁner- grained overbank deposits were deposited. These younger de- posits represent sedimentary package 2. Stage 3 Between ~50 ka and 30 ka, the Kalambo River again laterally migrated back and forth while undergoing minor aggra- dation. During this stage of lateral migration, channel bed erosion partially truncated or completely removed the top part of sedi- mentary package 2, so that preservation of this package is spatially patchy (i.e., thin or absent locally) but still younger, relatively coarse-grained channel and ﬁner-grained overbank deposits were deposited. Table 4 y p g This model provides new insights into the nature, timing, and controls of the river and ﬂoodplain processes involved in site for- mation. Previous studies at Kalambo Falls had identiﬁed the punctuated pattern of sedimentation in the basin and had sug- gested that this could be the result of periodic blocking and unblocking of the spillway gorge through which the Kalambo River ﬂows to reach the falls (e.g., Bond, 1969; Haldemann, 1969; Clark, 2001: 3e4). Our detailed ﬁeld surveys in August 2006, however, failed to ﬁnd any unequivocal ﬁeld evidence to support this hy- pothesis. On the southern side of the spillway gorge there are rounded and sub-rounded quartzite boulders at high elevations (>5 m) above the current channel. Percussion marks on these boulders may be consistent with ﬂuvial transportation, but these likely relate to much older phases of river activity rather than being evidence for proposed blockage events during the mid or late Pleistocene (Barham et al., 2015). No evidence for truncated debris ﬂows or rockfalls that could have blocked the gorge for extended periods of time were observed. Our model (Fig. 15a) illustrates how the sedimentary packages observed at Site C North could have resulted from ongoing meander dynamics, without necessarily requiring complex interpretations that invoke periodic blocking of the Kalambo River. At present, there is insufﬁcient evidence to clarify the inﬂuence of climatic change in each stage of river activity (Barham et al., 2015) but because meander dynamics can occur as an intrinsic part of ﬂuvial activity, climatic forcing is not necessary to explain the observed ﬂuvial succession. This model provides new insights into the nature, timing, and controls of the river and ﬂoodplain processes involved in site for- mation. Previous studies at Kalambo Falls had identiﬁed the punctuated pattern of sedimentation in the basin and had sug- gested that this could be the result of periodic blocking and unblocking of the spillway gorge through which the Kalambo River ﬂows to reach the falls (e.g., Bond, 1969; Haldemann, 1969; Clark, 5.1. Implications of the luminescence chronology for reconstructing ﬂuvial activity at Site C North Using geomorphological ﬁeld observations, sedimentological descriptions, and the luminescence ages (Table 4), a model for interpretation of the broad sequence of river and ﬂoodplain activity represented in the ﬂuvial succession at Site C North can be pro- posed (Fig. 15a). As our work focused on the succession in the central part of the Kalambo Basin only, this model should be viewed as a hypothesis that can be tested and perhaps reﬁned using data from other parts of the basin, including at the basin margins where the ﬂuvial succession interﬁngers with colluvial material (Clark, 2001). Six main stages of river and ﬂoodplain activity can be identiﬁed, as outlined below. Stage 1 Between ~500 ka and ~300 ka, the Kalambo River was characterised by lateral migration and minor vertical aggradation, leading to the deposition of relatively coarse-grained (sand, gravel) channel deposits and ﬁner-grained (clay, silt, sand) overbank 15. a) Schematic diagram illustrating a conceptual model for the development of the ﬂuvial succession found at Site C North in the central Kalambo Basin. Six sta y have led to the deposition and exposure of four main sedimentary packages, with packages 1e3 being especially rich in archaeological terms. b) Schematic diagra plications of this model for the generation and preservation of the archaeological record (adapted from Lewin and Macklin (2003) and Barham et al. 2015). Th terised by scattered artefacts and lags that nonetheless retain chronological coherence in vertical succession. G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 123 G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 Figure 15. a) Schematic diagram illustrating a conceptual model for the development of the ﬂuvial succession found at Site C North in the central Kalambo Basin. Six stages of river activity have led to the deposition and exposure of four main sedimentary packages, with packages 1e3 being especially rich in archaeological terms. b) Schematic diagram showing the implications of this model for the generation and preservation of the archaeological record (adapted from Lewin and Macklin (2003) and Barham et al. 2015). The record is characterised by scattered artefacts and lags that nonetheless retain chronological coherence in vertical succession. Figure 15. a) Schematic diagram illustrating a conceptual model for the development of the ﬂuvial succession found at Site C North in the central Kalambo Basin. 5.1. Implications of the luminescence chronology for reconstructing ﬂuvial activity at Site C North Six stages of river activity have led to the deposition and exposure of four main sedimentary packages, with packages 1e3 being especially rich in archaeological terms. b) Schematic diagram showing the implications of this model for the generation and preservation of the archaeological record (adapted from Lewin and Macklin (2003) and Barham et al. 2015). The record is characterised by scattered artefacts and lags that nonetheless retain chronological coherence in vertical succession. 5.3. Signiﬁcance of the Kalambo Falls archaeological record 5.3. Signiﬁcance of the Kalambo Falls archaeological record The details of the archaeological record at Site C North are described elsewhere (Barham et al., 2015), with the key ﬁnding being a Mode 2/3 transition preserved in sedimentary package 1 that has been dated to ~500e300 ka. The mode concept, as modi- ﬁed by Shea (2011), is used in this study to highlight basic tech- nological differences between archaeological horizons. This approach recognises that the sample sizes from Site C North are too small to make reliable attributions to the Stone Age industries deﬁned for Kalambo Falls (Clark, 1969, 2001). At Site C North, Mode 2 (Acheulean) is recognised by the occurrence of large cutting tools (>10 cm) that include bifacially shaped handaxes and cleavers. Mode 3 (Middle Stone Age) is recognised by the occurrence of prepared cores and less direct indicators including platform prep- aration (Barham et al., 2015). Mode 2 artefacts occur only in sedi- mentary package 1, whereas Mode 3 artefacts occur in sedimentary packages 1, 2, and 3, with a re-deposited mixture of Stone Age and Iron Age artefacts found in the late Holocene channel deposits that deﬁne the major part of package 4. These ﬁndings provide a springboard from which to initiate further archaeological investigations in the area and will help Kalambo Falls regain its place among the pre-eminent Stone Age sites in Africa. The combination of geomorphological, sedimento- logical, and geochronological approaches employed in this study also demonstrates the potential for investigations of other ﬂuvial archaeological sites, both in Africa and farther aﬁeld. In particular, where suitable quartz-rich deposits are present, the combined use of OSL and TT-OSL signals offers the potential for establishing chronological control perhaps extending back over half a million years. j p p g Until now, reliable radiometric ages have been lacking for the late Acheulean and the earliest Middle Stone Age at Kalambo Falls. The TT-OSL results from Site C North place the transition well within the Middle Pleistocene, and the site can now be added to the small number of other dated Mode 2/3 transitions in southern and eastern Africa (Beaumont and Vogel, 2006; Tryon et al., 2006; Porat et al., 2010) and in southwest Asia (Gopher et al., 2010). 5.2. Implications of ﬂuvial activity for the archaeological record have been preserved as lags (e.g., at the channel base or on point bars), the archaeological record thus still retains a stratigraphic coherence, with older artefacts being found in lower layers and younger artefacts in upper layers (Fig. 15b). In such situations, the age of the sedimentary package in which artefacts are found pro- vides a minimum age for those artefacts, and the age of the un- derlying erosively truncated sedimentary package provides a maximum age for those artefacts. The proposed model for ﬂuvial activity (Fig. 15a) has implica- tions for interpretations of the generation, preservation, and visi- bility of the associated archaeological record in the centre of the Kalambo Basin. For example, repeated lateral migration and partial or complete truncation of older deposits means that potential for reworking of artefacts has been high, with few if any preserved in primary contexts, as was recognised in earlier work at the site (e.g., Schick, 2001). Nonetheless, because most sedimentary packages have been subject to only partial truncation during later river ac- tivity and many artefacts may not be transported far and instead Recent incision (Stage 5; Fig. 15a) and subsequent lateral migration (Stage 6) has been vital for the visibility of the archaeo- logical record, for without these processes many artefacts would remain deeply buried and largely undetectable. Meander migration G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 124 chronologies, especially for older sites beyond the range of radio- carbon. Prior to this study, an appreciation of the signiﬁcance of the Kalambo Falls archaeological site had been restricted, particularly by the lack of a reliable chronology, thereby contributing to the site's marginalisation in discussions about the timing and impor- tance of the technological transition from the Acheulean to the Middle Stone Age (e.g., McBrearty, 2001). Using a combination of OSL and TT-OSL, we have provided a chronology for the ﬂuvial succession in the central part of the Kalambo Basin. This chronology reveals a punctuated depositional record, characterised by four temporally distinct sedimentary packages (age ranges of ~500e300 ka, ~300e50 ka, ~50e30 ka, and ~1.5e0.49 ka). Combining this chronology with insights derived from geomorphology and sedi- mentology has enabled development of a conceptual model that can be used to interpret the generation, preservation, and visibility of the archaeological record at Site C North. References Nascent regional differences such as this are only now becoming evident as a result of the development of radiometric dating methods. TT-OSL is of particular importance as it will enable ar- chaeologists to generate chronological frameworks in areas where the options for the use of other dating techniques are limited by geological factors and by poor organic preservation. These limita- tions apply in particular to many of the Stone Age sites in central and west Africa (Barham and Mitchell, 2008). 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Cambridge University Press, Cambridge, pp. 463e480. Haldemann, E.G., 1969. Chapter 2, Part I: Geological and physiographical setting of the Kalambo Falls prehistoric site. In: Clark, J.D. (Ed.), Kalambo Falls Prehistoric Site, Volume I. Cambridge University Press, Cambridge, pp. 20e46. Schreve, D.C., Bridgland, D.R., Allen, P., Blackford, J.J., Gleed-Owen, C.P., Grifﬁths, H.I., Keen, D.H., White, M.J., 2002. Sedimentology, palaeontology and archaeology of late Middle Pleistocene River Thames terrace deposits at Purﬂeet, Essex, UK. Quatern. Sci. Rev. 21, 1423e1464. Hu, G., Zhang, J.-F., Qiu, W.-L., Zhou, L.P., 2010. Residual OSL signals in modern ﬂuvial sediments from the Yellow River (HuangHe) and the implications for dating young sediments. Quatern. Geochronol 5, 187e193. Shea, J.J., 2011. Homo sapiens is as Homo sapiens was: behavioural variability versus “behavioral modernity” in Paleolithic archaeology. Curr. Anthropol 52, 1e35. Jacobs, Z., Duller, G.A.T., Wintle, A.G., 2006. Interpretation of single grain De dis- tributions and calculation of De. Radiation Measurements 41, 264e277. Jacobs Z Roberts R G 2007 Advances in optically stimulated luminescence (OSL) Sheppard, P.J., Kleindienst, M.R., 1996. Technological change in the Earlier and Middle Stone Age of Kalambo Falls (Zambia). Afr. Archaeol. Rev. 6. Conclusions Although ﬂuvial deposits such as terrace ﬂights and basin and valley ﬁlls can provide key records of early hominin activity, in- terpretations are commonly hampered by uncertainties in ﬂuvial process interpretations and by the difﬁculties in obtaining Beaumont, P.B., Vogel, J.C., 2006. On a timescale for the past million years of human history in central South Africa. S. Afr. J. Sci. 102, 217e228. Bond, G., 1969. Appendix A. The Geology of the Kalambo Falls Prehistoric Site. In: Clark, J.D. (Ed.), Kalambo Falls Prehistoric Site, Volume I. Cambridge University Press, Cambridge, pp. 197e214. G.A.T. Duller et al. / Journal of Human Evolution 85 (2015) 111e125 Characteristics of thermally transferred optically stimulated luminescence (TT-OSL) in quartz and its po- tential for dating sediments. Radiation Measurements 43, 1204e1218. Lee, C., Bada, J.L., Peterson, E., 1976. Amino-acids in modern and fossil woods. Na- ture 259, 183e186. Lewin, J., Macklin, M.G., 2003. Preservation potential for Late Quaternary river al- luvium. J. Quatern. Sci. 18, 107e120. Wilkins, J., Schoville, B.J., Brown, K.S., Chazan, M., 2012. Evidence for early hafted hunting technology. Science 388, 942e946. Lyons, R.G., Tooth, S., Duller, G.A.T., 2014. Late Quaternary climatic changes revealed by luminescence dating, mineral magnetism and diffuse reﬂectance spectros- copy of river terrace palaeosols: a new form of geoproxy data for the southern African interior. Quatern. Sci. Rev. 95, 43e59. Wintle, A.G., Murray, A.S., 2006. A review of quartz optically stimulated lumines- cence characteristics and their relevance in single-aliquot regeneration dating protocols. Radiation Measurements 41, 369e391. Woltering, M., Johnson, T.C., Werne, J.P., Schouten, S., Damste, J.S.S., 2011. Late Pleistocene temperature history of Southeast Africa: A TEX86 temperature re- cord from Lake Malawi. Palaeogeogr. Palaeoclimatol. Palaeoecol 303, 93e102. Macklin, M.G., Tooth, S., Brewer, P.A., Noble, P.L., Duller, G.A.T., 2010. Holocene ﬂooding and river development in a Mediterranean steepland catchment: the
https://openalex.org/W4390591206	https://www.intechopen.com/citation-pdf-url/88850	English	null	Current Perspective on the Treatment of <i>Helicobacter pylori</i> Infection	IntechOpen eBooks	2,023	cc-by	6,496	Chapter Current Perspective on the Treatment of Helicobacter pylori Infection Malika Taufiq and Maliha Taufiq Abstract Helicobacter pylori (H. pylori) is a gram-negative aerobic bacteria that infects humans and is responsible for causing more than 80% of cases of chronic gastritis, peptic ulcer disease, and non-cardia gastric cancer. Both peptic ulcer disease and gastric cancer lead to 1 million deaths annually. H. pylori is a pathogen responsible for these conditions, highlighting the importance of accurate diagnosis and eradication. Various invasive and non-invasive methods are available to diagnose with varying sensitivity and specificity. Invasive methods include endoscopy, culture, histology, rapid urease test, and molecular tests such as PCR. Non-invasive tests include serol- ogy, urea breath test, and stool antigen tests. However, there is growing concern about the increasing resistance of H. pylori to commonly available treatments. The lack of availability of invasive and non-invasive procedures to detect H. pylori world- wide, particularly in developing countries, are important concerns that need to be addressed in the future. Hence in the future, more emphasis should be placed on the development of an effective vaccine against H. pylori. Keywords: Helicobacter pylori, gastric cancer, peptic ulcer disease, triple therapy, resistance, surveillance 2. Prevalence and risk factors H. pylori is a common bacterial infection that has infected 4.4 billion people world- wide, almost half of the world’s population, with varying prevalences of 24–85% in different countries [1]. According to a systematic review, there is wide variation in the prevalence between regions and countries, with the highest prevalence in Africa (79%), Latin America and the Caribbean (65%), and Asia (59%). It is lowest in North America (37%) and Oceania (24.6%) [5]. It is almost ubiquitous in Southeast Asia. The prevalence varies among different countries within the same region, and it varies among different ethnic/racial groups [5]. H. pylori infection is chronic, and it is com- monly acquired during childhood. The exact means of acquisition is unknown. It is spread from person to person through oral-oral and oral-fecal transmission. Common risk factors include age, lower socioeconomic status, certain race/ethnic groups, poor hygienic conditions such as poor sanitation, lack of access to clean water and water contamination, people with a family history of infection, certain occupations such as sheep herding, healthcare, etc. [1]. 1. Introduction Helicobacter pylori (H. pylori) is a gram-negative aerobic bacteria, which was first discovered by Warren and Marshall in 1983. They proved that it can colonize and infect the highly acidic environment of the stomach. Later, they proved that H. pylori causes peptic ulcer disease (PUD) [1]. H. pylori is a causative agent of acute and chronic gastritis and a major predisposing factor for peptic ulcer disease, gastric cancer, and gastric lymphoma [1]. In 2018, H. pylori was responsible for 810,000 new cases of non-cardia gastric cancer worldwide [2], and about 8 in 10 cases of gastric cancer are due to H. pylori infection [2]. It infects almost half of the world’s popula- tion and is almost ubiquitous everywhere. More than 80% of PUD is caused by H. pylori, and the estimated lifetime risk of PUD in H. pylori-infected patients is approx- imately 15% [3]. Gastric cancer is the third leading cause of cancer-related death worldwide, and H. pylori is responsible for 74.7% of all non-cardia gastric cancer [3]. Gastric cancer and PUD together cause 1 million deaths annually [3]. Helicobacter 1 Tertiary Care – Medical, Psychosocial, and Environmental Aspects pylori infection is a widespread condition that often remains asymptomatic in 90% of cases. However, this serves to be a major disadvantage as this infection can progress and result in chronic gastritis. H. pylori has been identified as one of the main causes of chronic gastritis. The prolonged inflammation caused by H. pylori can lead to significant changes in the gastric mucosa such as loss of gland and normal epithelium being replaced by collagen tissue. All this can lead to atrophic gastritis. Moreover, chronic gastritis can lead to intestinal metaplasia, a condition where the gastric epithelial cell undergoes transformation to cells that partially resemble those typically found in the small or large intestine. Both atrophic gastritis and intestinal metaplasia commonly called as chronic atrophic gastritis (CAG) are precancerous conditions, which have the potential to evolve into gastric cancer (GC) [4]. 4. Clinical features The clinical presentation of H. pylori infection is highly variable. As many as 90% of individuals infected are asymptomatic, while the other 10% can present with dys- pepsia, which is defined as epigastric discomfort/pain lasting longer than one month, that may be associated with other symptoms such as nausea, early satiety, epigastric fullness, and bloating. This is often diagnosed as functional dyspepsia [6]. Symptoms correlate poorly with the severity of gastric mucosal injury seen on endoscopy, and diagnosis is often missed and not treated in time, leading to chronic gastritis or peptic ulcer disease. Due to the delay in diagnosis, peptic ulcers can present with complica- tions such as gastrointestinal bleeding and perforation [6]. Because H. pylori infection remains untreated, with time chronic inflammation can lead to atrophic gastritis, which is precancerous and can progress to gastric cancer [4]. At this stage, the patient often presents with alarm symptoms such as weight loss, anemia, dysphagia, recur- rent vomiting, and an epigastric/abdominal mass. Sometimes chronic gastritis can lead to other cancers, such as gastric MALT lymphoma, which presents with dyspep- sia and non-specific constitutional symptoms [6]. Rarely H. pylori may present with extragastrointestinal manifestations such as isolated iron deficiency anemia (IDA) and idiopathic thrombocytopenic purpura (ITP) [6]. 3. Pathogenesis H. pylori colonizes the stomach’s acidic environment by means of 6–8 sheathed flagella, which enable it to inhabit the gastric mucus layer. Various chemotactic fac- tors and metals are responsible for the mobility of H. pylori [1]. It contains intrinsic enzymes that play an important role in colonization. First, an enzyme called urease splits urea into carbon dioxide (CO2) and ammonia, which attenuates the acidity of the stomach environment. Second, the hydrogenase enzyme enables bacteria to use hydrogen as a source of energy. Various adhesion molecules and surface receptors help attach bacteria to gastric epithelial cells [1]. Various virulence factors, such as cytotoxin-associated gene A (Cag A) and non-Cag A factors like vacuolating cytotoxin (Vac A), duodenal ulcer promoting gene A (Dup A), outer inflammatory protein (Oip A), and gamma-glutamyl transpeptidase (GGT), induce changes in the epithelial cells, such as apoptosis, and act as proinflammatory agents [1]. Bacteria with certain virulence factors are more pathogenic than others and cause peptic ulcer disease and gastric cancer. In response to the colonization and attachment of bacte- ria, host cells initiate an inflammatory response mainly mediated by TH1 cells [1]. Various cytokines and interleukins are released, which are either pro-inflammatory or anti-inflammatory, leading to chronic inflammation [1]. H. pylori infection remains 2 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 asymptomatic except in some cases where it leads to peptic ulcer, chronic gastritis, atrophic gastritis, and eventually gastric cancer [1, 6]. asymptomatic except in some cases where it leads to peptic ulcer, chronic gastritis, atrophic gastritis, and eventually gastric cancer [1, 6]. • Population with a high incidence of gastric cancer A complete history and physical examination are essential for identifying risk fac- tors, symptoms, and signs associated with H. pylori infection [7]. These can include epigastric pain lasting for more than 4 weeks, or alarm symptoms like unexplained weight loss, progressive dysphagia, odynophagia, recurrent vomiting, abdominal pain, iron deficiency anemia (IDA), jaundice, gastrointestinal (GI) bleeding, and a family history of GI cancer. Following a thorough history and physical exam, appro- priate diagnostic investigations should be recommended based on the assessment. There are a variety of invasive and non-invasive techniques to appropriately make the diagnosis of an H. pylori infection. Patients under age 55 and without alarm symp- toms can be advised to go for noninvasive tests. Following are the different diagnostic investigations available in different clinical settings: • Urea breath test (UBT): UBT requires the ingestion of urea labeled with radioactive isotopes of Carbon 13 or 14. It has a sensitivity and specificity of approximately 100%. UBT can detect active infection and can be used to confirm eradication [7]. • Stool monoclonal antigen test: This test uses monoclonal antibodies and is as accurate as UBT’s when the validated test is used. This test is a cost-effective tool that can detect active infection and can be used to confirm eradication [7]. • Serological test: This test detects immunoglobulin G specific to H. pylori in the serum. However, it cannot distinguish between past and active infection. Serological testing can be used in cases of peptic ulcer disease (PUD) with GI bleeding or in patients with continuous NSAID use, or for mass screening [7]. • Endoscopy with biopsy: Endoscopy with biopsy can be especially useful to rule out cancer in patients with gastritis as well as to diagnose serious causes in patients aged 55 or older with one or more alarm symptoms [7]. It is also performed as a method of surveillance in patients with precancerous lesions or early gastric cancer. H. pylori can be detected using the rapid urease test (RUT) on biopsy specimens if the patient has not taken antibiotics for 4 weeks and proton pump inhibitors (PPIs) for 2 weeks. RUT is an accurate and cost-effective method [7]. If the above conditions are not met, H. pylori can be diagnosed using histology with or without RUT, culture, and polymerase chain reaction (PCR), which allows for susceptibility testing [7]. 5. Diagnosis According to the American College of Gastroenterology (ACG), H. pylori testing is indicated in the following cases: [6]. g All patients with active PUD • All patients with a previous history of PUD (unless it is documented that prior infection resolved) low-grade MALT lymphoma, h/o endoscopic resection of early gastric cancer • Patients with uninvestigated dyspepsia under the age of 60 years • Patient starting long-term NSAIDs • Patient starting long-term NSAIDs • Patients with unexplained iron deficiency anemia and adults with Idiopathic thrombocytopenic purpura • In addition to it experts panel recommend a few more indications where testing is appropriate • Patients residing in the same household as patients with proven active H. pylori infection. • Patient with family history of PUD and gastric cancer 3 Tertiary Care – Medical, Psychosocial, and Environmental Aspects • First-generation immigrants from high-prevalence areas • First-generation immigrants from high-prevalence areas • Population with a high incidence of gastric cancer 6. Treatment All treatment guidelines agree that the best approach to the treatment of H. pylori is to be successful in the first attempt, thereby avoiding retreatment and reducing costs, anxiety, and further promotion of resistant strains [8]. H. pylori, in contrast to other bacterial gastrointestinal infections, requires a combination of antibiotics. Various factors, such as the slow growth rate of H. pylori, bacterial virulence, the inability of the drug to reach an appropriate level in the gastric mucus layer, and 4 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 inactivation of antibiotics at low pH levels, make it difficult to eradicate with any single antibiotic [8]. Commonly used antibiotics for treatment include metronidazole, clarithromycin, amoxicillin, tetracycline, and bismuth. In some cases, third-line treatments may involve the use of ciprofloxacin, moxifloxacin, levofloxacin, furazoli- done, or rifabutin [8]. H. pylori treatment requires different combinations of the antibiotics mentioned above, combined with bismuth salt or an acid-suppressive agent, as none of the above-mentioned antibiotics can work as monotherapy [8]. The rationale for adding an acid-suppressive agent, either a proton pump inhibitor PPI or an H2 receptor blocker, to the regimen is that it increases gastric pH, hence extending the half-life of antibiotics and altering the microenvironment of the bacterium [8]. Moreover, some PPIs have antimicrobial activity, which helps reduce the side effects of the given antibiotics and improves compliance [2, 8]. Bismuth compounds have been used in the treatment of peptic ulcers since the 19th century. Colloidal bismuth sub- citrate, bismuth subsalicylate, and ranitidine bismuth citrate are commonly used as part of anti-H. pylori therapy. The mode of action of bismuth salts is complex and includes inhibition of protein synthesis, adenosine triphosphate (ATP), and cell wall, working synergistically with antibiotics and increasing the efficacy of anti-H. pylori therapy [8]. 6.1 Standard therapy The standard triple therapy for H. pylori treatment consists of amoxicillin/ clarithromycin and a PPI or metronidazole/clarithromycin with a PPI given daily for 7–14 days. Studies have shown that a 14-day duration of triple therapy is much more effective and achieves a higher eradication rate compared to a 7-day therapy [7]. However, this treatment has been shown to fail 20–30% of the time due to poor compliance, prescription errors, short treatment duration, or resistance [8]. Due to increasing resistance to clarithromycin, it is recommended as first-line therapy in places/regions where resistance is lower than 15% [1]. An alternative to standard triple therapy is to combine amoxicillin, metronidazole, and clarithromycin with a PPI for 10–14 days, known as a concomitant treatment, which has better eradication rates compared to standard triple therapy [1, 7]. Adding probiotic to standard triple therapy, specifically Saccharomyces boulardii and Lactobacillus, improves eradication rates, reduces adverse effects, and improves compliance [7]. 6.6 Rifabutin triple therapy Rifabutin triple therapy has also been highlighted as effective in H. pylori eradica- tion, and most guidelines recommend it as salvage therapy when first-line therapy fails [2, 6]. It consists of amoxicillin 1 g, omeprazole 40 mg, and rifabutin 50 mg for 14 days [2]. 6.2 Sequential therapy Sequential therapy consists of a five-day course of a PPI and amoxicillin twice a day, followed by a five-day course of a PPI with clarithromycin and metronidazole or tinidazole. The overall eradication rate for sequential therapy is 84%, with an eradica- tion rate of 74% in areas where clarithromycin resistance is prevalent [7]. Sequential therapy has been shown to be superior to standard triple therapy for a 7-day duration but not superior to standard triple therapy for a 14-day duration, bismuth quadruple therapy (BQT), or non-bismuth quadruple therapy [9]. Moreover, sequential therapy has comparable compliance, incidence of side effects, and therapy interruption rates compared to standard therapy [9]. It has shown positive outcomes in both pediatric and geriatric populations, with a trial enrolling patients over 65 years of age showing better outcomes in patients treated with sequential therapy compared to the standard therapy regimen [10]. 5 Tertiary Care – Medical, Psychosocial, and Environmental Aspects 6.3 Bismuth-based quadruple therapy Bismuth-based quadruple therapy is emerging as an alternative choice for first-line treatment in multiple studies. It includes a bismuth salt (subsalicylate or subcitrate) with metronidazole, tetracycline, and a PPI, all taken four times daily for 10–14 days. The eradication rate of a 10-day bismuth-based therapy was 90.4%, and a 14-day therapy had a rate of 97.1%, indicating that an increased duration correlated with bet- ter eradication of H. pylori infection [11]. This therapy is primarily used when penicil- lin cannot be used or when clarithromycin resistance is over 15% [12]. It is often used as salvage therapy when first-line therapy fails [6]. Longer treatment durations and consideration of metronidazole resistance are highlighted as important factors for achieving higher eradication rates [12]. 6.4 Non-bismuth-based quadruple therapy Non-bismuth-based quadruple therapy, also known as concomitant therapy, involves a combination of a PPI with amoxicillin, metronidazole, or tinidazole, and clarithromycin, given twice daily for 10–14 days. It has the highest eradication rates of about 90%, even in areas of high metronidazole and clarithromycin resistance [7]. However, the success rate of a 7-day concomitant therapy has decreased due to changes in clarithromycin resistance patterns [13]. 6.5 Levofloxacin-based triple therapy Levofloxacin-based triple therapy consists of a combination of a PPI and amoxicillin given twice daily, with levofloxacin given once daily. It can be used as salvage therapy and is better tolerated than bismuth-based quadruple therapy [7]. Levofloxacin can also be given as sequential therapy, consisting of a PPI with amoxicillin for 5–7 days, followed by a PPI with levofloxacin and nitroimidazole for 5–7 days, or as quadruple therapy, composed of levofloxacin, a PPI, nitazoxanide, and doxycycline administered for 7 or 10 days [14]. Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 BQT or levofloxacin triple therapy is the preferred treatment option if the patient was previously treated with clarithromycin-containing first-line therapy. Clarithromycin or levofloxacin-containing treatment is considered if the patient received first-line BQT [15]. Clarithromycin triple therapy should not be considered in second-line treatment [14]. Combinations that can be considered for salvage treatment are BQT, levofloxacin triple therapy, concomitant therapy, rifabutin triple therapy, and high-dose dual therapy [14]. Due to the limited number of antibiotics that are effective against H. pylori and the interpatient and local differences in primary and secondary antibiotic resistance, there is no standard third-line regimen, and treatment should be based on endoscopy with bacterial culture and susceptibility testing [6]. 7. Salvage therapy In case of first-line treatment failure or persistent symptoms, salvage therapy should be based on knowledge of previous antibiotics used and local resistance data [6]. Patients who experience treatment failure are either treated with second-line agents, prolonged treatment, or the addition of bismuth salts [6]. The most com- monly used second-line regimen consists of a combination of tetracycline, amoxicil- lin/metronidazole, a PPI, and bismuth given daily for 10 days [6]. 6 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 8. Prevalence and clinical consequences of antibiotic resistance The mean overall prevalence of resistance to metronidazole was 44%, ranging from 10% in Japan to 84% in Bangladesh and 88% 7 Tertiary Care – Medical, Psychosocial, and Environmental Aspects in Nepal [15]. Data from 2006 to 2015 showed that metronidazole resistance was higher than 40% in most countries except Japan, Myanmar, South Korea, Taiwan, and Thailand [14]. Metronidazole resistance in the Asia Pacific region was 47%, which falls between the reported rates from Europe (35%) and Latin America (50%) [15]. The mean overall prevalence of resistance to levofloxacin was 18%, ranging from 2% in Bhutan to 66% in Bangladesh [15]. Levofloxacin resistance increased from 2% before 2000 to 27% in 2011–2015, according to sub-analysis of the data [15]. Levofloxacin resistance in the Asia Pacific region was higher (21%) compared to Europe (13%) and Latin America (19%) [15]. p The mean overall prevalence of resistance to amoxicillin and tetracycline was 3% and 4%, respectively, reflecting lower use of these antibiotics in medical settings [15]. p The mean overall prevalence of resistance to amoxicillin and tetracycline was 3% and 4%, respectively, reflecting lower use of these antibiotics in medical settings [15]. These findings collectively suggest that clarithromycin-based triple therapy can be used as first-line treatment in countries where resistance is below 15% and bismuth- based quadruple therapy and non-bismuth-based quadruple therapy should be The mean overall prevalence of resistance to amoxicillin and tetracycline was 3% and 4%, respectively, reflecting lower use of these antibiotics in medical settings [15]. These findings collectively suggest that clarithromycin-based triple therapy can be These findings collectively suggest that clarithromycin-based triple therapy can be used as first-line treatment in countries where resistance is below 15% and bismuth- based quadruple therapy and non-bismuth-based quadruple therapy should be considered where clarithromycin resistance is over 15% [15]. y Due to the unavailability of molecular testing or culture and susceptibility testing in most parts of the world, empiric therapy is recommended [2]. However, empiric therapy should be based on knowledge of the patient’s previous antibiotic exposure, penicillin allergy, and local resistance rates. According to the ACG guideline, BQT, concomitant/non-bismuth quadruple therapy, and clarithromycin-based triple therapy are considered first-line therapy for H. pylori eradication [2]. Macrolide- based triple therapy is to be used when there is no previous exposure to it and in regions where local resistance is less than 15% [2]. 8. Prevalence and clinical consequences of antibiotic resistance g In summary, eradication is recommended in all patients with PUD and gastric cancer. First-line therapy should have an eradication rate of more than 80%. Since pretreatment susceptibility is rarely done in primary care settings, therapy is chosen empirically based on regional bacterial resistance patterns, local recommendations, knowledge of previous antibiotic use, drug allergy, and drug availability. 8. Prevalence and clinical consequences of antibiotic resistance One of the biggest challenges in H. pylori treatment is the growing antibiotic resistance against agents used in first-line treatment. Therefore, it is crucial to moni- tor the prevalence of resistance in local settings [6]. Numerous studies have shown that antibiotic resistance reduces the success rate of different anti-H. pylori therapies. However, the extent to which it clinically affects the outcomes depends on the differ- ent components used, the dose of each antibiotic, and the level of resistance present in H. pylori strains [8]. For example, studies have shown that success rates of nitro- imidazole-containing PPI-based triple therapy drop from 90% in susceptible strains to 73% in resistant strains. The addition of bismuth salt to nitroimidazole-containing triple therapy increases the success rate to 92% in nitroimidazole-susceptible strains compared to 83% in resistant strains [8]. Limited studies have shown that macrolide resistance substantially reduces the efficacy of macrolide-containing H. pylori therapy [8]. The efficacy/success rate of adding macrolide to triple therapy may decrease from 86% in susceptible strains to 25% in resistant strains [8]. Antibiotic resistance is a growing problem worldwide, and its prevalence varies in different countries within the Asia Pacific region [15]. Data from the first-ever comprehensive systematic review and meta-analysis of primary antibiotic resistance against H. pylori in the past 25 years showed that the mean overall prevalence of resistance to clarithromycin was 17%, ranging from 0% in Bhutan and Myanmar to 37% in Bangladesh [15]. Clarithromycin resistance increased from 7% before 2000 to 21% in 2011–2015, according to sub-analysis of the data [15]. In the Asia Pacific region, clarithromycin resistance was higher than 15% in countries such as Bangladesh, China, India, Iran, Japan, Nepal, New Zealand, Pakistan, Saudi Arabia, Singapore, South Korea, Turkey, and Vietnam. Clarithromycin resistance was less than 15% in countries such as Bhutan, Indonesia, Laos, Malaysia, Myanmar, Russia, Taiwan, and Thailand [15]. The southeastern Asia region has the lowest risk of clarithromycin resistance [15]. Due to the growing resistance over time in the Asia Pacific region, the efficacy of clarithromycin-based triple therapy, sequential/con- comitant therapy, was lower than 80% in countries with clarithromycin resistance over 20% [15]. Metronidazole resistance is common throughout the world, reflecting the frequent use of this antibiotic in medical settings [15]. 10. Benefits of eradication therapy Peptic ulcer disease: Eradication therapy heals most cases of PUD and greatly diminishes the risk of recurrent bleeding. A systematic review found that eradication therapy is more effective than antisecretory therapy without eradication in prevent- ing recurrent bleeding [8, 14]. Uninvestigated dyspepsia: It is one of the indications for the diagnosis and treat- ment of H. pylori infection according to ACG guidelines. Studies have shown that the “test and treat” strategy is cost-effective and useful compared to acid-suppressive treatment in patients with uninvestigated dyspepsia. However, it is not effective in patients aged above 55 with alarm symptoms and is subject to regional H. pylori prevalence [14]. p Functional dyspepsia or non-ulcer dyspepsia (FD): A Cochrane systematic review published in 2006 showed a small but statistically significant benefit of treating H. pylori infection in patients with FD, with a number needed to treat (NNT) of 14 [14]. py p , ( ) [ ] Chronic atrophic gastritis (CAG): H. pylori infection is the most common cause of atrophic gastritis (AG). Multiple studies had demonstrated a strong relationship between H. pylori infection and the development of chronic atrophic gastritis [4]. Based on a meta-analysis, the rate ratio of AG incidence in patients with vs. without H. pylori infection was 5.0 (95% CI 3.1–8.30), and AG incidence was very low (<1% annually) among H. pylori uninfected individuals, supporting the strong relationship between H. pylori and AG [4]. CAG is a precancerous condition that can progress to dysplasia, and gastric cancer (GC). The rate of progression of AG to adenocarcinoma is 0.1–0.3% per year but may be higher depending on AG severity, extent, concomi- tant IM, and other factors The vast majority of patients with AG have evidence of current or past H. pylori infection [4]. Therefore, eradication of H. pylori is of utmost importance among anyone infected. Successful treatment and eradication of H. pylori can lead to the restoration of normal gastric mucosa in some patients [4]. Although not all cases show improvement, numerous studies have demonstrated a significant reduction in the risk of gastric cancer associated with H. pylori eradication [4]. MALT lymphoma: MALT lymphoma is now largely supplanted by marginal zone B cell lymphoma of MALT type. For patients with MALT lymphoma who have H. pylori infection, studies have shown that the tumor regresses in 60–93% of patients after eradication. 9. Recent developments in H. pylori treatment Due to the lack of culture and sensitivity testing and growing resistance against clarithromycin and metronidazole, bismuth quadruple therapy is considered a reliable empiric choice of treatment. A recent systematic review and meta-analysis showed that both BQT and high-dose dual therapy achieve similar eradication rates, with high-dose dual therapy having better adherence and fewer side effects. High-dose dual therapy involves using a high dose of a proton pump inhibitor (PPI) with 3 g of amoxicillin given daily for 14 days. This regimen achieves eradication rates of around 70–89% in patients with one or more prior treatment failures, and all major guide- lines have recommended high-dose dual therapy [2]. Another development in H. pylori treatment is the approval of a new combination product containing omeprazole, rifabutin, and amoxicillin. In the ERADICATE Hp2 trial, this regimen successfully eradicated H. pylori in 84% of patients. The recom- mended treatment dose consists of 4 capsules to be taken 3 times daily. The daily dose of omeprazole is 120 mg, rifabutin is 150 mg, and amoxicillin is 3 g, which are given in equally divided capsules. The pack contains 12 identical capsules of the single combination product. In future research, the focus will be on developing narrow- spectrum agents that have specific targets, as numerous genomes of H. pylori have been sequenced and various newer drug therapies are under development [2]. 8 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 12. Role of probiotics in management Probiotics are given as an adjuvant treatment with eradication therapy as they improve eradication rates, reduce the side effects of antibiotics, and improve compli- ance. However, the strength of the evidence supporting their use is not strong enough because studies are either of weak quality or have a potential risk of bias. There is no strong evidence to suggest that single-strain probiotics work better than multiple- strain probiotics [1]. 13. Role of H. pylori vaccine Efforts to develop a vaccine against H. pylori have had little success over the last three decades. However, a large phase 3 clinical trial in China of an oral vaccine containing urease B has shown over 70% protection against infection. Nevertheless, the vaccine is not yet available, and more data is required before it can be widely used [1]. 11. Acid suppression with potassium-competitive acid blockers Vonoprazan is the first and most extensively studied potassium-competitive acid blocker (P-CAB). Vonoprazan has a rapid onset of action, longer duration of action, and more profound acid suppression compared to PPIs. It is currently undergoing phase 3 clinical trials in the USA and Europe, and so far, it has yielded promising results in both dual and triple therapy [2]. 10. Benefits of eradication therapy However, the response can be inconsistent, with some patients showing a delayed response or experiencing tumor relapse within one year of treatment [14]. Early gastric cancer: Studies have shown that the incidence of metachronous gas- tric cancer following endoscopic resection of gastric neoplasm was reduced following eradication therapy [14]. NSAIDs: H. pylori is an independent risk factor for NSAID-induced ulcers and ulcer-like bleeding. Eradication of H. pylori before starting NSAID treatment reduces the development of ulcers and the risk of ulcer bleeding, with a 57% reduc- tion in the incidence of peptic ulcers. The benefits are most prominent in NSAID- naive patients [14]. Asymptomatic individuals and gastric cancer: Evidence suggests that eradication reverses the gastric premalignant changes of gastric atrophy and intestinal meta- plasia, although conflicting findings exist. However, studies have shown that the incidence of gastric cancer among asymptomatic individuals is reduced after eradica- tion therapy vs. placebo or no treatment, with an NNT of 124. The estimated benefit in the population with a high risk of gastric cancer will be higher, with an estimated NNT of 15 [14]. 9 Tertiary Care – Medical, Psychosocial, and Environmental Aspects Iron deficiency anemia (IDA): Adults with iron deficiency anemia benefit from combining iron treatment with eradication treatment, resulting in an increase in hemoglobin (Hb), serum iron, and serum ferritin levels (p-value 0.00001) [14]. Iron deficiency anemia (IDA): Adults with iron deficiency anemia benefit from combining iron treatment with eradication treatment, resulting in an increase in hemoglobin (Hb), serum iron, and serum ferritin levels (p-value 0.00001) [14]. g p Idiopathic thrombocytopenic purpura (ITP): There is limited evidence from both randomized and nonrandomized trials that there is sustained improvement in platelet count after eradication of H. pylori in a proportion of adults with ITP. The American Society of Hematology suggests screening for H. pylori infection in adults with ITP and offering eradication therapy if they test positive for an active infection [14]. 14]. 14. Follow-up However, in places where advanced endoscopy with new imaging techniques is available, if an ulcer appears benign with regular margins, repeat endoscopy after eradication may not be necessary [16] Endoscopy is indicated 15. Conditions in which retesting may be considered In cases of non-ulcer dyspepsia (functional dyspepsia), which has a poor correla- tion with H. pylori, a test-and-treat strategy is typically used. However, if symptoms persist or recur following 6–12 months of treatment, retesting is indicated. For unin- vestigated dyspepsia, retesting is indicated if symptoms recur or persist after 4 weeks of treatment [16]. In most cases of chronic gastritis, successful eradication leads to the healing of gastritis and a reduced risk of developing complications [16]. g g p g p Certain risk factors may warrant surveillance in patients with H. pylori gastritis. These risk factors include: Presence of precancerous lesions: In some cases, H. pylori gastritis can progress to more severe forms of gastritis, such as atrophic gastritis or intestinal metaplasia. These conditions are considered precancerous as they increase the risk of developing gastric cancer. Surveillance endoscopy may be recommended in patients with these precancerous lesions to monitor for any signs of dysplasia or gastric cancer. Family history of gastric cancer: Patients with a family history of gastric cancer may have a higher risk of developing the disease themselves. In such cases, surveil- lance endoscopy might be considered to detect any early signs of gastric cancer. Persistent symptoms or alarm features: If a patient continues to experience persis- tent symptoms such as recurrent abdominal pain, bleeding, or unintended weight loss despite successful eradication of H. pylori, further evaluation with endoscopy may be necessary to investigate the underlying cause. 14. Follow-up After treatment, it is important to assess the success or failure of the treatment. The optimal testing time depends on the choice of treatment and the test used. However, it is generally recommended to retest after four weeks of stopping antibiotics and two weeks after discontinuing PPI therapy. Various methods, both invasive and non-inva- sive, can be used for retesting, including the UBT, stool antigen test, and endoscopy [16]. According to the ACG, UBT or stool antigen test should be performed to confirm the eradication of H. pylori [16]. Endoscopy is indicated after treatment of H. pylori infection in two conditions: complicated PUD and gastric ulcer. The most common complications of PUD are bleeding, perforation, and obstruction/stenosis, which require either endoscopy/surgery for initial management. Non-invasive methods 10 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 cannot be used in these cases because patients should be on a prolonged course of PPI, thus endoscopy is usually done to confirm eradication after treatment [16]. py y Gastric ulcer carries the risk of underlying malignancy, and therefore retesting with endoscopy and biopsy is necessary following eradication [16]. This not only confirms the eradication status with absolute certainty but also allows for the assess- ment of any underlying risk of malignancy by obtaining a biopsy from the margin of the ulcer. In cases of gastric ulcer, both before and after treatment biopsy is indicated to exclude malignancy [16]. However, in places where advanced endoscopy with new imaging techniques is available, if an ulcer appears benign with regular margins, repeat endoscopy after eradication may not be necessary [16]. Endoscopy is indicated after eradication in conditions such as for surveillance of premalignant lesions, early gastric cancer, familial gastric cancer, and gastric MALT lymphoma. The frequency of surveillance depends on the underlying conditions [16]. Gastric ulcer carries the risk of underlying malignancy, and therefore retesting with endoscopy and biopsy is necessary following eradication [16]. This not only confirms the eradication status with absolute certainty but also allows for the assess- ment of any underlying risk of malignancy by obtaining a biopsy from the margin of the ulcer. In cases of gastric ulcer, both before and after treatment biopsy is indicated to exclude malignancy [16]. Tertiary Care – Medical, Psychosocial, and Environmental Aspects Author details Malika Taufiq1* and Maliha Taufiq2 1 Mums Care Hospital, Lusaka, Zambia 2 Medical College, Aga Khan University, Karachi, Pakistan Address all correspondence to: drmalika76@gmail.com © 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), Tertiary Care – Medical, Psychosocial, and Environmental Aspects Author details Malika Taufiq1 and Maliha Taufiq2 1 Mums Care Hospital, Lusaka, Zambia 16. Recurrence Several factors have been implicated in the recurrence of H. pylori infections. These factors include coming from a low socioeconomic background (low income), poor hygiene, dining out in establishments, and undergoing invasive approaches for diagnosis and treatment [17]. 11 11 © 2023 The Author(s). Licensee IntechOpen. 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. 12 Current Perspective on the Treatment of Helicobacter pylori Infection DOI: http://dx.doi.org/10.5772/intechopen.114135 H. pylori infection. American Family Physician. 2015;91(4):236-242 H. pylori infection. American Family Physician. 2015;91(4):236-242 [1] De Brito BB, Da Silva FAF, Soares AS, Pereira VA, Santos MLC, Sampaio MM, et al. Pathogenesis and clinical management of Helicobacter pylori gastric infection. World Journal of Gastroenterology. 2019;25(37):5578-5589. DOI: 10.3748/wjg.v25.i37.5578 [8] Gerrits MM, van Vliet AH, Kuipers EJ, Kusters JG. Helicobacter pylori and antimicrobial resistance: Molecular mechanisms and clinical implications. The Lancet Infectious Diseases. 2006;6(11):699-709. DOI: 10.1016/ S1473-3099(06)70627-2 [2] Saleem N, Howden CW. Update on the Management of Helicobacter pylori infection. Current Treatment Options in Gastroenterology. 2020;18(3):476-487. DOI: 10.1007/ s11938-020-00300-3. Epub 2020 Jul 17 [9] Vaira D, Zullo A, Hassan C, Fiorini G, Vakil N. Sequential therapy for Helicobacter pylori eradication: The time is now! Therapeutic Advances in Gastroenterology. 2009;2(6):317-322. DOI: 10.1177/1756283X09343326 [3] Wang YK, Kuo FC, Liu CJ, Wu MC, Shih HY, Wang SS, et al. Diagnosis of Helicobacter pylori infection: Current options and developments. World Journal of Gastroenterology. 2015;21(40):11221- 11235. DOI: 10.3748/wjg.v21.i40.11221 [10] Zullo A, Gatta L, De Francesco V, Hassan C, Ricci C, Bernabucci V, et al. High rate of Helicobacter pylori eradication with sequential therapy in elderly patients with peptic ulcer: A prospective controlled study. Alimentary Pharmacology & Therapeutics. 2005;21(12):1419-1424. DOI: 10.1111/j.1365-2036.2005.02519.x [4] Shah SC, Piazuelo MB, Kuipers EJ, Li D. AGA clinical practice update on the diagnosis and management of atrophic gastritis: Expert review. Gastroenterology. 2021;161(4):1325-1332. e7. DOI: 10.1053/j.gastro.2021.06.078. Epub 2021 Aug 26 [11] Salazar CO, Cardenas VM, Reddy RK, Dominguez DC, Snyder LK, Graham DY. Greater than 95% success with 14-day bismuth quadruple anti- Helicobacter pylori therapy: A pilot study in US Hispanics. Helicobacter. 2012;17(5):382-390 [5] Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global prevalence of Helicobacter pylori infection: Systematic review and meta-analysis. Gastroenterology. 2017;153(2):420-429. DOI: 10.1053/j. gastro.2017.04.022. Epub 2017 Apr 27 [5] Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global prevalence of Helicobacter pylori infection: Systematic review and meta-analysis. Gastroenterology. 2017;153(2):420-429. DOI: 10.1053/j. gastro.2017.04.022. Epub 2017 Apr 27 [12] Graham DY, Lee SY. How to effectively use bismuth quadruple therapy: The good, the bad, and the ugly. Gastroenterology Clinics of North America. 2015;44(3):537-563. DOI: 10.1016/j.gtc.2015.05.003. Epub 2015 Jun 19 [6] Shatila M, Thomas AS. Current and future perspectives in the diagnosis and management of Helicobacter pylori infection. Journal of Clinical Medicine. 2022;11(17):5086. [11] Salazar CO, Cardenas VM, Reddy RK, Dominguez DC, Snyder LK, Graham DY. Greater than 95% success with 14-day bismuth quadruple anti- Helicobacter pylori therapy: A pilot study in US Hispanics. Helicobacter. 2012;17(5):382-390 [9] Vaira D, Zullo A, Hassan C, Fiorini G, Vakil N. Sequential therapy for Helicobacter pylori eradication: The time is now! Therapeutic Advances in Gastroenterology. 2009;2(6):317-322. DOI: 10.1177/1756283X09343326 [1] De Brito BB, Da Silva FAF, Soares AS, Pereira VA, Santos MLC, Sampaio MM, et al. Pathogenesis and clinical management of Helicobacter pylori gastric infection. World Journal of Gastroenterology. 2019;25(37):5578-5589. DOI: 10.3748/wjg.v25.i37.5578 H. pylori infection. American Family Physician. 2015;91(4):236-242 DOI: 10.3390/ jcm11175086 [13] Huang T, Yang S, Tai W, Liang C, Kuo C, Yao C, et al. The efficacies of non-bismuth containing quadruple [7] Fashner J, Gitu AC. Diagnosis and treatment of peptic ulcer disease and 13 Tertiary Care – Medical, Psychosocial, and Environmental Aspects therapies in the treatment of first-line anti-Helicobacter pylori across 4-year time interval with changing antibiotics resistance. Biomedical Journal. 2021;44(6):S275-S281. DOI: 10.1016/j. bj.2020.11.012 [14] Chey WD, Leontiadis GI, Howden CW, Moss SF. ACG clinical guideline: Treatment of Helicobacter pylori infection. American Journal of Gastroenterology. 2017;112(2):212-239. DOI: 10.1038/ajg.2016.563. Epub 2017 Jan 10. Erratum in: Am J Gastroenterol. 2018 Jul;113(7):1102 [14] Chey WD, Leontiadis GI, Howden CW, Moss SF. ACG clinical guideline: Treatment of Helicobacter pylori infection. American Journal of Gastroenterology. 2017;112(2):212-239. DOI: 10.1038/ajg.2016.563. Epub 2017 Jan 10. Erratum in: Am J Gastroenterol. 2018 Jul;113(7):1102 [15] Kuo YT, Liou JM, El-Omar EM, Wu JY, Leow AHR, Goh KL, et al. Primary antibiotic resistance in Helicobacter pylori in the Asia-Pacific region: A systematic review and meta- analysis. Lancet Gastroenterología y Hepatología. 2017;2(10):707-715. DOI: 10.1016/S2468-1253(17)30219-4. Epub 2017 Aug 7 [16] Kuipers EJ. When is endoscopic follow-up appropriate after Helicobacter pylori eradication therapy? Gastroenterology Clinics of North America. 2015;44(3):597-608. DOI: 10.1016/j.gtc.2015.05.006. Epub 2015 Jun 19 [16] Kuipers EJ. When is endoscopic follow-up appropriate after Helicobacter pylori eradication therapy? 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https://openalex.org/W2327809899	https://eprints.glos.ac.uk/4565/1/Facet%20Theory%20and%20the%20mapping%20sentence.pdf	English	null	Facet Theory and the Mapping Sentence As Hermeneutically Consistent Structured Meta-Ontology and Structured Meta-Mereology	Frontiers in psychology	2,016	cc-by	4,719	This is a peer-reviewed, final published version of the following document and is licensed under Creative Commons: Attribution 4.0 license: This is a peer-reviewed, final published version of the following document and is licensed under Creative Commons: Attribution 4.0 license: Hackett, Paul M ORCID: 0000-0002-9365-8084 (2016) Facet Theory and the Mapping Sentence As Hermeneutically Consistent Structured Meta-Ontology and Structured Meta- Mereology. Frontiers in Psychology, 7. doi:10.3389/fpsyg.2016.00471 Official URL: http://dx.doi.org/10.3389/fpsyg.2016.00471 DOI: http://dx.doi.org/10.3389/fpsyg.2016.00471 EPrint URI: https://eprints.glos.ac.uk/id/eprint/4565 Disclaimer The University of Gloucestershire has obtained warranties from all depositors as to their title in the material deposited and as to their right to deposit such material. The University of Gloucestershire makes no representation or warranties of commercial utility, title, or fitness for a particular purpose or any other warranty, express or implied in respect of any material deposited. The University of Gloucestershire makes no representation that the use of the materials will not infringe any patent, copyright, trademark or other property or proprietary rights. The University of Gloucestershire accepts no liability for any infringement of intellectual property rights in any material deposited but will remove such material from public view pending investigation in the event of an allegation of any such infringement. PLEASE SCROLL DOWN FOR TEXT. PERSPECTIVE published: 31 March 2016 doi: 10.3389/fpsyg.2016.00471 PERSPECTIVE Edited by: Richard Stöckle-Schobel, Ruhr-University Bochum, Germany Reviewed by: John G. Holden, University of Cincinnati, USA Patrick Mair, Harvard University, USA Correspondence: Paul M. W. Hackett paul_hackett@emerson.edu An earlier version of this paper (Koval and Hackett, 2015) was presented at the 14th international Facet Theory Conference. Fordham University, New York, August 2015. An earlier version of this paper (Koval and Hackett, 2015) was presented at the 14th international Facet Theory Conference. Fordham University, New York, August 2015. Keywords: ontology, mereology, facet theory, mapping sentence, meta-ontology, meta-mereology Specialty section: This article was submitted to Theoretical and Philosophical Psychology, a section of the journal Frontiers in Psychology Received: 09 January 2016 Accepted: 17 March 2016 Published: 31 March 2016 Specialty section: This article was submitted to Theoretical and Philosophical Psychology, a section of the journal Frontiers in Psychology Facet Theory and the Mapping Sentence As Hermeneutically Consistent Structured Meta-Ontology and Structured Meta-Mereology Paul M. W. Hackett 1 School of Communication, Emerson College, Boston, MA, USA, 2 Department of Philosophy, University of Oxford, Oxford, UK When behavior is interpreted in a reliable manner (i.e., robustly across different situations and times) its explained meaning may be seen to possess hermeneutic consistency. In this essay I present an evaluation of the hermeneutic consistency that I propose may be present when the research tool known as the mapping sentence is used to create generic structural ontologies. I also claim that theoretical and empirical validity is a likely result of employing the mapping sentence in research design and interpretation. These claims are non-contentious within the realm of quantitative psychological and behavioral research. However, I extend the scope of both facet theory based research and claims for its structural utility, reliability and validity to philosophical and qualitative investigations. I assert that the hermeneutic consistency of a structural ontology is a product of a structural representation’s ontological components and the mereological relationships between these ontological sub-units: the mapping sentence seminally allows for the depiction of such structure. INTRODUCTION When thinking about the world around us it is commonplace and may even seem natural to sub-divide our experiences in attempting to achieve better understanding. The practice of partitioning research content has a long history dating back to at least the time of the ancient, classical philosophers, where such well-known examples include ontologies by Aristotle (1975) and Plato (Harte, 2002). During the subsequent millennia, categorial ontologies have been developed by a wide range of psychologists and philosophers, each of who have concerned themselves with attempting to understand the basic components of human existence (see for example: in psychology, Piaget and Inhedler, 1969, Kelly, 2013; in philosophy, Chisholm, 1996). Given the multitude of ontologies and other componential existential models that exist, the question may be asked as to whether a meta-ontology may be developed that speaks about how ontologies may be understood in structurally theoretical terms. Moreover, questions may also be posed as to the possibilities of developing a meta- mereological structure, which explicates the combined relations of the meta-ontology. During Received: 09 January 2016 Accepted: 17 March 2016 Published: 31 March 2016 Edited by: Richard Stöckle-Schobel, Ruhr-University Bochum, Germany 3I am using meta-ontology in the sense of theory that underlies a generic framework for the constitution of interpretative consistency of a research domain, whilst not imposing an external structure to either the content under investigation or to the interpretation itself. DEFINING TERMS In the title of this essay I have employed three phrases that qualify my understanding of the requirements of categorial research investigations: hermeneutic consistency, structured meta-ontology, and structured meta-mereology. These expressions have been carefully selected to emphasize what I believe a qualitative facet theory approach is able to achieve and an initial review of these terms will explicate the nature of the ontology/mereology in which I am interested. In the title of this essay I have employed three phrases that qualify my understanding of the requirements of categorial research investigations: hermeneutic consistency, structured meta-ontology, and structured meta-mereology. These expressions have been carefully selected to emphasize what I believe a qualitative facet theory approach is able to achieve and an initial review of these terms will explicate the nature of the ontology/mereology in which I am interested. Citation: Hackett PMW (2016) Facet Theory and the Mapping Sentence As Hermeneutically Consistent Structured Meta-Ontology and Structured Meta-Mereology. Front. Psychol. 7:471. doi: 10.3389/fpsyg.2016.00471 March 2016 \| Volume 7 \| Article 471 Frontiers in Psychology \| www.frontiersin.org Hermeneutically Consistent Structured Meta-Ontology and Meta-Mereology Hackett HERMENEUTIC CONSISTENCY Hermeneutical is an adjective that implicates and focuses ontologies as being interpretative tools. Hermeneutically consistent implies that the ontology I oﬀer is reliable in terms of the structure and the interpretation of its content. In the usage of the phrase hermeneutic consistency, hermeneutical refers to a speciﬁc interpretive methodology as understood through the writing of Heidegger (1962) and Gadamer (2013). These authors were interested in knowledge and truth and in their work the phrase hermeneutic consistency refers to the ability to achieve a coherent explanation of an informational source. Many other philosophers, especially epistemologists, are interested in knowledge and truth and the coherence of explanations about sources of information2. However, Heidegger and Gadamer are of particular import as it may be claimed that their inﬂuence has spread more widely than some other scholars. For instance, both Heidegger and Gadamer are commonly cited within sociological, psychological and perhaps most importantly to this paper, within research design lecture series and textbooks. Furthermore, the hermeneutical process is of great importance within disciplines that seek interpretation of complex events (as an illustration see: Osborne, 2007; Porter and Robinson, 2011) who provide introductory accounts of hermeneutical processes in reading scripture. In the same way, facet theory based interpretations are also concerned with the interpretative interplay between an event and those experiencing and attempting to understand these occurrences. Ontology is the study and formal explication of a domain of content in terms of its more fundamental or basic categorial components as these may be understood at this fundamental level and as their meaning may be further revealed through consideration of more sub-ordinate, particular, or evident categorial entities. I use the term meta-ontology to imply that the qualitative ontology I propose constitutes an ontology about ontologies rather than being an ontology of a speciﬁc or substantive content area3. My use of this term refers to an ontology of the diﬀerent, often instrumentalist, ontologies that diﬀerent disciplines of enquiry adopt to characterize and delimit their frameworks. Furthermore, the term structured ontology and structured mereology respectively bring together the concepts of ontology and mereology (or the underlying nature of experience) within a determinate structural template under the deﬁnition of ontology I have provided. The next term in my title is mereology. 1In this paper I present facet theory and the mapping sentence as a philosophical/qualitative approach to the study of behavior. Criticisms of inadequacy can be made of analytic systems of formal logic (the mapping sentence may be seen as one such system) in their diﬃculty in diﬀerentiating meaning such as those embodied in sarcasm and irony versus sincerity where formally these may be indistinguishable [see for example the work of Gibbs (e.g., Gibbs and Colston, 2012) and the collection of writings by (Gibbs and Colston, 2007)]. However, the mapping sentence would address this diﬀerence by the inclusion of elements of sincerity and sarcasm within a content facet of degree of genuineness. Furthermore, there have been many developments, such as self-organizing systems and impredicative declarations which are of interest to the development of system based deﬁnition but beyond the scope of this article. The interested reader is guided toward the writing of Turvey and Moreno (2006). 2This ability is studied or thought about in many disciplines and contexts other than philosophical ONTOLOGY this essay I provide answers to these questions1, however, I will initially clarify the precise terms of my exposition. Ontology refers to the basic components underlying nature of experience, and structured ontology explicates such understanding within a determinate composition. Ontology has slightly dissimilar meanings when used within the diﬀerent disciplines that have incorporated ontology into part of their lexicon and way of thinking. For example: in philosophy— ontology is a branch of metaphysics concerned with the nature of being; within logic—ontology is the set of entities that a given theory assumes beforehand; in technology—ontology provides a systematic explanation of existence; within information and computer sciences—ontology is the rigorous designation of existent components (sorts, characteristics) and their inter- associations. From these deﬁnitions it can be seen that to some extent there are common elements in what ontology is taken to mean. Ontology may therefore appear to refer to being and components of existence, which are perhaps instantiated by a scholar prior to consideration of a content area. Given the diﬀerences in the use of the term ontology I wish to escape any possible confusion that may arise by providing a precise deﬁnition and understanding of ontology: MEREOLOGY Mereology is concerned with attempts to understand the relationships between, and implications of, part-to-whole and part-to-part associations within a categorial system or ontology. Mereology is deﬁned within metaphysics as: “... any theory of part hood or composition.” (Harte, 2002, p7). However, as with the term ontology, mereology is understood in slightly diﬀerent manners dependent upon the discipline of usage (e.g., philosophy, science, logic, mathematics, semantics). I wish to avoid possibilities of confusion and misinterpretation and I therefore provide my own deﬁnition of mereology as follows: March 2016 \| Volume 7 \| Article 471 Frontiers in Psychology \| www.frontiersin.org 2 Hermeneutically Consistent Structured Meta-Ontology and Meta-Mereology Hackett Mereology is the systematic and explicit investigation, analysis and resulting understanding of the relationships within a structured ontology, in terms of the part to part, part to whole, part to context, part to background, and part to observation range, relationships. Shye, 1995; Canter, 1985a,b; Shye, 1978; Shye and Amar, 1985; Shye and Elizur, 1994). After having used facet theory in a traditionally quantitative manner, Hackett (2013, 2014) has, over the past few years, developed a qualitative facet theory6. During the course of this brief essay I oﬀer a qualitative7 facet theory approach as an instantiation of a meta- ontology and meta-mereology. In this paper I evaluate facet theory, and its major instrument the mapping sentence, as a qualitative and philosophical stance toward the understanding of behavior. A meta-mereology is a mereology that is concerned with the nature of mereologies rather than the content of any particular or speciﬁc mereology4. Structured meta-mereology implicates an interest in the conﬁguration of mereological relationships. I must provide one ﬁnal deﬁnition that applies to my speciﬁcations of both ontology and mereology. On these understandings, ontologies and mereologies exist where and when: The philosophical and theoretical bases of facet theory along with qualitative facet theory approach to research design, data collection and analysis is best understood and reported using the mapping sentence. A mapping sentence is a formal statement of a research domain which includes the respondents, sub- categories of the research content along with the range over which observations will be made, in the structure of a sentence written in normal prose. The mapping sentence is both the major tool of facet theory research design and analysis and also a series of structural/spatial hypotheses. As Canter (1985b) says: “... MEREOLOGY a piece of facet research is a process of reﬁnement, elaboration and validation of a mapping sentence.” (p266): I will be using a mapping sentence in precisely these terms in this paper. Philosophically, the mapping sentence is a structural ontology and in application to any substantive area of research and understanding may also be seen as a mereological device. Related to the notion of the mapping sentence is that a mereology is a compositional identity, where composition is the relation between a whole and its speciﬁc parts, in which parts form the whole and where the whole is nothing more than its parts: the whole is its parts and parts may only be understood within the whole (see, Cotnoir and Baxter, 2014). Context and background are essential and inherent components of the existence and realization of the structured ontological/mereological system, where changes in background and context would result in signiﬁcant diﬀerences in the structured ontology/mereology, and where the speciﬁcation of a diﬀerent range of observations would signiﬁcantly alter the content of the structured ontology/mereology and the nature of knowledge embodied within such structure. So far I have provided a limit to the scope of my essay and in the following sections I oﬀer facet theory and the mapping sentence as a means for achieving a structured ontology/mereology under the constraints of these deﬁnitions. I advance my ontology/mereology under the belief that if a researcher understands the components of the behaviors of interest and the interrelationships between these components, a greater appreciation of the total behavior may result. 5Borg (1978, p65) deﬁned facet theory as: “... a general methodology for investigation in the social sciences: it provides a general framework for the precise deﬁnition of an universe of observations, which is directly related to both the speciﬁcation of the various elements of empirical studies (stimuli, subjects, responses) and to theories about the structure of those observations.” 6Hackett has carried-out qualitative analyses of speciﬁc research domains using mapping sentences as a framework for the conception and design of research projects. He has then progressed by analysing qualitative and conceptual data within a facet theory mind-set to allow theory development. This has required Hackett to use facet theory as a philosophical perspective that he has taken when viewing the subject matter of his research into human behavior (Hackett, 2013, 2014). 4I employ term meta-mereology in the same sense as I use meta-ontology (see footnote 2). QUALITATIVE FACET THEORY AND THE MAPPING SENTENCE In qualitative facet theory and within a facet theoretical philosophy two central theses arise from the above deﬁnitions of ontology and mereology: Louis Guttman originated facet theory with an implicitly point of view that understand human activities and knowledge about such activities as being formed of discrete components (Guttman, 1947; Levy, 1994). Guttman (1959, p130) deﬁned a facet as “... a set that is a component of a Cartesian product.” and in his authoritative text, Canter (1985a, p22) states how a facet is constituted as a “... labeling of a conceptual categorization underlying a group of observation.” Facet theory has been deﬁned as, “a strategy for research in psychology and other sciences that study complex behavioral systems. Facet Theory centers on the formalization of research contents and on intrinsic data analysis for the purpose of discovering stable laws and conducting theory- based measurements in those sciences”5. When taken together, a speciﬁed structured ontology and a mereological account of this structure form what is known as a mapping sentence. For any speciﬁed area of interest, a mapping sentence provides a hermeneutically consistent account of a domain of interest. Thus, facet theory and speciﬁcally the mapping sentence is well characterized through the use of the terms structural ontology and mereology with the explicit intent of developing hermeneutically consistent knowledge. Facet theory has traditionally been based in quantitative research approaches and the statistical analysis (e.g., Borg and 7In facet theory the term qualitative has been used to mean a qualitatively arranged facet rather than a linear or quantitatively ordered facet: This is not the sense of the word that I will employ. I use qualitative to imply rich observational, non- numerical information. Subsequently I analyse data to establish reliable and valid interpretative hermeneutics. March 2016 \| Volume 7 \| Article 471 Frontiers in Psychology \| www.frontiersin.org 3 Hackett Hermeneutically Consistent Structured Meta-Ontology and Meta-Mereology FIGURE 1 \| Mapping sentence for Lowe’s four-category ontology. FIGURE 1 \| Mapping sentence for Lowe’s four-category ontology. FIGURE 1 \| Mapping sentence for Lowe’s four-category ontology. 8Aristotle’s categories are: 1, Substance (o ´υσ´ια); 2, Quantity (πoσ´oν); 3, Quality (πoι´oν); 4, Relation (πρ´oζ); 5, Place (πo ˜υ); 6, Time (π´oτε); 7, Being-in-a-position (κε˜ιθαι); 8, Having (˝εχειν); 9, Action (πoιε˜ιν); 10, Aﬀection (π´ασχειν). From these Categories I developed a mapping sentence for Aristotle’s categorial system (Hackett, 2014). 9Lowe’s ontology has appeared in several slightly varied iterations all of which Lowe portrayed as an ontological square. A HERMENEUTICALLY CONSISTENT TEMPLATE In this paper I am claiming that the mapping sentence is at the heart of traditional, philosophical and qualitative explorations employing a facet theory outlook in both exploratory and conﬁrmatory research. The mapping sentence is the basis for investigations, structural hypothesis testing and theory generation and as a stand-alone research approach. Mapping sentences specify research domains allowing the deﬁnition of the domain’s sub-aspects and sub-aspect interrelationships availing appreciation of the domain’s content. To further illustrate a qualitative application of a mapping sentence in Figure 2 I provide a mapping sentence of the theoretical content of this essay. This qualitative/philosophical mapping sentence demonstrates the hermeneutic consistency of understanding that arises from non-numerical research that is organized through using a mapping sentence. EXAMPLE OF A QUALITATIVE MAPPING SENTENCE arrangement) of Lowe’s ontology. When Lowe’s ontology is modeled in a mapping sentence the interplay of elements is stressed and by clearly explicating a possible mereology of elements the active role of the reader is also emphasized. Furthermore, the mapping sentence requires the researcher to consider the nature of the context of the evaluation and background features that may aﬀect content. In earlier research I have demonstrated the utility of a non- numerically based facet theory that employs the conceptual rigor that the mapping sentence has provided in my investigation of the mereology of Aristotle’s Categories (Aristotle, 1975)8. This mapping sentence oﬀers an account of The Categories that clearly displays Aristotle’s ontology and uniquely a potential mereological relationship between the Categories parts-to-parts and parts-to-whole and in so doing oﬀers further exploration of Aristotle’s ontology. In ﬁgure 1 I provide a mapping sentence for a more contemporary ontology by Lowe (2007) in his four-category ontology9. Lowe’s ontology embodies the notion that the world may be understood as comprising three distinct types of objects, two kinds of events, two modes and three attributes. Lowe settled with this structure as he believes that this four-category ontology provides “a uniquely satisfactory metaphysical foundation for the natural sciences” (Lowe, 2007 Page 16). TAKE IN FIGURE 1. ABOUT HERE The mapping sentence for Lowe’s ontology in ﬁgure 1 oﬀers a transparent modeling of Lowe’s conceptions of the basics of existence. Uniquely, the mapping sentence demonstrates not only the ontology’s structure but also the interplay (or mereological 9Lowe’s ontology has appeared in several slightly varied iterations all of which Lowe portrayed as an ontological square. 8Aristotle’s categories are: 1, Substance (o ´υσ´ια); 2, Quantity (πoσ´oν); 3, Quality (πoι´oν); 4, Relation (πρ´oζ); 5, Place (πo ˜υ); 6, Time (π´oτε); 7, Being-in-a-position (κε˜ιθαι); 8, Having (˝εχειν); 9, Action (πoιε˜ιν); 10, Aﬀection (π´ασχειν). From these Categories I developed a mapping sentence for Aristotle’s categorial system (Hackett, 2014). TAKE IN FIGURE 2 ABOUT HERE In this mapping sentence the range facet delimits the substantive concern of the mapping sentence, which in this case is the extent to which a mapping sentence structured ontology can March 2016 \| Volume 7 \| Article 471 Frontiers in Psychology \| www.frontiersin.org 4 Hackett Hermeneutically Consistent Structured Meta-Ontology and Meta-Mereology FIGURE 2 \| Mapping sentence for the hermeneutic consistency of a mapping sentence. FIGURE 2 \| Mapping sentence for the hermeneutic consistency of a mapping sentence. have provided support for claims regarding the potential of qualitative or philosophical research that is undertaken within a facet theory framework. I have claimed utility for the use of a mapping sentence as a purely philosophical outlook when attempting to understand human experience by oﬀering a mapping sentence as a philosophically coherent approach to understanding Lowe’s ontology and as a tool to investigate the hermeneutical consistency of research. avail a hermeneutically consistent understanding of a content domain. Returning to the start of the mapping sentence, person (x) is taken to be any individual reading and understanding the mapping sentence. Continuing along the sentence, the combinatorial arrangements of the two content facets are determinants of the values observed in the range. In this sentence: the ontology facet speciﬁes the content of the mapping sentence ontology to be—facets (with sub-divisions of facet elements); background (which lists background characteristics of the instantiation of the ontology); range which speciﬁes the epistemological/characteristics of the observations that constitute the mapping sentence’s logic. Thus, the mereology facet characterizes the nature of the relationships that are extant within the mapping sentence ontology as being either part-to- part (facet/facet element-to-facet/facet element) or part-to-whole (facet/facet element-to-mapping sentence). It is my contention that the hermeneutic consistency of a structural ontology is a product of a structural representation’s ontological components and the mereological relationships between these ontological units: the mapping sentence seminally allows for the depiction of such structure. Finally, I claim facet theory and mapping sentences form a precise though ﬂexible framework for the designing research and writing within philosophical and qualitative psychological research10. CONCLUSIONS The author conﬁrms being the sole contributor of this work and approved it for publication. I commenced by proposing that understanding a content domain may result from sub-dividing the domain into relevant categories. I then noted how facet theory has achieved a category-based epistemological exposition of many research areas under a quantitative research rubric. In this paper I 10I would like to thank the associate editor of Frontiers for suggesting that the facet theory approach may be used in future research to thematically investigate the philosophy of science and especially the structure of scientiﬁc theories. Canter, D. (ed.). (1985a). Facet Theory: Approaches to Social Research. New York, NY: Springer Verlag. Canter, D. (1985b). “How to be a facet researcher,” in Facet Theory: Approaches to Social Research, ed D. Canter (New York, NY: Springer Verlag), 265–276. Chisholm, R. M. (1996). A Realistic Theory of Categories: An Essay on Ontology. Cambridge: Cambridge University Press. Cotnoir, A. J., and Baxter, D. L. M. (eds.). (2014). Composition as Identity. Oxford: Oxford University Press. Gadamer, H. G. (2013). Truth and Method. New York, NY: Bloomsbury Academic. REFERENCES “Partial-order scalogram analysis by base coordinates and lattice mapping of the items by their scalogram roles,” in Facet Theory: Approaches to Social Research, ed D. Canter (New York, NY: Springer Verlag), 277–298. Shye, S., and Amar, R. (1985). “Partial-order scalogram analysis by base coordinates and lattice mapping of the items by their scalogram roles,” in Facet Th h l h d ( k Guttman, L. (1959). “Introduction to facet design and analysis,” in Proceedings of the Fifteenth International Congress of Psychology, Brussels–1957. Amsterdam: North Holland, 130–132. Theory: Approaches to Social Research, ed D. Canter (New York, NY: Springer Verlag), 277–298. Hackett, P. M. W. (2013). Fine Art and Perceptual Neuroscience: Field of Vision and the Painted Grid. New York, NY: Psychology Press. Shye, S., and Elizur, D. (1994). Introduction to Facet Theory: Content Design and Intrinsic Data Analysis in Behavioral Research (Applied Social Research Shye, S., and Elizur, D. (1994). Introduction to Facet Theory: Content Design and Intrinsic Data Analysis in Behavioral Research (Applied Social Research Methods). Thousand Oaks, CA: Sage Publications, Inc. Hackett, P. M. W. (2014). Facet Theory and the Mapping Sentence: Evolving Philosophy, Use and Application. Basingstoke: Palgrave. Hackett, P. M. W. (2014). Facet Theory and the Mapp Methods). Thousand Oaks, CA: Sage Publications, Inc. Philosophy, Use and Application. Basingstoke: Palgrave. Turvey, M. T., and Moreno, M. A. (2006). Physical metaphors for the mental lexicon. Ment. Lex. 1, 7–33. doi: 10.1075/ml.1.1.04tur Harte, V. (2002). Plato on Parts and Wholes: The Metaphysics of Structure. Oxford: Oxford University Press. Heidegger, M. (1962). Being and Time. New York, NY: Harper Perennial Modern Classics. Conﬂict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Kelly, G. A. (2013). A Theory of Personality: The Psychology of Personal Constructs. New York, NY: W. W. Norton and Company. Copyright © 2016 Hackett. 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 accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Koval, E., and Hackett, P. M.W. (2015). REFERENCES Canter, D. (ed.). (1985a). Facet Theory: Approaches to Social Research. New York, NY: Springer Verlag. Aristotle (1975). Aristotle’s Categories and de Interpretatione. Oxford: Oxford University Press. Canter, D. (1985b). “How to be a facet researcher,” in Facet Theory: Approaches to Social Research, ed D. Canter (New York, NY: Springer Verlag), 265–276. Chisholm, R. M. (1996). A Realistic Theory of Categories: An Essay on Ontology. Cambridge: Cambridge University Press. I. (1978). “Some basic concepts in facet theory,” in Geometric Rep Borg, I. (1978). “Some basic concepts in facet theory,” in Geometric Representation of Relational Data, ed J. Lingoes (Ann Arbor, MI: Mathesis Press). of Relational Data, ed J. Lingoes (Ann Arbor, MI: Mathesis Press) Borg, I., and Shye, S. (1995). Facet Theory: Form and Content (Advanced Quantitative Techniques in the Social Sciences). Thousand Oaks, CA: Sage Publications, Inc. Cotnoir, A. J., and Baxter, D. L. M. (eds.). (2014). Composition as Identity. Oxford: Oxford University Press. Gadamer, H. G. (2013). Truth and Method. New York, NY: Bloomsbury Academic. March 2016 \| Volume 7 \| Article 471 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 5 Hermeneutically Consistent Structured Meta-Ontology and Meta-Mereology Hackett Lowe, E. G. (2007). The Four-Category Ontology: A Metaphysical Foundation for Natural Science. Oxford: Oxford University Press. Gibbs, R. W., and Colston, H. L. (eds.). (2007). Irony in Language and Thought: A Cognitive Science Reader. New York, NY: Routledge. g g Gibbs, R. W., and Colston, H. L. (2012). Interpreting Figurative Meaning. Cambridge: Cambridge University Press. Osborne, G. R. (2007). The Hermeneutical Spiral: A Comprehensive Introduction to Biblical Interpretation. Westmont, IL: IVP Academic. Piaget, J., and Inhedler, B. (1969). The Psychology of the Child. New York, NY: Basic Books. Guttman, L. (1947). “Scale and intensity analysis for attitude, opinion and achievement in new methods in applied psychology,” in Proceedings of the Maryland Conference on Military Contributions to Methodology in Applied Psychology held at the University of Maryland, November 27–28, 1945, under the auspices of the Military Division of the American Psychological Association, ed G. A. Kelly (College Park, MD: University of Maryland). Porter, S. E., and Robinson, J. C. (2011). Hermeneutics: An Introduction to Interpretive Theory. Grand Rapids, MI: Wm. B. Eerdmans Publishing Co. Shye, S. (1978). Theory Construction and Data Analysis in the Behavioral Sciences. San Francisco, CA: Jossey-Bass. Shye, S., and Amar, R. (1985). Frontiers in Psychology \| www.frontiersin.org REFERENCES “Hermeneutic consistency, structured ontology and mereology as embodied,” in Facet Theory and the Mapping Sentence, paper presented at the fourteenth International Facet Theory Conference, Fordham University, New York, NY. Levy, S. (ed.). (1994). Louis Guttman on Theory and Methodology: Selected Writings, Dartmouth Benchmark Series. Aldershot: Dartmouth. March 2016 \| Volume 7 \| Article 471 Frontiers in Psychology \| www.frontiersin.org 6
https://openalex.org/W3094703244	https://opus.lib.uts.edu.au/bitstream/10453/145027/2/energies-13-05713-v2.pdf	English	null	A Review of Energy Management Assessment Models for Industrial Energy Efficiency	Energies	2,020	cc-by	13,360	Received: 25 September 2020; Accepted: 29 October 2020; Published: 1 November 2020 Abstract: The necessity to ensure energy eﬃciency in the industries is of signiﬁcant importance to attain reduction of energy consumption and greenhouse gases emissions. Energy management is one of the eﬀective features that ensure energy eﬃciency in the industries. Energy management models are the infancy in the industrial energy domain with practical guidelines towards implementation in the organizations. Despite the increased interest in energy eﬃciency, a gap exists concerning energy management literature and present application practices. This paper aims to methodologically review the energy management assessment models that facilitate the assessment of industrial energy management. In this context, the minimum requirements model, maturity model, energy management matrix model, and energy eﬃciency measures characterization framework are discussed with implications. The study concludes with interesting propositions for academia and industrial think tanks delineating few further research opportunities. Keywords: energy management; industrial energy eﬃciency; energy management practices; assessment model energies energies energies www.mdpi.com/journal/energies Review A Review of Energy Management Assessment Models for Industrial Energy Eﬃciency A S M Monjurul Hasan and Andrea Trianni * School of Information, Systems, and Modelling, Faculty of Engineering and IT, University of Technology Sydney, 81 Broadway, Ultimo 2007, Australia; asmmonjurul.hasan@uts.edu.au * Correspondence: andrea.trianni@uts.edu.au Received: 25 September 2020; Accepted: 29 October 2020; Published: 1 November 2020 Energies 2020, 13, 5713; doi:10.3390/en13215713 agement Assessment Models ciency * ulty of Engineering and IT, University of Technology Sydney, hasan@uts.edu.au 2020; Published: 1 November 2020 ency in the industries is of signiﬁcant importance to eenhouse gases emissions. Energy management is one ciency in the industries. Energy management models n with practical guidelines towards implementation nterest in energy eﬃciency, a gap exists concerning ication practices. This paper aims to methodologically dels that facilitate the assessment of industrial energy uirements model, maturity model, energy management res characterization framework are discussed with sting propositions for academia and industrial think unities. energy eﬃciency; energy management practices; st entities for consuming energy [1], is responsible for p-rising of energy expenses, stringent environmental aped increased demand to the reduction of energy ustries [3]. In this context, ensuring energy eﬃciency strial processes that must be addressed as a priority. tation of energy management practices can provide e optimum usage of energy resources maintaining the e energy costs [4–6]. eing developed to endorse energy eﬃciency in the ction of greenhouse gas emissions, and productivity ﬃciency still remains unattained [9,10]; with low easures [11,12] because of certain barriers [13,14], otential. There are multiple studies conducted at using the barriers to adopt energy eﬃciency in the are also found towards energy-eﬃcient technology ﬃciency gap has been conferred, keeping the relevance it has also incorporated behavioral issues [24]. e mostly studied through theoretically or conceptually, d in an empiric way [25]. Academic studies have been archers about energy management practices and their agement Assessment Models ciency * ulty of Engineering and IT, University of Technology Sydney, hasan@uts.edu.au 2020; Published: 1 November 2020 ency in the industries is of signiﬁcant importance to eenhouse gases emissions. Energy management is one ciency in the industries. Energy management models n with practical guidelines towards implementation nterest in energy eﬃciency, a gap exists concerning ication practices. This paper aims to methodologically dels that facilitate the assessment of industrial energy uirements model, maturity model, energy management res characterization framework are discussed with sting propositions for academia and industrial think unities. energy eﬃciency; energy management practices; st entities for consuming energy [1], is responsible for p-rising of energy expenses, stringent environmental aped increased demand to the reduction of energy ustries [3]. In this context, ensuring energy eﬃciency strial processes that must be addressed as a priority. tation of energy management practices can provide e optimum usage of energy resources maintaining the e energy costs [4–6]. eing developed to endorse energy eﬃciency in the ction of greenhouse gas emissions, and productivity ﬃciency still remains unattained [9,10]; with low easures [11,12] because of certain barriers [13,14], otential. There are multiple studies conducted at using the barriers to adopt energy eﬃciency in the are also found towards energy-eﬃcient technology ﬃciency gap has been conferred, keeping the relevance it has also incorporated behavioral issues [24]. e mostly studied through theoretically or conceptually, d in an empiric way [25]. Academic studies have been archers about energy management practices and their 1. Introduction The industrial sector, being one of the largest entities for consuming energy [1], is responsible for 30% of total carbon emission [2]. Further, the up-rising of energy expenses, stringent environmental restrictions, and fossil fuel depletion have shaped increased demand to the reduction of energy consumption and its associated costs in the industries [3]. In this context, ensuring energy eﬃciency is one of the signiﬁcant mainstays of the industrial processes that must be addressed as a priority. Energy eﬃciency gains through the implementation of energy management practices can provide multiple beneﬁts to an organization ensuring the optimum usage of energy resources maintaining the desired energy productivity level and reduce the energy costs [4–6]. The energy management programs are being developed to endorse energy eﬃciency in the industries for facilitating energy savings, reduction of greenhouse gas emissions, and productivity beneﬁts [7,8]. However, industrial energy eﬃciency still remains unattained [9,10]; with low implementation rates of energy eﬃciency measures [11,12] because of certain barriers [13,14], although research has shown its immense potential. There are multiple studies conducted at local, regional, national, and multinational focusing the barriers to adopt energy eﬃciency in the industries [15–19]. On the contrary, the drivers are also found towards energy-eﬃcient technology adoption by several studies [20–22]. The energy eﬃciency gap has been conferred, keeping the relevance on technical aspects and appliances [23], whilst it has also incorporated behavioral issues [24]. Energy management and energy services are mostly studied through theoretically or conceptually, whilst energy management practices are studied in an empiric way [25]. Academic studies have been conducted regionally and beyond by many researchers about energy management practices and their Energies 2020, 13, 5713; doi:10.3390/en13215713 2 of 21 Energies 2020, 13, 5713 characterization [15,26–28]. Energy management practices, as well as energy services, are perceived as signiﬁcant explanations, and few eﬀorts are paid to depict them including the assessment model to facilitate industrial think tank focusing particular set of actions for improved energy management [24]. It is notable to mention that research mainly acquainted the idea of an “extended energy eﬃciency gap”, expressing the gap abide by technical as well as managerial components. In addition, a vast portion of unexplored market potential namely “energy service gap” exists because of high operating cost at the industrial application phase [29], even though energy services speak for a favorable market-centric resolution for improved energy eﬃciency [30]. 1. Introduction So far, the avenues between integration of energy management with production systems are unexplored. Further, energy management into industrial decision-making process is not discussed thoroughly till now. Therefore, it is imperative to explore the domain of energy management to support industrial decision-makers pointing to the speciﬁed actions which are required to minimize the energy management lagging aspects, still keeping mind the multi-dimensional context and complexity of industrial energy management systems [31,32]. Given the introductory context, the paper aims to methodologically review the energy management assessment models that facilitate the assessment of industrial energy management. Notably, this study does not consider energy generation part and conﬁnes its focus to energy management framework only to help the industrial decision-making process covering energy consumption aspects in the industries. This study is novel considering the fact that there has not been any study focusing on energy management 4.0 in the industrial decision-making process and comprehends the energy management framework to the best of authors’ knowledge. In this study, we have worked to synthesize this gap in the greater interest of academia. By doing the review, we want to highlight future research avenues having nexus with energy management and industrial energy eﬃciency. Interestingly, all of the present research gaps fall into the big area, which is energy management 4.0 in industrial decision making. On another note, this study would help the industrial managers and engineers by ﬁguring out improvement options in their energy management activities and supply chain system. In addition, the available options for policymakers to address energy management regulations are also incorporated in this study. The rest of this paper is designed as follows: an introduction to the energy management concept is presented in Section 1. Section 2 describes the methodology. Section 3 provides the descriptive results of reviewing the literature on energy management assessment model. Subsequently, this paper concludes with explaining and incorporating the results in Section 4. Section 5 presents the concluding remarks. 2. Methodology A systematic and rigorous review process was conducted in this paper. The primary focus of such reviews is to point out the related available studies established on pre-formulated research queries to synthesize the conclusion based on the evidence [33]. It is notable to mention that the systematic review features substantial leverages contrast to conventional narrative approaches of literature work. The conventional review does not apprehend formal methodological approaches, whilst the systematic review incites to minimize research biases through the adoption of search strategies, preordained inquiry string, and inclusion and elimination criterion [33]. Moreover, the comprehensive documentation nature of review enhances the clarity of review as well as facilitates subsequent replication [34]. In this paper, the relevant literature search methodology comprised of scientiﬁc literature sources, mainly the “Scopus” and “Web of Science” as both of the sources are well accepted in academia for their research quality and reliability [35]. We checked the online databases indexed in “Scopus” and “Web of Science” to identify the articles based on our keyword. In this research, the selected keywords to sort out the literature are “Energy Management”, “Industry”, “Energy Management Model”, “Energy Management Practices”, and “Energy Eﬃciency”. Nonetheless, there was no speciﬁc starting timeframe for searching the literature in the database, though attempts were made to consider the recent researches. Table 1 presents the selection basis of the literature review. 3 of 21 Energies 2020, 13, 5713 Table 1. Selection basis of the literature review. Table 1. Selection basis of the literature review. Heading Remark Research domain Energy; Engineering; Management Search string Industrial Energy Management; Energy management Practice; Energy Management Framework Publication Type The academic journals, conference proceedings, and book chapters. Working papers are not considered due to their review process state and reliability issue [36]. The included publications are Elsevier, Springer, IEEE Xplore, MDPI, Taylor & Francis, John Wiley & Sons, and Emerald. Availability Available online Area Industry Relevance Articles articulate energy management; energy eﬃciency proceedings at the institutional perspective Time Focus on the recent researches Each of the selected articles has been checked manually for content analysis in stage 2, the “screening” process. During the screening process, expulsion criteria that are followed in this research are presented in Table 2. Articles were discarded in this stage based on the criterion EXC 1, EXC 2. In stage 3, a backward review was conducted to reconsider relevant articles based on our selected keywords. management and energy mana very critical and decisive to the 3.1. Energy Management Deﬁnition any concept related to energy management and its framework additional resolutions and aspects were also introduced that were not considered in the initial phase. 3. Results and Analysis 3.1. Energy Management Definition Deﬁning energy management is signiﬁcant when it comes to the point at energy management modelling or energy system practices implementation. Energy management concept is speciﬁed by many studies that incorporate multiple arenas. The prime areas covered by multiple studies to deﬁne energy management are energy consumption, strategic aspect, the involvement of managerial perspective, and people relevancy [25]. Defining energy management is significant when it comes to the point at energy management modelling or energy system practices implementation. Energy management concept is specified by many studies that incorporate multiple arenas. The prime areas covered by multiple studies to define energy management are energy consumption, strategic aspect, the involvement of managerial perspective, and people relevancy [25]. The German Federal Environment Agency defined energy management as the inclusion of planned and execution of actions to ensure predefined performance by a minimum amount of energy input [37]. B.L. Capehart has characterized the term energy management as the proficient and effective usage of energy towards maximization of profits and increasing reasonable positions [38]. O’Callaghan et al., defined the energy management as the application of resources in regards of supply, conversion, and utilization which integrates monitoring, measurement, archiving, critical examination and analyzation, control, and rerouting of energy as well as material flows through the The German Federal Environment Agency deﬁned energy management as the inclusion of planned and execution of actions to ensure predeﬁned performance by a minimum amount of energy input [37]. B.L. Capehart has characterized the term energy management as the proﬁcient and eﬀective usage of energy towards maximization of proﬁts and increasing reasonable positions [38]. O’Callaghan et al., deﬁned the energy management as the application of resources in regards of supply, conversion, and utilization which integrates monitoring, measurement, archiving, critical examination and analyzation, control, and rerouting of energy as well as material ﬂows through the systems for ensuring minimal energy usage and achieve meaningful goals [39]. To deﬁne energy management, Bunse et al. focused on the inclusion of control, supervision, and improvement activities towards energy eﬃciency [6]. On the contrary, Ates et al. strengthened on the combination of techniques, activities, and managerial processes that leads to reduce energy cost and anthropogenic emissions [40]. One of the studies by Abdelaziz et al. 2. Methodology The following stage consists of the exclusion of articles based on the criterion EXC 2, EXC 3, EXC 4, EXC 5, EXC 6, and EXC 7. Finally, the last step of methodology replicates the content analysis of selected articles. The entire methodological steps are illustrated in Figure 1. In the phase of analyzing the content, it was essential to distinguish between energy management and energy management assessment framework/model. Therefore, the situation was very critical and decisive to the inclusion of such speciﬁcation in this study. Nonetheless, discarding any concept related to energy management and its framework additional resolutions and aspects were also introduced that were not considered in the initial phase. Table 2. Exclusion criterion of the literature. Exclusion Heading Remark EXC 1 The article published not in English EXC 2 The article uses “Energy management” term only in title and does not incorporate in any energy management framework or model in an elaborated form EXC 3 The article uses “Energy management” only as a part of the future research direction or future perspective EXC 4 The article uses “Energy Management” just as a cited term EXC 5 Articles deals only with drivers, barriers to energy management practices in the industries EXC 6 Availability of full texts EXC 7 Working papers Table 2. Exclusion criterion of the literature. 4 of 21 Energies 2020, 13, 5713 E i 13 Energies 2020, 13, x FOR PEER REVIEW 4 of 23 Figure 1. The methodological steps followed in the research. Identification •Identifying relevant journal and conference articles indexed in “Web of Science” and “Scopus”. The journal articles identified in this stage were 335. Screening •Articles were eliminated due to EXC 1, EXC 2. The discarded number of of articles were 54. The number of remaining articles for further consideration was (335-54)= 281. Backward Review •Reviewing the previous literature for reconsideration based on the keywords of the research. Screening •Articles were eliminated based on exclusion criterion (presented in Table 2). The number of excluded articles were 281 at this stage. The number of remainning article was (281-189)= 92. Content Analysis •Analysis of the articles identified manually related to research context Figure 1. The methodological steps followed in the research. g Figure 1 The methodological steps followed in the research Identification •Identifying relevant journal and conference articles indexed in “Web of Science” and “Scopus”. The journal articles identified in this stage were 335. 2. Methodology Screening •Articles were eliminated due to EXC 1, EXC 2. The discarded number of of articles were 54. The number of remaining articles for further consideration was (335-54)= 281. Backward Review •Reviewing the previous literature for reconsideration based on the keywords of the research. Screening •Articles were eliminated based on exclusion criterion (presented in Table 2). The number of excluded articles were 281 at this stage. The number of remainning article was (281-189)= 92. Content Analysis •Analysis of the articles identified manually related to research context Figure 1. The methodological steps followed in the research. •Identifying relevant journal and conference articles indexed in “Web of Science” and “Scopus”. The journal articles identified in this stage were 335. Identification Screening •Reviewing the previous literature for reconsideration based on the keywords of the research. Backward Review •Articles were eliminated based on exclusion criterion (presented in Table 2). The number of excluded articles were 281 at this stage. The number of remainning article was (281-189)= 92. •Articles were eliminated based on exclusion criterion (presented in Table 2). The number of excluded articles were 281 at this stage. The number of remainning article was (281-189)= 92. Screening Content Analysis Figure 1. The methodological steps followed in the research. Figure 1. The methodological steps followed in the research. Energies 2020, 13, 5713 Energies 2020, 13, 5713 Energies 2020, 13, 5713 In academic literature, energy management is portrayed as a holistic combination of applying resources, conversion, and application of energy [16,20,25,32]. The system involves checking, auditing, recording, scrutinizing, and more importantly controlling the energy ﬂows to ensure the minimum consumptions of energy but to achieve maximum energy productivity [16,42]. Academicians have pointed some of the minimal prerequisites for implementation and operation of energy management in the industries [27,40,41,43,44]. Table 3 illustrates the requirements toward energy management with speciﬁcations whether the requirements are considered full, partly, or not under consideration. Table 3. Minimal prerequisites for energy management in the industries. This table is adopted from Schulze et al. [32]. Minimum Prerequisite Abdelaziz et al. [41] Christoﬀersen et al. [44] Thollander and Ottosson [27] McKane et al. [43] Ates and Durakbasa [40] Long-term strategic plan; inclusion of energy policy; energy saving targets. Energy activities by dedicated responsibilities and actions × × × Acquaintance of energy management team led by the energy manager × × Policies and proceedings × × Energy audit to explore energy-saving features × × × Planning and implementation of an explicit energy-saving program Identiﬁcation of key performance indicators × × × × Meter and monitoring of energy consumption × × Energy reporting × × × Top management commitment × × × × Employee engagement in energy management activities × Abbreviations: (Full Consideration); (Partial Consideration); × (Not Considered). Table 3. Minimal prerequisites for energy management in the industries. This table is adopted from Schulze et al. [32]. Minimal prerequisites for energy management in the industries. This table is adopted from Abbreviations: (Full Consideration); (Partial Consideration); × (Not Considered). It becomes discernible by analyzing the minimum requirements for energy management from Table 3 that the sets of minimum requirements elucidated in the studies contrast in the number of elements as well as conformation of the individual features. In addition, it shows indistinctness on the conclusiveness of the list of minimum requirements whether it is suitable to describe a fully developed energy management. By analyzing earlier contributions on the topic, we can note the lack of a comprehensive conceptual framework about energy management. management and energy mana very critical and decisive to the 3.1. Energy Management Deﬁnition promoted energy management focusing on energy optimization strategy that incorporates compelling the energy demand [41]. A comprehensive deﬁnition of energy management has been proposed by Schulze et al. that incorporates all necessary energy management elements and energy management practices in the industries [32]. 5 of 21 Energies 2020, 13, 5713 Therefore, in this study, we respond to this research gap by complying a review of academic journal publications in the area of industrial energy management and use its results to propose future research avenues to explore further. Energies 2020, 13, 5713 6 of 21 3.2. Approaches to Energy Management Models There are research streams which are considered in academia as well as the industries to assess the energy management models. The streams can be categorized as “Minimum requirements”, “Maturity models”, and “Energy management matrixes” [25]. Furthermore, there is assessment tool namely “Energy Management Measures Characterization Framework”, so to shape the energy management aspects accordingly”. This is practice based, therefore basing on energy management practices with characteristics. 3.2.1. Minimum Requirements The ISO 50001 standard that deals with energy management issues is incorporated at the ﬁrst stream and thus apprehends guidelines to enable energy management system [45]. Enabling the organizations towards energy eﬃciency is the primary purpose of ISO 50001 Energy Management System standard. The standard is reviewed and published by the ISO/TC 301 Technical Standardization Committee, Energy Management, and Energy Saving in 2018 [45]. The protocol has a high level of hierarchical structure consists of ten chapters with a homologous architecture. The ISO 50001 standard is a consistent improvement framework which consists of “Plan-Do-Check-Act” at organizational practices. Table 4 presents the phases that are comprehended at ISO 50001 Energy Management System standard. However, it does not apprehend the critical assessment of the enterprises’ eﬀectiveness for a taken initiative of particular energy management practice. In addition, the initial stream incorporates primary endeavor to evaluate energy management, maintaining the limit of analysis [40,44]. Table 4. The continual phases of ISO 50001 Energy Management System standard [45] Phase Remark Plan To apprehend the organizational context; incorporation of energy policy; incorporation of energy management team; consideration of actions towards risks and opportunities; conduct of energy review; identiﬁcation of signiﬁcant energy uses and establishment of energy performance indicators; energy baseline; objectives and energy targets; necessary action plans to improve energy performance in accordance with the organization’s energy policy. Do Implementation of the action plans; operation and maintenance controls, and communication; ensuring competence in energy domain i.e., energy performance in design and procurement. Check Monitor; quantify; analyzation; evaluation; audit and conducting management review of energy performance as well as energy management system. Act Activities to address non-conformities and continuation for improving energy performance. Table 4. The continual phases of ISO 50001 Energy Management System standard [45]. 3.2.2. Maturity Models [56,57]. 3.2.4. Energy Eﬃciency Measures (EEM) Characterization Framework 3.2.4. Energy Efficiency Measures (EEM) Characterization Framework The EEM characteristics are delved by the fourth research stream [58]. The energy efficiency measures characterization framework is important to formulize in the context of information sharing both for the policy and decision-makers about energy efficiency measures. Thanks to improved knowledge and information on industrial energy efficiency measures. Indeed, the policymakers could have enhanced support to develop the operative policies for endorsing energy efficiency at the industries. In addition, the improved knowledge on energy efficiency measures characteristics can articulate in-depth comprehension of the bottlenecks that hindering the implementation of energy ffi i [59] I d d hi i i i f f l i d li k The EEM characteristics are delved by the fourth research stream [58]. The energy eﬃciency measures characterization framework is important to formulize in the context of information sharing both for the policy and decision-makers about energy eﬃciency measures. Thanks to improved knowledge and information on industrial energy eﬃciency measures. Indeed, the policymakers could have enhanced support to develop the operative policies for endorsing energy eﬃciency at the industries. In addition, the improved knowledge on energy eﬃciency measures characteristics can articulate in-depth comprehension of the bottlenecks that hindering the implementation of energy eﬃciency processes [59]. Indeed, this is an interesting fact for resolution and policy makers. efficiency processes [59]. Indeed, this is an interesting fact for resolution and policy makers. Fleiter et al. exhibited detailed and thorough narratives of characterizations that facilitated understandings of the endorsement process for EEMs [58]. The framework encompasses twelve diverse features of energy efficiency measures which are emanated from the field of technical, relative advantage, and informational perspective. Worrell et al. characterized and grouped the energy efficiency measures into multiple attributes such as waste, emission, operation and maintenance, y p g p y Fleiter et al. exhibited detailed and thorough narratives of characterizations that facilitated understandings of the endorsement process for EEMs [58]. The framework encompasses twelve diverse features of energy eﬃciency measures which are emanated from the ﬁeld of technical, relative advantage, and informational perspective. Worrell et al. characterized and grouped the energy eﬃciency measures into multiple attributes such as waste, emission, operation and maintenance, productivity, working environment, among others, where the secondary beneﬁts are listed [60]. On the contrary, Trianni et al. devised a framework to explore energy management practices [59]. 3.2.2. Maturity Models This second stream solicits a systematic perspective for assessing energy management in the organization [8] that includes the analysis for the requisite steps to enact energy management system [46]. Continuous improvement options are one of the signiﬁcant features of maturity model. Therefore, the maturity model is accepted and popular in academia as well as industries since the development of the capability maturity model (CMM) [47–49]. The maturity models help the institutional enterprises surmount the austerity and enhance the quality by measuring institutional maturity based on particular or multiple domains with the help of predeﬁned rules [50,51]. However, the maturity models are single dimensioned that focus either on objects maturity or process maturity, whilst the process maturity levels are dominant than the object-based model [52]. In one of the studies, Bojana et al. presented the 7 of 21 e , rity, Energies 2020, 13, 5713 y the maturity mod maturity stages of energy management at activity levels [53]. Figure 2 exhibits the levels considered in maturity models for energy and utility management. studies, Bojana et al. presented the maturity stages of energy management at activity levels [53]. Figure 2 exhibits the levels considered in maturity models for energy and utility management. Figure 2. The levels in maturity models for energy and utility management (Source: [54]). Initial Managed Defined Quantitatively Managed Optimized Figure 2. The levels in maturity models for energy and utility management (Source: [54]). Defined Figure 2 The levels in maturity models for energy and utility management (Source: [54]) Figure 2. The levels in maturity models for energy and utility management (Source: [54]). g 3 2 3 E M M i 3.2.3. Energy Management Matrixes 3.2.3. Energy Management Matrixes The energy management matrixes are incorporated with the third stream [55,56], which confers multiple similarities with the maturity model. It offers an insight into the present approach to energy issues in a company and helps the management to improve energy efficiency by integrating feedback. It also shows the substantial improvement potential in energy efficiency that is achievable by technical activity alone. However, the application of the energy management matrix in a wider range of industrial organizations has acknowledged manifold activities towards improvement of energy management practice. In addition, it puts the hitherto isolated technologically-based attempts to improve energy efficiency in a more effective management framework, often for the first time. The high standpoint from an analytical perspective, maturity concept conversion into a sophistication level along with a self-appraisal approach based on organization’s perspective are the common points of energy matrixes with maturity models. Hence, no additional benefits are provided from these models in terms of approaches and aspects considered for reasoning. However, introducing assessment models have brought an amelioration that incorporates detailed activity list considered as energy management practices, whilst critical factors have not been addressed for evaluation The energy management matrixes are incorporated with the third stream [55,56], which confers multiple similarities with the maturity model. It oﬀers an insight into the present approach to energy issues in a company and helps the management to improve energy eﬃciency by integrating feedback. It also shows the substantial improvement potential in energy eﬃciency that is achievable by technical activity alone. However, the application of the energy management matrix in a wider range of industrial organizations has acknowledged manifold activities towards improvement of energy management practice. In addition, it puts the hitherto isolated technologically-based attempts to improve energy eﬃciency in a more eﬀective management framework, often for the ﬁrst time. The high standpoint from an analytical perspective, maturity concept conversion into a sophistication level along with a self-appraisal approach based on organization’s perspective are the common points of energy matrixes with maturity models. Hence, no additional beneﬁts are provided from these models in terms of approaches and aspects considered for reasoning. However, introducing assessment models have brought an amelioration that incorporates detailed activity list considered as energy management practices, whilst critical factors have not been addressed for evaluation [56,57]. [56,57]. 3.2.4. Energy Eﬃciency Measures (EEM) Characterization Framework Minimum requirements [56,57]. 3.2.4. Energy Eﬃciency Measures (EEM) Characterization Framework An inclusive view on energy eﬃciency measures integrating the recent applicable perspectives is encompassed in this framework for industrial decision-makers. The framework has inferred in specifying energy alongside the environmental and ﬁnancial aspects. Moreover, the impact on production system, including the application aspects and interaction with other systems of energy eﬃciency measures are also considered in the framework. Another noteworthy feature of the framework is the inclusion of corporate involvement, which is important for industrial decision-makers and policy delegates. Moreover, the inclusion of the attribute set related to non-energy beneﬁts is one of the salient features that has been neglected in the earlier characterization framework. Nonetheless, analytical factors of energy management activities are not portrayed comprehensively. Lung et al. aﬃrm about the impact of additional savings and productivity beneﬁts stemming from energy eﬃciency initiatives 8 of 21 Energies 2020, 13, 5713 resulting in more compellingly. The authors focused on the methodology to characterize the attributes of productivity beneﬁts as well as ancillary savings into a payback forecasting framework [61]. Another model has been proposed in a contemporary study by Trianni et al. in the domain of characterization framework to assess industrial energy management, focusing on the benchmarking of energy management practices [25]. In this model, three elements have been considered that are energy management practice lists followed by speciﬁc baseline for benchmarking the performances and optimal threshold adoption in the assessment. The notable aspects of this model are the energy management practice adoption evaluation and more comprehensiveness output compared to the other models. More importantly, it features elaborate energy management approaches and capabilities assessment to an indistinct evaluation of energy management practices. On the contrary, Sorrell [62] and Benedetti et al. [63] have considered three-dimensional classiﬁcation framework focusing to energy service contracts. The framework of Sorrell is customer perspective based and consisted of “Scope”, “Depth”, and “Finance” dimension. Benedetti et al. considered “Scope”, “Intangibility of the Contract”, and “Degree of Risk”. g The synopsis of the existing management assessment models is presented in Table 5. 9 of 21 Energies 2020, 13, 5713 y management assessment models. The table is an aggrandized approach of Trianni et al. [25]. Narration Remark Reference cy, energy saving goals arding energy-saving projects gy eﬃcient purchases, speciﬁc ty and tasks. Functioning ecially the employees by cating. This model consider the energy management as a comprehensive management system. [56,57]. 3.2.4. Energy Eﬃciency Measures (EEM) Characterization Framework Focused on policy, energy saving goals and speciﬁc energy saving projects. However, the model does not integrate the energy manager concept. Furthermore, there is no clear guideline about top or mid-level management support to achieve energy savings. Though, involvement of employee to energy saving related work are suggested. [44] e basis for instructions. bility; policy; legitimate concern nergy performance index; energy ergy management blueprint; usness; communication; sition; operation and control; nalysis; compliance evaluation ; in-house audit of the energy , corrective as well as vernance; management review. ISO 50001 incorporates nineteen characteristics in the framework. Precisely, management commitment and energy manager are also inclined to the model. Moreover, the framework integrates the employee involvements and documentation and records for further assessment. [45] ments from the (27)’s set by oceedings; inclusion of dedicated This model is an extended version of Christoﬀersen et al. [44]. The model integrates energy metering, energy policy, energy manager, saving target and saving projects focusing on energy. [40] Table 5. Synopsis of the existing energy management assessment models. The table is an aggrandized approach of Trianni et al. [25]. Model Narration Model Narration Category This model consider the energy management as a comprehensive management system. Focused on policy, energy saving goals and speciﬁc energy saving projects. However, the model does not integrate the energy manager concept. Furthermore, there is no clear guideline about top or mid-level management support to achieve energy savings. Though, involvement of employee to energy saving related work are suggested. Signiﬁcant features: energy policy, energy saving goals (quantitative) or aspirations regarding energy-saving projects and their implementation. Energy eﬃcient purchases, speciﬁc allotment of energy responsibility and tasks. Functioning engagement of stakeholders, specially the employees by apprising, persuading, and educating. “Plan-Do-Check-Act” cycle is the basis for instructions. Preconditions: management liability; policy; legitimate concern and obligations; energy audit; energy performance index; energy baseline; energy targets, and energy management blueprint; proﬁciency, training and consciousness; communication; archiving; energy services acquisition; operation and control; monitoring, measurement and analysis; compliance evaluation maintaining the legal necessities; in-house audit of the energy management system; aberration, corrective as well as precautionary action; archive governance; management review. Alteration of the merest requirements from the (27)’s set by adding the metering of major proceedings; inclusion of dedicated energy manager at the industry. 10 of 21 10 of 21 Energies 2020, 13, 5713 Table 5. Conts. Remark Model Narration Category Model Narration Remark Reference Maturity Models Five stages: preliminary, arrange, delineate, managed in quantitative form and reformed; Novel process avenues are regulated towards progress focusing on environmental aspect; Four maturity phases: practice enactment, standardization of practice, performance management and recurring phase for improvement. The model used Capability Maturity Model Integration (CMMI) as a reference framework and extended to environmental management context. It comprised of particular procedures for energy as well as resource management. No clear guideline about dedicated energy manager. [54] Instructions to attain improved energy eﬃciency and amenability with energy management standards especially ISO 50001. Energy management actions are categorized into ﬁve maturity phases subsequent to the PDCA cycle. The framework adapts manifold energy management practices based on PDCA cycle. Notably, top management support is incorporated in the framework. Energy management roles are characterized. However, no clear indication about energy manager inclusion in the process. [64] Five levels: Emerging, Deﬁne, Integration, Optimization and Novelty; four sections on the basis of PDCA cycle, 16 pillars, and 63 sub-pillars. The model allows 5 attribution promulgation for each sub-pillar to evaluate the maturity. Energy management review along with action plan are integrated to the framework. Model Narration In addition, competence building feature is also included. [65] Primary features for the energy consumption management keeping alignment to ISO 50001. Five phases: initial, intermittent, planning, supervisory and optimal. 5 dimensions that are portrayed as requisite for success: consciousness, information, and expertise (utmost signiﬁcant); methodological proposition; energy performance management and archiving system; institutional architecture; alignment with strategy. The tool is not incorporated with inclusion of energy manager. [8] Incorporation with ISO 50001; knowledge base creation for self-assessment along with monitoring and improvement. The levels are depicted for each ISO 50001 process instilled by Eric et al. [54]. The assessment tool includes top management commitment, and energy manager appointment with other manifold energy management practices. [53] Salient features are the assessment of compelling factors for energy management adoption, contribution towards a better understanding of suitable energy management conﬁguration with the help of evaluation of maturity level. The model considers inclusion of energy manger, precisely a dedicated energy management team. In addition, top management support is integrated with the considered attributes in the model. [46] Incorporation of qualitative metrics; assessment model implies on PDCA cycle; inclusion of SWOT analysis tool, incorporation of global energy management team and external peers. Incorporates their application speciﬁc purposes which are descriptive, prescriptive, and comparative. Features with manifold energy management practices along with energy manager. [66] Category Five stages: preliminary, arrange, delineate, managed in quantitative form and reformed; Novel process avenues are regulated towards progress focusing on environmental aspect; Four maturity phases: practice enactment, standardization of practice, performance management and recurring phase for improvement. Instructions to attain improved energy eﬃciency and amenability with energy management standards especially ISO 50001. Energy management actions are categorized into ﬁve maturity phases subsequent to the PDCA cycle. Five levels: Emerging, Deﬁne, Integration, Optimization and Novelty; four sections on the basis of PDCA cycle, 16 pillars, and 63 sub-pillars. The model allows 5 attribution promulgation for each sub-pillar to evaluate the maturity. Salient features are the assessment of compelling factors for energy management adoption, contribution towards a better understanding of suitable energy management conﬁguration with the help of evaluation of maturity level. Incorporation of qualitative metrics; assessment model implies on PDCA cycle; inclusion of SWOT analysis tool, incorporation of global energy management team and external peers. 11 of 21 Energies 2020, 13, 5713 11 of 21 Table 5. Conts. EEMs characterization framework Model Narration Remark Model Narration Category Model Narration Remark Reference Consists of three features: (1) energy eﬃciency features (2) energy eﬃciency maturity levels; (3) implementation method which is accustomed from ZED scheme especially for SMEs. Seven dimensions: management obligation, arrangement and procedure, compliance of regulation and ﬁscal enticements, archiving system, product and procedure innovation, in-house communication, and ethos. Consists of nineteen characteristics. Total number of nineteen energy eﬃciency characteristics are integrated in the model. In addition, management commitment is segregated into two sections in the form of strategic priority and energy policy. [67] Energy Management Matrixes Five levels of energy management matrixes to address six institutional aspects that are policy, organization, motivation, information scheme, marketing, and ﬁnancing. Top management support is fully integrated into the framework under policy section. Energy managerial role included in organizational structure. [68] Five levels of energy management matrixes to assess six institutional issues that are energy management scheme; organization; staﬀinspiration; tracking, supervision and reporting systems; staﬀconsciousness/training and promotion, and ﬁnancing. Energy manager feature is integrated with a proposition of organizational structure. Moreover, energy management is considered comprehensively in this framework. [55] Five levels of energy management matrixes to assess six institutional issues which are policy or speciﬁc guidelines, coordinating, training, evaluation of performance, communication, and ﬁnancing. Valuation model exploring the subsequent aspects reﬂected as energy management practice: policy and legislation, energy blueprint, organizational formation; regulation; acquisition strategy, ﬁnancing scheme, observation, and analysis of energy consumption, setting of goal; identiﬁcation of possible options; staﬀinvolvement and training; operational process; communications. The Carbon Trust guidelines comprised of ﬁve aspects. Inclusion of dedicated energy manager is not integrated to this model. However, the model incorporates senior management commitment to enhance energy eﬃciency related initiatives. [56] 12 of 21 12 of 21 Energies 2020, 13, 5713 Table 5. Conts. Table 5. Conts. on Remark Reference es considered as energy appointment, energy policy; collection lishment of yardstick or hnical perspective and e scope, improvement technical procedures and ion; formation of a g, capacity building, nt of result, recapitulation nition, and receiving The ENERGY STAR guideline clearly emphasizes on appointment of energy director with dedicated energy team. In addition, the model looks to establish baselines for measuring energy performance. [57] ed. Each characteristic are haracter “Relative te of return, introductory neﬁts of non-energy. 4. Discussion Energies 20 The energy management frameworks were mainly researched to adopt energy management practices at the technical levels in the industries. However, the reviewed papers emphasized the energy management system, ISO 50001, and PDCA cycle, while some studies suggested holistic approaches towards industrial energy eﬃciency. 4. Discussion The energy management frameworks were mainly researched to adopt energy management practices at the technical levels in the industries. However, the reviewed papers emphasized the energy management system, ISO 50001, and PDCA cycle, while some studies suggested holistic The framework proposed by Christoﬀersen et al. was stood out on the Danish industries and emphasized on multiple factors, mostly energy policy, goals and capstone projects aimed at energy savings. Regulation, external relations, company characteristics, and organizational internal condition are the main out-layers of the model to frame the energy management. However, the company size and energy intensity are two factors that can be considered to categorize the industries to apply or analyze the model [44]. The main features proposed by Christoﬀeren et al. align with ISO 50001: 2011 standard though this model has been replaced by ISO 50001: 2018 [58]. The earlier model encompassed energy management system implementation based on PDCA cycle and enlisted few prerequisites that include mainly management liability, policy, energy audit, energy performance indexing, energy management blueprint, documentation, and so forth. One of the major changes in the recent model is the PDCA cycle modiﬁcation. “Checking” was the center in the earlier version, whilst “Leadership” became the focus of all cycle components. Figure 3 represents the revised PDCA cycle of ISO 50001:2018. In the minimum requirement segment, the model proposed by Ates et al. comprehended conventional streams towards energy management. One of the signiﬁcant features is the inclusion of energy manager, whilst ISO 40001 (environmental permit) also act as an enabling feature along with ISO 50001 [40]. energy management system, ISO 50001, and PDCA cycle, while some studies suggested holistic approaches towards industrial energy efficiency. The framework proposed by Christoffersen et al. was stood out on the Danish industries and emphasized on multiple factors, mostly energy policy, goals and capstone projects aimed at energy savings. Regulation, external relations, company characteristics, and organizational internal condition are the main out-layers of the model to frame the energy management. However, the company size and energy intensity are two factors that can be considered to categorize the industries to apply or analyze the model [44]. Model Narration er is attributed by gap among core cter “Information context” planning and execution nd ﬁeld wise One of the salient features of this framework is inclusion of non-energy beneﬁts. Energy manager is not integrated into the framework. [58] payback time, application ce stream and energy n is attributed by waste n. Production is attributed , and operation and attributes are energy mentation easiness, success corporate level, distance rity. Interaction-related ct eﬀects. Corporate involvement is one of the notable attributes and considered as signiﬁcant for industrial decision-makers. The need for analyzing energy eﬃciency measures as per diﬀerent perspectives is highlighted; precisely having the aspects in grouped for providing more inclusive view on the pertinent outlooks distinguishing the energy eﬃciency measures. [59] Remark Model Narration Category Model exploring the succeeding features considered as energy management practice: energy director appointment, incorporation of energy team, apply of energy policy; collection of information and management, establishment of yardstick or threshold, analysis, assessing from technical perspective and energy audits; exploring and setting the scope, improvement option estimation, goal setting; deﬁne technical procedures and targets, roles and resources determination; formation of a communication plan, awareness raising, capacity building, inspire, trail and monitor; measurement of result, recapitulation of action plan; maintain internal recognition, and receiving external appreciation. Three main characteristics are considered. Each characteristic are divided into sub-divisions. The ﬁrst character “Relative advantage” is attributed by internal rate of return, introductory expenses, reimbursement time, and beneﬁts of non-energy. “Technical context” the second character is attributed by modiﬁcation type, impact opportunity, gap among core processes, and Lifetime. The last character “Information context” is attributed by transaction expenses, planning and execution knowledge, Dissemination progress, and ﬁeld wise applicableness. Economic characterization consists of payback time, application costs. Energy is attributed with resource stream and energy saving. Environmental characterization is attributed by waste minimization and emission contraction. Production is attributed by productivity, working environment, and operation and maintenance. Implementation related attributes are energy saving strategy, types of action, implementation easiness, success probability, community engagement in corporate level, distance among key processes, and audit regularity. Interaction-related characterization is attributed by indirect eﬀects. 13 of 21 Energies 2020, 13, 5713 13 of 21 4. Discussion Energies 20 In addition, there is very 14 of 21 Energies 2020, 13, 5713 little contribution on the risk management and opportunities of energy eﬃciency from an integral and strategic point of view, including the planning and control of product lines, process design, projects, and business approaches [69]. Notably, the fruitful operation of the energy management system requires the integrated deployment of planned, tactical, and operational levels that align the business culture with sustainable attainment. In this context, the vision that the organization plans to form should be linked to energy eﬃciency strategy with organization’s survival plan in the market. Additionally, it is necessary to make explicit reference to newly adapted technical features through peer to peer energy management platform for optimizing the integration of energy management system component with the variable energy demand [70,71]. Moreover, an integrated perspective to control of operational features of each process are required to explore linked to energy eﬃciency [69]. little contribution on the risk management and opportunities of energy eﬃciency from an integral and strategic point of view, including the planning and control of product lines, process design, projects, and business approaches [69]. Notably, the fruitful operation of the energy management system requires the integrated deployment of planned, tactical, and operational levels that align the business culture with sustainable attainment. In this context, the vision that the organization plans to form should be linked to energy eﬃciency strategy with organization’s survival plan in the market. Additionally, it is necessary to make explicit reference to newly adapted technical features through peer to peer energy management platform for optimizing the integration of energy management system component with the variable energy demand [70,71]. Moreover, an integrated perspective to control of operational features of each process are required to explore linked to energy eﬃciency [69]. In the energy management maturity model segment, the model proposed by Ngai et al. based on capability maturity model integration (CMMI), an extension of capability maturity model incorporated five levels according to the behavioral exhibition of the industries [54]. The levels are determined by performance area of key processes [72]. The achievement goals of key process areas must be specified for individual level for further actions. Notably, the propositions of CMM framework has been applied at multiple process enhancement programs in order to achieve the desired quality in the production system [73]. 4. Discussion Energies 20 One limitation of this model is inadequate implementation time, having only one factory for consideration. However, the authors have affirmed the acceptability of the model because of prior implementation of management practices. On the contrary, Antunes et al. emphasized the PDCA cycle to design the energy management framework [64]. Additionally, the authors implied the model with ISO 50001 and incorporated energy management practices also. Notable to mention that Finnerty et al. also designed the framework based on the PDCA cycle, keeping on focus on energy management practices [66]. The model proposed by Introna et al. is comprised of ﬁve dimensions and enables the feature of self-evaluation for the industries towards energy management practices. The dimensions are characterized by identifying the necessary elements in energy management consumption segment of the industries [8]. On the contrary, Jovanovi´c et al. focused on ISO 50001 processes as well as PDCA phases, keeping the knowledge base in the model EMMM50001 [53]. The EMMM50001 establishes the relation to EUMMM maturity levels, maintaining ISO 50001 speciﬁcations and PDCA phases. Notably to mention that EMMM50001 links the CMMI criteria, also maintaining the ISO 50001. It can be observed that the majority of the maturity models emphasized on similar type of characteristics and areas to evaluate the energy management in an organization by a systematic set of commendations. However, the narrated models demand more time and resources to perform as per their characterization. In addition, looking at the scientiﬁc literature, all of the frameworks studied to see the requirements for providing a continuous development path following the PDCA approach and ISO 50001. Notably, few of the maturity models incorporate the implication of dedicated energy manager and top management support. In contrast, Antunes et al. [64] aﬃrm on top management support whilst not integrating the energy manager in the framework. The framework by Introna et al. [8] also did not complied with the energy manager. Nonetheless, Jovanovi´c and Filipovi´c [53] and Finnerty et al. [66] considered top management support along with the energy manager in their framework. Gordic’ et al. applied the energy management matrixes model in the Serbian car manufacturer industries and critically analyzed the existing energy management system with the model [55]. 4. Discussion Energies 20 The main features proposed by Christofferen et al. align with ISO 50001: 2011 standard though this model has been replaced by ISO 50001: 2018 [58]. The earlier model encompassed energy management system implementation based on PDCA cycle and enlisted few prerequisites that include mainly management liability, policy, energy audit, energy performance indexing, energy management blueprint, documentation, and so forth. One of the major changes in the recent model is the PDCA cycle modification. “Checking” was the center in the earlier version, whilst “Leadership” became the focus of all cycle components. Figure 3 represents the revised PDCA cycle of ISO 50001:2018. In the minimum requirement segment, the model proposed by Ates et al. comprehended conventional streams towards energy management. One of the significant features is the inclusion of energy manager, whilst ISO 40001 (environmental permit) also act as an enabling feature along with ISO 50001 [40]. Figure 3. The “Plan-Do-Check-Act” cycle adopted in ISO 50001: 2018 (Source: [45]). Figure 3. The “Plan-Do-Check-Act” cycle adopted in ISO 50001: 2018 (Source: [45]). Figure 3 The “Plan-Do-Check-Act” cycle adopted in ISO 50001: 2018 (Source: [45]) Figure 3. The “Plan-Do-Check-Act” cycle adopted in ISO 50001: 2018 (Source: [45]). Looking at the minimum requirement focused model, it is observed that all the energy management initiatives are not integrated into the frameworks. Christoffersen et al. [44] considered energy management as a comprehensive management system. However, the model does not integrate the energy manager concept. Furthermore, there is no clear guideline about top or mid-level management support to achieve energy savings. Though, the involvement of employee to energy- Looking at the minimum requirement focused model, it is observed that all the energy management initiatives are not integrated into the frameworks. Christoﬀersen et al. [44] considered energy management as a comprehensive management system. However, the model does not integrate the energy manager concept. Furthermore, there is no clear guideline about top or mid-level management support to achieve energy savings. Though, the involvement of employee to energy-saving related works are suggested. Nonetheless, The ISO 50001 model is a signiﬁcant protocol [69] along with the proposition by Ates and Durakbasa [40], manifold aspects are still to be explored regards of operational activities in the industrial energy management domain. For instance, the principles of sustainability and integral management need to be presented at the protocol. 4. Discussion Energies 20 Notably to mention that the energy management matrixes models proposed by Gordic’ et al., Carbon Trust and Energy Star encompass all key areas to assess the energy management practices in the model, with having few modifications at the individual version. On the contrary, Fleiter et al. [58] and Trianni et al. [52] emphasized on a characterization based model where both of the models are incorporated with speciﬁc attributes. The characterization scheme has some implications on policy design and assessment. However, formalization of the groups with categorized attributes enables the option towards relevant aspects identifying the energy eﬃciency measures. In addition, Trianni et al. contend a comprehensive scenario on EEMs focusing on the relevant aspects of industrial energy management [52]. One of the critical factors, “corporate involvement” 15 of 21 Energies 2020, 13, 5713 for industrial decision-makers is also implied, hence allowing additional feature and an increase in the applicability of the model. In another proposed framework, Trianni et al. incorporated energy management practice-based approach. However, the authors acknowledge more compatible space for the SMEs within the model, as SMEs are sought to be entitled to more attention, considering their cumulative energy consumption percentile [74]. In a recent study, Tina et al. persuade the signiﬁcance of SMEs and the policy implications in the peripheral of the industrial energy sector [74]. Referring to the SMEs, Prashar [67] proposes an energy eﬃciency maturity assessment framework that emphasizes SMEs. Notably, the author argues that the common energy eﬃciency framework approach does not facilitate fully to the SMEs; hence, a customized maturity framework is signiﬁcant. The author considered steel re-rolling mill sector of India as the contextual sphere for the proposed framework. Few of the studies on characterization the energy efficiency measure focuses on financial features. Notably to mention that these models do not frame the energy efficiency measures comprehensively, rather offer some framework without characterizing the energy efficiency measures in-depth. In one of the studies by Pye and McKane, they state that quantification of the accumulated benefits of energy efficiency scheme supports the enterprises perceive the monetary opportunities of EEMs financing [5]. The energy savings features act not as the singular primary driver for the industrial decision process; hence, the authors persuade that energy savings be viewed as a factor of the holistic approach towards energy efficiency programs. 4. Discussion Energies 20 Skumatz studied the methods to find out the attributes of EEMs and established the scheme to measure both of the positive and negative secondary benefits stemming from industrial energy efficiency schemes [75,76]. On the contrary, Mills and Rosenfeld provided a framework to understand multiple benefits of energy efficiency initiatives and grouped the attributes into the better interior environment, noise lessening, savings of labor and time, improved supervision of procedure, convenience, water savings and waste reduction, and benefits due to downsizing of equipment [77]. The majority of studies on energy eﬃciency measures, beneﬁts, and initial characterization frameworks propose few interesting reﬂections. However, a lack of consistency on the attributes grouping within existing categories from the methodological perspective is observed. It is found that the same attributes are grouped in diﬀerent categories by diﬀerent researchers. Moreover, the attributes are categorized and then aggregated again within other segments by diﬀerent researcher. For instance, “reduced noise” and “improved indoor environment” are framed in two diﬀerent categories in [77], whereas “reduced noise level” as categorized in “working environment” segment. On the other note, the decision-making process is a grey area keeping mind about the stakeholders. Nonetheless, the earlier characterization framework did not incorporate the energy eﬃciency measure implications in a comprehensive way. To be precise, the inclusion of non-energy beneﬁts is not incorporated into the characterization framework. Notably, the inclusion of non-energy features in the modeling factors would double the cost-eﬀective potential for energy-eﬃciency enhancement, likened to an analysis eliminating those beneﬁt [60]. However, few attributes (e.g., improved air quality, better worker safety, reduction of noise level, and improved working situation) are there in the characterization framework, which are diﬃcult to quantify [76]. Therefore, speculation is required to articulate the beneﬁts into a comparable cost ﬁgure, and hence the assessment turns out to be rather subjective [60]. The study by Ngai et al. [54] features energy management with particular process areas in the manufacturing industries. In this study, few guidelines are oﬀered to conduct analysis for organizational maturity improvement in terms of energy along with the management of utility resources. However, the integration of energy management into production process has not been complied comprehensively. This is a signiﬁcant avenue that needs be to address with utmost attention in future studies considering the technical implications oﬀered by Industry 4.0. 5. Concluding Remarks and Future Research Avenues The paper attributes a review of research works on the energy management model for energy management practices in the industries. Multiple models have been compiled and structured, maintaining the narrations. Moreover, the energy management frameworks were synthesized emanate from the ﬁndings in order to facilitate energy management in the industries by oﬀering necessary benchmarks to the industrial experts. The review ﬁndings show that the narrated models can support an organization to assess energy management and incorporate insightful contribution to energy eﬃciency initiatives. Nonetheless, some of the studies did not comply with a full methodological description and exhibited shorter model validation. In addition, a gap exists between the theoretical concept and practical implementation of energy management and its practices. Precisely, majority approaches remain unsuccessful in replicating and scope of actions distinct in energy management due to the certain barriers [27,66,85]. Moreover, most of the models have looked the energy management as a single unit function, whilst it is a combination of multi-dimensional approaches with the involvement of several functional units in the industries. Let us not forget about the multi-dimensional operational activities in the industries which are conjugal part with energy management. Notably, multi-dimensional approaches are critical to support the process and operational oriented program [86]. Therefore, a comprehensive operational approach should be considered by integrating all the relevant energy flows. It infers to all forms of energy, including externally supplied energy sources as well as internal energy flows. Interestingly, relating the energy management into the operational framework integrates the resource efficiency also at the manufacturing level. The raw or auxiliary material consumption might be of interest, considering the direct and indirect impact on energy and resource efficiency in the manufacturing process. Moreover, keeping mind about the non-static nature of energy consumption, the dynamic consumption feature might unveil manifold resource optimization aspects [87]. Unfortunately, the integration of energy management into operational activities have been little explored. It becomes even more imperative while we look to adopt Industry 4.0 keeping in mind about the manifold complex technical features consist of Internet of Things (IoT), big data, cloud computing, and so on in the industrial plants. Many scholars predict that the exponential progress in the promises of manifold technical features oﬀered by Industry 4.0 might aﬀect the production activities in the industries inclusively. 4. Discussion Energies 20 Notable to mention here, is that increasing the eﬃciency at the production processes is one of the salient features of Industry 4.0 [78]. The deployment of smart machinery oﬀers diverse beneﬁts which primarily includes manufacturing productivity and waste reduction [79]. Therefore, it is worth observing the energy management characteristics linked with production process through the lens of Industry 4.0. Energies 2020, 13, 5713 16 of 21 16 of 21 Nonetheless, energy management towards industrial energy eﬃciency has been widely discussed in academia, and several barriers are still persistent in the energy management domain. The identiﬁcation of barriers is important because it hampers or slows down the adoption of energy eﬃciency measures [80]. Several studies have addressed the barriers which cover energy-intensive industries to SMEs and include regional, national, and transnational perspectives [15,26,27,81–84]. However, most comprehensive studies focusing on energy management have been discussed without really looking at the integration of energy management into production and operation management. An imperative avenue, therefore, lies to be further explored in future within this research domain. 5. Concluding Remarks and Future Research Avenues In addition, there are high chances of modiﬁcation in the traditional industrial actions that cover the processing of elements and material, grinding, and assemble/ dismantle. This is emphasized in Industry 4.0 concept and its implementation, where we pursue to pool the common features with the enormous potential of digital technology [88]. However, it is understood as a necessary incremental approach aimed at further optimizing the energy system without seeking to disrupt it in principle. In the energy eﬃciency domain, the energy management and its practices have already inﬂuenced the production scenario in a broader aspect, and this inclination should remain as long as we allow the nexus between Industry 4.0 and energy eﬃciency. On the other hand, energy productivity investments in present as well as the recent technologies must be conveyed through the implementation of energy management and its practices [89]. Energy management practices and energy services are acknowledged as fundamental solutions; the diminutive eﬀort is being paid in characterizing them [24]. Notably, assessment models for supporting the industrial decision-makers Energies 2020, 13, 5713 17 of 21 emphasizing detailed activities for better energy management is deﬁcient. Therefore, it is imperative to consider the energy management in multiple aspects keeping mind about the complex nature of industrial energy system [31]. Interestingly, energy management has implications on asset maintenance, e.g., on maintenance procedures. As energy management includes the control of energy-consuming devices to optimize energy consumption, manual toggling on and oﬀdevices depending on requirement is a rudimentary custom of energy management. The initiation of mechanical and electrical equipment (e.g., timers for programmed toggling, bimetallic strip thermostat, pneumatic and electrical transmission system, and so on) provided means for early energy management schemes in the form of automatic temperature control. Nowadays, the inclusion of direct digital control in energy management has retroﬁt beneﬁts that allow device monitoring linked to maintenance procedure, thanks to energy management and its practices. The comprehensive data recommend that while energy management does improve the accuracy and response of a system in the industries, the energy management routines facilitate partially asset maintenance [90]. It infers to monitoring or log building equipment performance while consuming the energy resulting increasing magnitude of all beneﬁts covering maintenance and cost avoidance beneﬁt. Unfortunately, much of the energy management studies have bypassed this retroﬁt fact while focusing on the energy management framework. Author Contributions: Conceptualization, A.S.M.M.H. and A.T.; methodology, A.S.M.M.H. and A.T.; formal analysis, A.S.M.M.H. and A.T.; investigation, A.S.M.M.H. and A.T.; writing—original draft preparation, A.S.M.M.H. and A.T.; writing—review and editing, A.S.M.M.H. and A.T.; visualization, A.T.; supervision, A.T. All authors have read and agreed to the published version of the manuscript. References 2020, 248, 119263. [CrossRef] 8. Introna, V.; Cesarotti, V.; Benedetti, M.; Biagiotti, S.; Rotunno, R. Energy Management Maturity Model: An organizational tool to foster the continuous reduction of energy consumption in companies. J. Clean. Prod. 2014, 83, 108–117. 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In addition, the narrated models have little explored the sustainability feature integrated with energy eﬃciency, pointing to the optimization of resource utilization [91]. We must consider the paradigm that allows industrial energy management eﬀective for the companies. In this context, it would be certainly interesting to visualize the energy management through Industry 4.0 technologies and solutions, may contribute to improved sustainability performances of the companies. If Industry 4.0 is expected to unveil enormous directions not only to energy management but also the sustainability ﬁeld, the challenge deﬁnitely lies on the integrational aspects with energy–industry–sustainability nexus. Therefore, the future research avenues should reﬂect the energy management framework complying the diverse directions and encompassing the operational management, Industry 4.0 along with sustainability features. Author Contributions: Conceptualization, A.S.M.M.H. and A.T.; methodology, A.S.M.M.H. and A.T.; formal analysis, A.S.M.M.H. and A.T.; investigation, A.S.M.M.H. and A.T.; writing—original draft preparation, A.S.M.M.H. and A.T.; writing—review and editing, A.S.M.M.H. and A.T.; visualization, A.T.; supervision, A.T. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2956803025	http://ejournal.iba.ac.id/index.php/jemasi/article/download/11/11	Indonesian	null	ANALISIS UKURAN PERUSAHAAN DAN PERTUMBUHAN ASET TERHADAP STRUKTUR MODAL PERBANKAN UMUM PEMERINTAH DI BURSA EFEK INDONESIA	Jemasi	2,017	cc-by-sa	4,332	JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 ABSTRACK This study aims to determine the effect of firms and asset growth on capital structure government general banking in Indonesia Stock Exchange. The problem in this research how to influence firm size and asset growth on capital structure government general banking in BEI? Data analysis, autocorrelation, multicolinearity and heterocedasticity test. The population is the entire company government general banking in BEI as many as 4 companies, with the selected sample of 4 companies through the sampling census. The results showed firm size and asset growth have influence on capital structure government general banking in Indonesia Stock Exchange (BEI). Keyword: Firm Size, Asset Growth, Capital Structure ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh ukuran perusahaan dan pertumbuhan aset terhadap struktur modal perbankan umum pemerintah di Bursa Efek Indonesia. Permasalahan dalam penelitian ini bagaimana pengaruh ukuran perusahaan dan pertumbuhan aset terhadap struktur modal perbankan umum pemerintah di BEI? Model analisis data dengan multiple regresion melalui uji asumsi klasik yaitu uji normalitas data, autokorelasi, multikolinearitas dan uji heterokedastisitas. Populasi yaitu seluruh perusahaan perbankan umum pemerintah di BEI sebanyak 4 perusahaan, dengan sampel terpilih sebanyak 4 perusahaan melalui sensussampling. Hasil penelitian menunjukkan bahwa ukuran perusahaan dan pertumbuhan aset mempunyai pengaruh terhadap struktur modal perbankan umum pemerintah di Bursa Efek Indonesia (BEI). Oleh: Emma Lilianti Fakultas Ekonomi Universitas PGRI Jalan Jenderal Ahmad Yani Lorong Gotong Royong Palembang Oleh: Emma Lilianti Fakultas Ekonomi Universitas PGRI Jalan Jenderal Ahmad Yani Lorong Gotong Royong Palembang PENDAHULUAN Keberadaan sebuah perusahaan tidak hanya melakukan kegiataan yang sifatnya individual. Perusahaan perlu terus mengembangkan bisnisnya dari segi jenis produk dan besarnya skala perusahaan. Untuk itu perusahaanharus mampu bersaing dalam dunia usaha,misalnya dengan membuat inovasi barupadaproduknyasehingga meningkatkan tingkat penjualan. Perusahaan perlu melakukan perluasan usaha, meningkatkan kualitas 182 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 sumber daya manusia, dan lainnya. Untuk melakukan semua itu dibutuhkan dana yang sangat besar. Pemenuhan kebutuhan dana dapat dilakukan dengan pendanaan internal maupun eksternal. Sumber dana internal, yaitu sumber dana yang dibentuk atau dihasilkan sendiri di dalam perusahaan, misalnya dana yang berasal dari keuntungan yang tidak dibagikan atau laba yang ditahan didalam perusahaan. Makin besar dana intern yang berasal dari laba ditahan akan semakin memperkuat posisi keuangan perusahaan dalam menghadapi kesulitan keuangan diwaktu-waktu mendatang. Laba ditahan ini dapat digunakan oleh perusahaan sebagai cadangan untuk menghadapi kerugian yang timbul dimasa datang, untuk melunasi hutang perusahaan, untuk menambah modal kerja atau pun untuk ekspansi perusahaan di masa datang. Selain sumber dana dari internal, sumber dana yang lain adalah sumber eksternal, yaitu sumber dana yang berasal dari tambahan penyertaan modal pemilik atau penerbitan saham baru, penjualan obligasi, dan kredit dari bank. Penetapan sumber dana dianggap penting karena masing-masing sumber dana tersebut memiliki tingkat risiko yang berbeda-beda. Untuk itu manajer keuangan diharapkan mampu menerapkan pemilihan alternatif sumber dana yang paling tepat. Dalam hal ini, perusahaan perlu mempertimbangkan apakah dananya dipenuhi dari modal sendiri, hutang, atau kombinasi keduanya. Penggunaan dana yang berasal dari hutang maupun ekuitas akan membantu perusahaan dalam pengembangan perusahaan. Bank merupakan lembaga perantara keuangan dimana rata-rata orang sering berinteraksi,Mishkin (2012:9). Salah satu hal yang penting yang dihadapi manajer keuangan adalah bagaimana seharusnya perusahaan mendapatkan dana untuk aktiva, apakah meminjam dengan menggunakanhutang atau menjual saham, Atmajaya (2014 :8). Bank juga merupakan pengumpul dana dan penyalur kredit berarti bank dalam operasinya mengumpulkan dana kepada SSU dan menyalurkan kredit kepada DSU, Hasibuan (2013:2). Tuntutan terhadap perusahaan/industri sektor perbankan semakin besar dan luas, perusahaan/perbankan diharapkan tidak hanya mementingkan kepentingan manajemen dan pemilik modal (investor dan kreditor) tetapi juga karyawan, konsumen serta masyarakat. Perusahaan mempunyai tanggung jawab sosial terhadap pihak-pihak di luar manajemen dan pemilik modal. Terkadang perusahaan melalaikannya dengan alasan 183 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 bahwa mereka tidak memberikan kontribusi terhadap kelangsungan hidup perusahaan. Bank adalah pengumpul dana dan penyalur kredit berarti bank dalam operasinyamengumpulkan dana kepada SSU dan menyalurkan kredit kepada DSU, Hasibuan (2012:2). Penelitian Seftianne dan R. PENDAHULUAN Handayani berjudul faktor-faktor yang mempengaruh istruktur modal pada perusahaan publik sector manufaktur menemukan bahwa ukuran perusahaan dan pertumbuhan perusahaan berpengaruh signifikan terhadap faktor modal. Yovin, D. dkk. menyatakan pertumbuhan aset mempengaruhi Struktur Modalpada Perusahaan Foodsand Beverages yang Terdaftar di Bursa Efek Indonesia, sedangkan ukuran perusahaan tidak berpengaruh signifikan. Selanjutnya penelitian Manopo dkk mengemukakan ukuran perusahaan dan pertumbuhan aset mempengaruhi Struktur Modal Perbankan yang Go Publik di BEI. Berdasarkan latar belakang penelitian di atas, rumusan masalah dalam penelitian adalah bagaimana pengaruh ukuran perusahaan dan pertumbuhan aset terhadap struktur modal Bank Umum Pemerintah di Bursa Efek Indonesia? Pengertian Struktur Modal Copeland dan Weston (2010) mengungkapkan keputusan untuk memilih sumber pembiayaan merupakan keputusan bidang keuangan yang paling penting bagi perusahaan. Sumber pembiayaanatau sumber pedanaan suatu perusahaan dapat dilihat di sisi pasiva dari neraca perusahaan sedangkanpenggunaan dana dapat di lihat pada sisi aktiva dari neraca perusahaan. Aset yang dimilikiperusahaan menunjukkan penggunaan bersih dari dana sedangkan hutang dan modal sendirimencerminkan sumber dananya (Husnan, 2011). Struktur modal adalah komposisi ekuitas dan pinjaman dalam pembiayaan proyek. Komposisiekuitas dan pembiayaan proyek akan menghasilkan biaya modal rata-rata yang berbeda apabilakomposisinya berbeda. Untuk itu, pembahasan tentang struktru modal mnenjadi penting karena tidaksaja menyangkut biaya modal yang harus dibayar kembali oleh investor, tetapi menjadi barometerkeuntungan. Pembahasan mengenai teori struktur modal ini banyak dilakukan oleh Modiglani danMiller dalam kaitan rencana titik optmila dari komposisi ekuitas dan pinjaman yang akan 184 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 menghasilkan keuntungan kamsimal. Keuntungan maksimal disini diasumsikan pada tingkat IRR konstan, Yusgiantoro (2010: 145) Faktor-Faktor yang Mempengaruhi Struktur Modal Copeland dan Weston (2010) mengungkapkan penentuan struktur modal perlu mempertimbangan hal-hal yaitu, tujuan perusahaan, tingkat leverage, kemampuan dana intern, pemusatan pemilikan dan pengendalian suara, batas kredit, besarnya perusahaan, pertumbuhan aset perusahaan, stabilitasearnings, biaya modal sendiri, biaya hutang, tarif pajak, perkiraan tingkat inflasi dan kemampuan dana. Faktor-faktor yang berpengaruh dalam pengambilan keputusan struktur modal adalah: 1. Stabilitas penjualan. Perusahaan dengan penjualan yang relatif stabil dapat lebih aman memperoleh lebih banyak pinjaman dan menanggung beban tetap yang lebih tinggi dibandingkan dengan perusahaan yang penjualannnya tidak stabil. 1. Stabilitas penjualan. Perusahaan dengan penjualan yang relatif stabil dapat lebih aman memperoleh lebih banyak pinjaman dan menanggung beban tetap yang lebih tinggi dibandingkan dengan perusahaan yang penjualannnya tidak stabil. 2. Struktur aset. Perusahaan yang asetnya sesuai untuk dijadikan jaminan kredit cenderung lebih banyak menggunakan banyak utang. Aset multiguna yang dapat digunakan oleh banyak perusahaan merupakan jaminan yang baik, sedangkan aset yang hanya dapat digunakan untuk tujuan tertentu tidak begitu baik untuk dijadikan jaminan. 3. Leverage operasi. Jika hal-hal lain tetap sama, perusahaan dengan leverage operasi yang lebih kecil cenderung lebih mampu untuk memperbesar leverage keuangan karena ia akan mempunyai risiko bisnis yang lebih kecil. 4. Tingkat pertumbuhan. Jika hal-hal lain tetap sama, perusahaan yang tumbuh dengan pesat harus lebih banyak menggunakan modal eksternal. Lebih jauh lagi, biaya pengambangan untuk penjualan saham biasa jauh lebih besar daripada biaya untuk penerbitan surat utang, yang mendorong perusahaan untuk lebih banyak mengandalkan utang. Namun, pada saat yang sama perusahaan yang tumbuh dengan pesat sering menghadapi ketidakpastian yang lebih besar, yang cenderung mengurangi keinginannya untuk menggunakan utang. 5. Profitabilitas. Seringkali pengamatan menunjukan bahwa perusahaan dengan tingkat pengembalian yang tinggi atas investasi menggunakan utang yang relatif kecil. 6. Pajak. Bunga merupakan beban yang dapat dikurangkan untuk tujuan perpajakan, dan pengurangan tersebut sangat bernilai bagi perusahaanyang terkena tarif pajak yang tinggi. Karena itu makin tinggi tarif pajak perusahaan, makin besar manfaat penggunaan utang. 7. Pengendalian. Pengaruh utang lawan saham terhadap posisi pengendalian manajemen dapat mempengaruhi struktur modal. Apabila manajemen saat ini mempunyai hak untuk mengendalikan perusaahaan tetapi sama sekali tidak diperkenankan untuk membeli saham tambahan, mereka mungkin akan memilih utang untuk pembiayaan baru. Di lain pihak, manajemen memutuskan untuk menggunakan ekuitas jika kondisi keuangan perusahaan sangat lemah sehingga penggunaan utang dapat membawa perusahaan pada risiko kebangkrutan, karena jika perusahaan jatuh bangkrut, para 185 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 manajer tersebut akan kehilangan pekerjaan. Faktor-Faktor yang Mempengaruhi Struktur Modal Tetapi, jika utangnya terlalu kecil mereka akan menghadapi risiko pengambilalihan. Teori Struktur Modal Agency Theory, teoriini dikemukakan oleh Michael C. Jensen dan William H. Meckling pada tahun 1976. Jensen dan William (dalam Horne dan Wachowicz, 2007:482) berpendapat bahwa manajemen merupakan agen dari pemegang saham, sebagai milik perusahaan. Para pemegang saham berharap agen bertindak atas kepentingan mereka sehingga mendelegasikan wewenang kepada agen. Telah diketahui bahwa para manajer mungkin memiliki tujuan pribadi yang bersaing dengan tujuan memaksimalkan kekayaan pemegang saham. Manajer diberi kekuasaan oleh para pemilik perusahaan, yaitu pemegang saham untuk membuat keputusan dimana hal ini menciptakan potensi konflik kepentingan yang dikenal sebagai teoriagensi (Brigham dan Houston, 2011:26). Menurut pendekatan ini, struktur modal disusun sedemikian rupa untuk mengurangi konflik antar berbagai kelompok kepentingan (Mamduh, 2015:91). Penyebab konflik antara manajer dengan pemegang saham berkaitan dengan aktivitas pencarian dana dan bagaimana dana tersebut diinvestasikan. Menurut teori ini, semakin tidak stabil pendapatan perusahaan dan semakin besarnya profitabilitas mengalami kebangkrutan, maka masalah yang berhubungan dengan hutang semakin besar. Trade-off Theory, teori ini mengasumsikan bahwa struktur modal perusahaan merupakan hasil trade-off dari keuntungan pajak dengan menggunakan hutang dengan biaya yang akan timbul sebagai akibat penggunaan hutang tersebut (Sartono,2010). Esensitrade- off theory dalam struktur modal adalah menyeimbangkan manfaat dan pengorbanan yang timbul sebagai akibat penggunaan hutang. Sejauh manfaat lebih besar, tambahan hutang masih diperkenankan. Apabila pengorbanan karena penggunaan hutang sudah lebih besar, maka tambahan hutang sudah tidak diperbolehkan. Teori ini menjelaskan sebuah perusahaan yang struktur modalnya tanpa menggunakan hutang dengan keseluruhan menggunakan hutang adalah perusahaan yang dalam keadaan buruk. Perusahaan tanpa menggunakan hutang dalam modalnya akan membayar pajak yang lebih besar daripada perusahaan yang menggunakan hutang. Hal tersebut akan mempengaruhi nilai perusahaan. Nilai perusahaan dengan menggunakan hutang akan lebih besar dibanding perusahaan tanpa menyertakan hutang dalam modalnya. 186 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 Perusahaan dengan modal keseluruhan hutang dikatakan buruk karena dalam setiap hutang terdapat bunga hutang yang dibayarnya. Dengan keseluruhan hutang dalam modal perusahaan, dalam setiap keuntungan perusahaan tersebut akan menggunakan labanya untuk membayar bunga. Tentu keadaan tersebut tidak menguntungkan bagi sebuah perusahaan. Perusahaan dapat melakukan perhitungan mengenai struktur modal optimal dengan mempertimbangkan peningkatan nilai perusahaan dan biaya yang akan muncul. Pecking Order Theory, teori ini disebut sebagai pecking order karena teori ini menjelaskan mengapa perusahaan akan menentukan hierarki sumber dana yang paling disukai. Komponen Struktur Modal Komponen yang menjadi penyusunan dalam komposisistrukturmodalterdiridari hutang jangka panjang, saham biasa, saham preferen dan laba ditahan. Liabilitas tidak lancer adalah k e w a j i b a n y a n g j a n g k a waktunya adalah panjang umumnya lebih dari sepuluh tahun (Sartono,2010:238). Hutang jangka panjang ini pada umumnya digunakan untuk membiayai perluasan perusahaan (ekspansi) atau modernisasi dari perusahaan. Komponen-komponen hutang jangka panjang ini terdiri dari: (a) Hutang hipotik adalah bentuk hutang jangka panjang yang dijamind engan aktiva tidak bergerak (tanah dan bangunan), (b) Obligasi adalah sertifikat yang menunjukan pengakuan bahwa perusahaan meminjam uang dan menyetujui untuk membayarnya kembali dalam jangka waktu tertentu. Pelunasan atau pembayaran kembali obligasi dapat diambil dari penyusutan aktiva tetap yang dibelanjai dengan pinjaman obligasi tersebut dan dari keuntungan. Teori Struktur Modal Secara ringkas teori tersebut menyatakan bahwa (Brealey dan Myers dalam Husnan, (2011:3): (a) Perusahaan menyukai internal financing (pendanaan dari hasil operasi perusahaan), (b) Perusahaan mencoba menyesuaikan rasio pembagian dividen yang ditargetkan, dengan berusaha menghindari perubahan pembayaran dividen secara drastis, (c) Kebijakan dividen yang relatif segan untuk diubah, disertai dengan fluktuasi profitabilitas dan kesempatan investasi yang tidak bias diduga, mengakibatkan bahwa dana untuk investasi, meskipun pada kesempatan yang lain, mungkin kurang. Apabila dana hasil operasi kurang dari kebutuhan investasi, maka perusahaan akan mengurangi saldo kas untuk menjual sekuritas yang dimiliki, dan (d) Apabila pendanaan dari luar (eksternal financing) diperlukan, maka perusahaan akan menerbitkan sekuritas yang paling aman terlebih dahulu yaitu dimulai dengan penerbitan obligasi, kemudian diikuti oleh sekuritas, baru akhirnya apabila masih belum mencukupi, saham baru diterbitkan. AsymmetricInformation Theory atau ketidaksamaan informasi menurut Brigham dan Houston (2011:35), adalah situasi dimana manajer memiliki informasi yang berbeda (yang lebih baik) mengenai prospek perusahaan dari pada yang dimiliki investor. Keadaan ini memungkinkan para manajer menggunakan informasi yang diketahuinya untuk mengambil keputusan, khususnya keputusan pendanaan perusahaan. Dalam asymmetric information theory, menjelaskan bahwa ukuran perusahaan berpengaruh terhadap lebih tertutup atau terbukanya perusahaan untuk membagi informasi kepada pihak luar. Perusahaan kecil menganggap bahwa membagi informasi kepada pihak pemberi pinjaman atau modal membutuhkan biaya yang besar. Hal ini 187 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 menghambat penggunaan pendana an eksternal dan meningkatkan kecenderungan bagi perusahaan kecil untuk menggunakan modal perusahaan. SignalingTheory, menurut Brigham dan Houston (2011:39), signal atau isyarat adalah suatu tindakan yang diambil manajemen perusahaan yang memberi petunjuk bagi investor tentang bagaimana manajemen memandang prospek perusahaan. Perusahaan dengan prospek yang menguntungkan akan mencoba menghindari penjualan saham, termasuk penggunaan hutang yang melebihi target struktur modal yang normal. Perusahaan dengan prospek yang kurang menguntungkan akan cenderung untuk menjual sahamnya. Pengumuman emisi saham oleh suatu perusahaan umumnya merupakan suatu isyarat (signal) bahwa manajemen memandang prospek perusahaan tersebut suram. Apabila suatu perusahaan menawarkan penjualan saham baru, lebih sering dari biasanya, maka harga saham akan menurun, karena menerbitkan saham baru berarti memberi isyarat negative yang kemudian dapat menekan harga saham sekalipun prospek perusahaan cerah. Berdasarkan rumusan masalah di atas, maka kerangka pemikiran dalam penelitian: Hipotesis Penelitian Hipotesis Penelitian Berdasarkan kerangka pemikiran diatas, peneliti merumuskan hipotesis sebagai berikut : H1 : Ukuran perusahaan berpengaruh positif terhadap struktur modal pada Bank Umum Pemerintah di BEI. H2 :Pertumbuhan Asetberpengaruh positif terhadap struktur modal pada Bank Umum Pemerintah di BEI. H3 : Ukuran perusahaan dan pertumbuhan aset terhadap struktur modal pada Bank Umum Pemerintah di BEI. p Berdasarkan kerangka pemikiran diatas, peneliti merumuskan hipotesis sebagai berikut : H1 : Ukuran perusahaan berpengaruh positif terhadap struktur modal pada Bank Umum Pemerintah di BEI. H2 :Pertumbuhan Asetberpengaruh positif terhadap struktur modal pada Bank Umum Pemerintah di BEI. H3 : Ukuran perusahaan dan pertumbuhan aset terhadap struktur modal pada Bank Umum Pemerintah di BEI. Tabel 1. Definisi Operasional Variabel Variabel Definisi Indikator Proksi Ukuran Perusahaan X1 Ukuran atau besarnya asset yang dimiliki perusahaan.Variabel ukuran perusahaan dalam penelitian ini diukur dengan menggunakan nilai logaritma natural (Ln)dari total aset 1. Aset lancar 2. Aset tetap LOG Total Aset Pertumbuha n Aset X2 selisih antara jumlah aset periode ini dengan periode sebelumnya dibandingkan dengan aset periode sebelumnya 1. Aset t 2. Aset t-1 G = Aset t −Aset t −1 Aset t −1 Struktur Modal (Y) Rasio ini menunjukan perbandingan antara jumlah liabilitas tidak lancar dan liabilitas lancar yang diberikan para kreditur terhadap jumlahmodalsendiriyang diberikanolehpemilik perusahaan 1. Liabilitas Lancar 2. Liabilitas tidak Lancar 3. Ekuitas DER = Total Liabilitas Total Ekuitas Sumber: Copeland dan Weston (2010) Kerangka Pemikiran Berdasarkan rumusan masalah di atas, maka kerangka pemikiran dalam penelitian: 188 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 H1 H2 H3 Gambar 1. Kerangka Pemikiran Struktur Modal Ukuran Perusahaan Pertumbuhan Aset Ukuran Perusahaan Struktur Modal Pertumbuhan Aset H3 Gambar 1. Kerangka Pemikiran H3 Gambar 1. Kerangka Pemikiran Gambar 1. Kerangka Pemikiran Hipotesis Penelitian Populasi dan Sampel Populasi dalam penelitian ini adalah 4 perusahaan sektor perbankan yang terdapat di BEI. Teknik Sampel yang digunakan sampling jenuh/sensus adalah teknik penentuan 189 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 sampel bila semua anggota populasi digunakan sebagai sampel. Hal ini sering dilakukan bila jumlah populasi relatif kecil, kurang dari 30 sampel. sampel bila semua anggota populasi digunakan sebagai sampel. Hal ini sering dilakukan bila jumlah populasi relatif kecil, kurang dari 30 sampel. Tabel 2. Sampel Penelitian No Kode Nama Emiten 1 BBTN PT. Bank Tabungan Negara (persero) Tbk 2 BBRI Pt. Bank Rakyat Indonesia (persero) Tbk 3 BBNI PT. Bank Negara Indonesia (Persero) Tbk 4 BMRI PT. Bank Mandiri (Persero) Tbk Sumber: http://idx.co.id Tabel 2. Sampel Penelitian HASIL PENELITIAN DAN PEMBAHASAN Hasil Penelitian Hasil perhitungan ukuran perusahaan, pertumbuhan aset dan struktur modal perbankan umum pemerintah tahun 2011 s.d 2015 adalah sebagai berikut: Tabel.3. Tabel.3. Perhitungan size perusahaan,Growth aset dan struktur modal perbankan umum pemerintah di BEI Tahun 2011 s.d 2015 Bank Tahun Log Aset Pertumbuhan Aset Struktur Modal BNI 2011 8,476 0,030 6,90 2012 8,523 0,115 6,66 2013 8,587 0,161 7,11 2014 8,620 0,077 5,59 2015 8,634 0,035 5,81 BRI 2011 8,813 0,140 8,43 2012 8,741 0,152 7,50 2013 8,797 0,136 6,89 2014 8,904 0,281 7,21 2015 8,888 0,036 6,57 BTN 2011 7,950 1,044 11,77 2012 8,048 1,254 9,87 2013 8,118 1,274 10,35 2014 8,160 1,102 10,84 2015 8,193 1,079 11,22 Mandiri 2011 8,742 0,106 7,81 2012 8,803 0,152 7,31 2013 8,865 0,153 7,26 2014 8,932 0,166 7,16 2015 8,961 0,069 6,90 Sumber: www.idx.co.id, diolah Tahun 2017 Perhitungan size perusahaan,Growth aset dan struktur modal perbankan umum pemerintah di BEI Tahun 2011 s.d 2015 190 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 Tabel.4 Hasil Uji Normalitas Data Hasil Uji Normalitas Data Size.Firm Growth.Aset DER N 20 20 20 Normal Parametersa,b Mean 8,5878 ,3781 7,9580 Std. Deviation ,32366 ,46361 1,82590 Most Extreme Differences Absolute ,182 ,376 ,249 Positive ,139 ,376 ,249 Negative -,182 -,226 -,124 Kolmogorov-Smirnov Z ,814 1,683 1,114 Asymp. Sig. (2-tailed) ,521 ,007 ,167 Sumber: Hasil pengolahan data dengan SPSS versi 22, Tahun 2017 Dari tabel 4 menunjukkan bahwa nilai Asymp Sig. (2-tailed) dari ukuran perusahaan sebesar 0,521, pertumbuhan aset sebesar 0,007, dan struktur modal sebesar 0,167. Nilai Asymp Sig. (2-tailed) tersebut > 0,05, maka dapat diambil kesimpulan bahwa variabel pertumbuhan perusahaan dan ukuran perusahaan serta struktur modal secara statistik telah terdistribusi secara normal dan layak digunakan sebagai data untuk penelitian. Hasil Uji Asumsi Klasik Pengujian jenis ini digunakan untuk menguji asumsi, apakah model regresi yang digunakan dalam penelitian ini layak atau tidak, uji asumsi klasik terdiri atas: Hasil Uji Normalitas Data Uji normalitas bertujuan untuk menguji apakah dalam model regresi, variabel pengganggu atau residual memiliki distribusi normal atau tidak. Uji normalitas data dilakukan dengan uji One-Sample Kolmogrov-Smirno Test dengan bantuan software SPSS 22. Berikut hasil dari pengujian normalitas dapat dilihat pada tabel dibawah ini: Tabel.4 Tabel.5 Hasil Uji Autokorelasi Dari tabel 5 tertera nilai DW sebesar 1,935 dengan demikian nilai DW berada antara (1,55 sampai 2,46), maka dapat disimpulkan bahwa tidak terjadi masalah autokorelasi dalam penelitian ini. Hasil Uji Multikolinearitas Hasil Uji Multikolinearitas Model Collinearity Statistics Tolerance VIF 1 (Constant) Size.firm ,238 4,193 Growth.aset ,238 4,193 Sumber: Hasil pengolahan data dengan SPSS versi 22 Dari tabel 5 dapat dilihat bahwa nilai VIF dari model analisis pada penelitian ini <10 dan nilai tolerance > 0,10. Nilai VIF ukuran perusahaan dan pertumbuhan aset sebesar 4.193 dan nilai tolerance ukuran perusahaan dan pertumbuhan asetsebesar0,238, maka dapat disimpulkan bahwa tidak terjadi multikolonieritas pada penelitian ini. Hasil Uji Multikolinearitas Pengujian ini dilakukan untuk menguji ada tidaknya kolerasi antara sesama variabel independen yang ada dalam model regresi dengan melihat tolerance dan Variance inflation Factor (VIF). Nilai tolerance yang rendah sama dengan VIF yang tinggi (karena VIF = 1/tolerance). Berikut hasil pengujian dapat dilihat pada tabel dibawah ini: Hasil Uji Autokorelasi Uji autokorelasi bertujuan untuk menguji apakah dalam suatu model regresi linear ada korelasi antara kesalahan pengganggu pada periode t-1 (sebelumnya). Pengujian ini dilakukan dengan uji Durbin-watson testdengan bantuan SPSS 22. Berikut hasil pengujian dapat dilihat pada tabel dibawah ini: 191 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 Tabel.5 Hasil Uji Autokorelasi Model R R Square Adjusted R Square Std. Error of the Estimate Durbin-Watson 1 ,915a ,837 ,817 ,78052 1,935 Sumber: Hasil pengolahan data dengan SPSS versi 22, Tahun 2017 Tabel 6. Hasil Uji Multikolinearitas Hasil Uji Model Analisis Regresi linier berganda, yaitu metode statistik yang digunakan untuk mengetahui pengaruh variabel terikat terhadap variabel bebas, dapat dilihat pada tabel berikut ini: 192 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 Tabel 7 Hasil Uji Regresi Linear Berganda Tabel 7 Hasil Uji Regresi Linear Berganda Model Unstandardized Coefficients Standardized Coefficients B Std. Error Beta 1 (Constant) 6,305 9,993 Size.firm ,033 1,133 ,006 Growth.aset ,622 1,791 ,320 Sumber: Hasil pengolahan data dengan SPSS versi 22 Dari tabel diatas dapat disusun persamaan regresi linier berganda sebagai berikut: Y = 6,305 + 0,033 X1 + 0,622 X2 Y = 6,305 + 0,033 X1 + 0,622 X2 sarkan model regresi ini terlihat bahwa : Berdasarkan model regresi ini terlihat bahwa : Berdasarkan model regresi ini terlihat bahwa : a. Konstanta sebesar 6,305 menunjukan bahwa jika variabel independen (ukuran perusahaan dan pertumbuhan aset) dianggap konstan (bernilai nol) maka struktur modal akan tetap ada sebesar 6,305. a. Konstanta sebesar 6,305 menunjukan bahwa jika variabel independen (ukuran perusahaan dan pertumbuhan aset) dianggap konstan (bernilai nol) maka struktur modal akan tetap ada sebesar 6,305. b. Koefisien regresi adalah sebesar 0,033 artinya apabila ukuran perusahaan mengalami kenaikan sebesar satu satuan maka akan menyebabkan struktur modal meningkat sebesar 0,033 satuan. c. Koefisien regresi pertumbuhan aset adalah sebesar 0,622 artinya apabila pertumbuhan aset mengalami kenaikan sebesar satu satuan maka akan menyebabkan struktur modal meningkat sebesar 0,622 satuan. Hasil Uji Hipotesis a. Hasil Uji Hipotesis F Hasil Uji Hipotesis a. Hasil Uji Hipotesis F Uji Simultan (Uji F) digunakan untuk menguji pengaruh variabel ukuran perusahaan dan pertumbuhan aset terhadap struktur modal. Tabel 8. Hasil Uji Hipotesis F Model Sum of Squares Df Mean Square F Sig. 1 Regression 52,988 2 26,494 43,489 ,000a Residual 10,357 17 ,609 Total 63,344 19 Sumber: Hasil pengolahan data dengan SPSS versi 22 Tabel 8. Hasil uji F menunjukkan bahwa nilai sig F sebesar 0,000 < sig α 0,05 artinya terdapat pengaruh ukuran perusahaan dan ukuran perusahaan terhadap struktur modal perbankan umum pemerintah di BEI, artinya H0 ditolak. 193 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 Tabel.9 Hasil Uji koefisien t Tabel.9 Hasil Uji koefisien t Model t Sig. 1 (Constant) ,631 ,536 Size.firm ,029 ,057 Growth.aset 1,580 ,000 Sumber: Hasil pengolahan data dengan SPSS versi 22 Hasil Uji koefisien t j Model t Sig. 1 (Constant) ,631 ,536 Size.firm ,029 ,057 Growth.aset 1,580 ,000 Sumber: Hasil pengolahan data dengan SPSS versi 22 Model t Sig. 1 (Constant) ,631 ,536 Size.firm ,029 ,057 Growth.aset 1,580 ,000 Sumber: Hasil pengolahan data dengan SPSS versi 22 Dari Tabel 9 variabel ukuran perusahaan menunjukkan nilai sig t sebesar 0,057 < sig α = 0,05 dan variabel pertumbuhan aset menunjukkannilaisig t sebesar 0,000≤ sig α = 0,05. Maka H0 ditolak dan Ha diterima, artinya terdapat pengaruh ukuran perusahaan dan pertumbuhan aset secara parsial terhadap struktur modal perbankan umum pemeerintah di BEI. b. Uji Koefisien Regresi (Uji t) Uji koefisien regresi digunakan untuk melihat tingkat signifikan masing-masing koefisien regresi. Uji koefisien regresi digunakan untuk melihat tingkat signifikan masing-masing Tabel.9 Hasil Uji koefisien t KESIMPULAN Hasil uji F menunjukkan bahwa nilai sig F sebesar 0,000< sig α 0,05 artinya terdapat pengaruh ukuran perusahaan dan pertumbuhan aset terhadap struktur modal perbankan umum pemerintah di BEI. Hasil uji t menunjukkan ukuran perusahaan dengan nilai sig t sebesar 0,057< sig α = 0,05 dan variabel pertumbuhan aset menunjukkan nilai sig t sebesar 0,000 ≤ sig α = 0,05. Maka H0 ditolak dan Ha diterima, artinya terdapat pengaruh ukuran perusahaan dan pertumbuhan asset secara parsial terhadap struktur modal perbankan umum pemerintah di BEI. Pembahasan Hasil penelitian menunjukkan hasil bahwa secara bersama-sama dan parsial ukuran perusahaan dan pertumbuhan aset berpengaruh signifikan terhadap struktur modal perbankan umum pemerintah di BEI. Hasil temuan ini mendukung penelitian Manopo dkk. (2011) yang menyatakan bahwa ukuran perusahaan mempengaruhi secara signifikan perbankan umum pemerintah. Dalam setiap penggunaan sumber dana baik dari modal sendiri atau modal asing, pasti mempunyai biaya modal yang berbeda-beda dan tingkat risiko yang berbeda pula. Setiap perusahaan baik perusahaan besar ataupun kecil pasti akan menggunakan sumber dana yang lebih aman terlebih dahulu (pendanaan secara internal), daripada menggunakan sumber dana dari luar. Selain itu, didukung dengan kondisi ekonomi yang tidak stabil mengakibatkan setiap perusahaan memiliki kebijakan masing-masing dalam menentukan struktur modalnya. Perusahaan besar dapat dengan mudah untuk menganalisis pasar modal, akan lebih mudah tergabung dan memahami pasar modal dan efek-efek yang diperjualbelikan. Kemudahan dalam mengakses pasar modal membuktikan bahwa perusahaan memiliki fleksibilitas dan kemampuan untuk mendapatkan dana yang dibutuhkan. Semakin besar suatu perusahaan maka 194 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 kecenderungan penggunaan dana eksternal juga akan semakin besar karena semakin besar peluang untuk melakukan ekspansi perusahaan dan peningkatan jumlah produk. Hasil pengujian regresi menunjukkan bahwa pertumbuhann aset berpengaruhsignifikan terhadap struktur modal. Bertentangan dengan penelitian Sansoethan (2015) yang menyatakan bahwa pertumbuhan aset tidak berpengaruh terhadap struktur modal. Pengaruh positifyang ditemukan dalam penelitian ini menunjukkan berarti semakin tinggi pertumbuhan aset maka struktur modal semakin tinggi. Sebaliknya pertumbuhan asset tidak berpengaruh signifikan terhadap struktur modal, berarti bahwa perubahan peningkatan maupun penurunan suatu asset yang diperoleh perusahaan setiap waktu tidak akan mempengaruhi pihak manajemen dalam pengambilan keputusan pendanaan dalam rangka pemenuhan kebutuhan dana perusahaan. Hasil penelitian in itidak sesuai dengan pecking ordertheory yang menyatakan bahwa perusahaan dengan tingkat pertumbuhan aset yang tinggi akan melakukan ekspansi dengam cara menggunakan dana eksternal berupa hutang. Juga menurut signaling theory, perusahaan dengan tingkat pertumbuhan aset yang tinggi cenderung menggunakan tingkat hutang yang lebih besar dalam struktur modal. Hasibuan Malayu S.P. 2007. Dasar Dasar Perbankan, Penerbit Bumi Aksara Jakarta Copeland, Rhomas. E dan J. Fred Weston. (2011). Manajemen Keuangan. Edisi Kedelapan. Julid 2:Binarupa Aksara DAFTAR PUSTAKA Brigham, E.F. dan J.F. Houston. (2010). Fundamentals of Financial Management. 8th ed.Harcourt College Publishers. Orlando. Terjemahan H. Wibowo. 2006. Manajemen Keuangan. Edisi Kedelapan. Erlangga.Jakarta. Copeland, Rhomas. E dan J. Fred Weston. (2011). Manajemen Keuangan. Edisi Kedelapan. Julid 2:Binarupa Aksara Copeland, Rhomas. E dan J. Fred Weston. (2011). Manajemen Keuangan. Edisi Kedelapan. Julid 2:Binarupa Aksara Hasibuan Malayu S.P. 2007. Dasar Dasar Perbankan, Penerbit Bumi Aksara Jakarta sibuan Malayu S.P. 2007. Dasar Dasar Perbankan, Penerbit Bumi Aksara Jakarta 195 JEMASI Vol.13 No.2, Jul-Des 2017, hal. 182-196 Husnan, S.(2011). Manajemen Keuangan Teori dan Penerapan. Edisi Keempat. BPFE. Yogyakarta. Mamduh, M.H.(2015).Manajemen Keuangan Internasional. BPFE. Yogyakarta. Manopo, Fimber, Widy. (2013). Faktor-Faktor yang Mempengaruhi Struktur Modal Perbankan yang Go Publik di BEI. Jurnal EMBA Vol.1 (3) Juni 2013. Hal:653-663 Mishkin Frederic S. (2012).Ekonomi Uang, Perbankan,dan Pasar Keuangan. Indeks Sansoethan, Kusuma Dithya. (2015) Faktor-Faktor yang Mempengaruhi Struktur Modal Pada Perusahaan Makanan dan Minuman. Jurnal Ilmu & Riset Akuntansi Vol 5 (1) Januari 2015. Hal: 9-31 Seftianne dan R.Handayani. (2011). Faktor-faktor yangMempengaruhi Struktur Modal pada Perusahaan Publik Sektor Manufaktur. Jurnal Bisnis dan Akuntansi 13 (1): 39-56. Sartono, Agus. (2010). Manajemen Keuangan Teori dan Aplikasi. Edisi Ke-Empat. Yogjakarta: BPFE. Yovin, D. dan N. P.S. Suryantini. (2012). Faktor-faktor yang Berpengaruh terhadap Struktur Modal pada Perusahaan Foods and Beverages yang Terdaftar di Bursa Efek Indonesia. E-Jurnal Manajemen Universitas Udayana 1 (2) 196
https://openalex.org/W2969469667	https://www.redalyc.org/journal/5606/560662202001/560662202001.pdf	English	null	Leadership Discourses of Keyae in Islamic Boarding School	Research, Society and Development	2,019	cc-by	7,306	Research, Society and Development ISSN: 2525-3409 ISSN: 2525-3409 rsd.articles@gmail.com Universidade Federal de Itajubá Brasil Research, Society and Development ISSN: 2525-3409 ISSN: 2525-3409 rsd.articles@gmail.com Universidade Federal de Itajubá Brasil Research, Society and Development ISSN: 2525-3409 ISSN: 2525-3409 rsd.articles@gmail.com Universidade Federal de Itajubá Brasil PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative Leadership Discourses of Keyae in Islamic Boarding School Discursos de liderança de Keyae no internato islâmico Discursos de liderazgo de Keyae en el internado islámico Salamet salametsal29@gmail.com STKIP PGRI Sumenep, Indonesia hp://orcid.org/0000-0002-1411-5576 Arqom Kuswanjono kuswanjonoarqom@gmail.com Gajah Mada University, Indonesia hp://orcid.org/0000-0001-6592-478X Ridwan Ahmad Sukri ridwansukriah@gmail.com Gajah Mada University, Indonesia hp://orcid.org/0000-0002-6250-1834 Abstract: is study is aimed to discuss the discourse of Keyae in Pesantren (Islamic Boarding School) and its relevance toward Islamic values in Madura, especially. Aerward, this study is conducted by involving the participators in Annuqayah Islamic Boarding School, Guluk-Guluk, Sumenep which is assumed to be representative, and have relevance in protecting Islamic values in Madura. e data which is obtained based on Michel Foucault’s discourse analysis. e reason to choose the theory aims to understand the conditions that supporting the emergence of leadership discourse of Keyae, form and its operational, discontinue and relational, archeology of leadership discourse, and reveal or analyze critics on leadership discourses of Keyae in Madura, speciﬁcally in social mechanism. erefore, based on the analysis, this study shows that leadership of Keyae in Islamic Boarding School in Madura is not merely as an agent of religious movement. However, it is as social, politic, culture, economic, and education transformations. Redalyc: https://www.redalyc.org/ articulo.oa?id=560662202001 Keywords: discourse analysis, Islamic boarding school, Keyae, Madura. Resumo: Este estudo tem como objetivo discutir o discurso das Escolas de Internação Islâmica e sua relevância para os valores islâmicos em Madura, especialmente. Posteriormente, este estudo é realizado envolvendo os participantes do Annuqayah Islamic Boarding School, Guluk-Guluk, Sumenep, que é considerado representativo e tem relevância na proteção dos valores islâmicos em Madura. Os dados obtidos são baseados na análise do discurso de Michel Foucault. A razão para escolher a teoria visa compreender as condições que sustentam a emergência do discurso de liderança de Keyae, forma e sua operacional, descontinua e relacional, arqueologia do discurso de liderança, e revelar ou analisar críticas sobre discursos de liderança de Keyae em Madura, especiﬁcamente em mecanismo social. Portanto, com base na análise, este estudo mostra que a liderança do internato islâmico em Madura não é apenas um agente do movimento religioso. No entanto, é como transformações sociais, políticas, culturais, econômicas e educacionais. Palavras-chave: análise do discurso, internato islâmico, Keyae, Madura. Resumen: Este estudio tiene como objetivo discutir el discurso de Keyae en Pesantren (Colegio de Internos Islámico) y su relevancia para los valores islámicos en Madura, especialmente. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative Leadership Discourses of Keyae in Islamic Boarding School This work is licensed under Creative Commons Attribution 4.0 International. Research, Society and Development, vol. 8, no. 11, 2019 Research, Society and Development, vol. 8, no. 11, 2019 Universidade Federal de Itajubá, Brasil Received: 05 July 2019 Revised: 04 August 2019 Accepted: 06 August 2019 Published: 24 August 2019 DOI: https://doi.org/10.33448/rsd- v8i11.1380 Redalyc: https://www.redalyc.org/ articulo.oa?id=560662202001 Redalyc: https://www.redalyc.org/ articulo.oa?id=560662202001 1. Introduction e term Keyae basically used as honor title for an expert in Islamic religion given by Madura societies. e term then developed not only for the leader of Islamic Boarding School, but there are some categories such as Keyae as Islamic Boarding School leaders, Keyae as teacher for reading Koran which is mostly known as Keyaji and Keyae as paranormal (performing medication using supernatural or spiritual power). However, the general and mostly known by Madura society is Keyae as Islamic Boarding School leader which has important roles in spreading Islamic religion in Madura. Keyae in Indonesian religion discourse is more emphasizing as leader in Islamic Boarding School as educated Muslim and humiliate his/her life only for Allah, as well as spreading and deepen religion lesson through Islamic classic book sources or Islamic education activity traditionally (Dhoﬁr, 1982: 55-56). Besides Keyae, another term that used for calling Keyae is Ulama (Dirdjosanjoto, 1999: 20). In the strategic position in social dynamics, Keyae has the same position with educated people and rich people in such society (Turmudi, 2004: 1). However, the central position of Keyae as religion, social, and politic leader (in the government structure) in Indonesia has been performed since kingdom era (Dirdjosantoso in Ummatin, 2002: 30). Basically, the position of Keyae in Madura is given position by genetic factor. Hence, this genetic factor becomes primary discourse concern for Keyae leadership. Moreover, the existence of Keyae and Islamic Boarding School cannot be separated, since Islamic Boarding School is a place where Keyae spreading Islamic religion and perform their leadership. e inﬂuence of Keyae is not merely in religion, but also as agent in cultural strengthen in society. Further, Keyae also stand for informal leader who has strong power in its community, even, most of Keyae in Madura are being politic ﬁgure. It is also happened in Madura society in which Keyae has role as guidance for the society. e phenomenon of Keyae in Islamic Boarding School describe how powerful Keyae is, so therefore Keyae will have double roles and has attractiveness to discuss and trace from various perspective. For Madura society, Keyae became a role model and central position to complain about any issues faced by them. Almost all the problems faced by the people in Madura, is assumed that it would be easy to resolve when asking for guidance from Keyae. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative Leadership Discourses of Keyae in Islamic Boarding School Posteriormente, este estudio se lleva a cabo involucrando a los participantes en el internado islámico Annuqayah, Guluk-Guluk, Sumenep, que se supone representativo, y tiene relevancia en la protección de los valores islámicos en Madura. Los datos que se obtienen en base al análisis del discurso de Michel Foucault. La 1 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 razón para elegir la teoría tiene como objetivo comprender las condiciones que apoyan la aparición del discurso de liderazgo de Keyae, su forma y su operativa, discontinua y relacional, arqueología del discurso de liderazgo, y revelar o analizar críticos sobre los discursos de liderazgo de Keyae en Madura, especíﬁcamente en mecanismo social Por lo tanto, según el análisis, este estudio muestra que el liderazgo de Keyae en el internado islámico en Madura no es simplemente un agente del movimiento religioso. Sin embargo, se trata de transformaciones sociales, políticas, culturales, económicas y educativas. Palabras clave: análisis del discurso, internado islámico, Keyae, Madura. 1. Introduction us, many personal issues and internal conﬂicts between communities are solved through Keyae intervention. 2 Salamet, et al. Leadership Discourses of Keyae in Islamic Boarding School From Foucault point of view, the phenomenon of Keyae leadership in Madura should be understood from relations power which dominates it. Further, Foucault (in Bertens, 2001: 307-319) explain that power exist everywhere, since power is one dimension from relation, absolute, and do not depend on human awareness. However, power is seen merely as a strategy. e strategy exist everywhere and including system, regulation, and structure. Power do not comes from outside, rather, it is determine the structure, regulation, and relation from inside which is making all things possibly happened. As referred to Forcault perspective, Keyae leadership in Madura is in line with the discourse, then discourse and power will always relational. Keyae leadership in Islamic Boarding School in this context can be deﬁned as power that is understood as regulation system and normalization societies life. Keyae view as leadership that is not hierarchy, meanwhile it is spread and operational in social mechanisms. Regarding to explanation of the roles of Keyae in Madura, this study is intended to discuss leadership discourse of Keyae and its relevance toward Islamic values in Madura especially in Islamic Boarding School using Michael Foucault perspective. e leadership discourse mostly discussed based on history side. Yet, there is no discussion from Michel Foucault archaeology and genealogy. e archaeology is quite diﬀerent with idea history. At least, there are four principal which diﬀers between archaeology analyses with history. Firstly, archaeology do not discuss about thinking and representation in discourses. Rather, archaeology is more discussing on discourse as practical based on its regulation. Secondly, archaeology do not try to look for linier or gradual correlation between discourses, but try to search the characteristic of the discourse. irdly, archaeology do not discuss about individual concern or ouveres. Archaeology concerns on the types of discursive practical which is relating to the individual ouvere. Fourthly, archaeology does not investigate discourse but it is more concern on arrested systematic as discourse object (Ritzer, 2003: 72). Meanwhile, genealogy analysis in reading phenomenon of Keyae Islamic Boarding School leadership in Madura will try to see how the relation of leadership in dominate, control, and lead the society. 1. Introduction Hence, from the explanation, the leadership phenomenon of Keyae in Islamic Boarding School in Madura will be discussed from the perspective of Michel Foucault theory. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative PDF generated from XML JATS4R by Redalyc Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 e Archaeology of Knowledge basically reﬂects Foucault’s intuitive thinking, which tries to change the research methodology with speculative improvisation, yet still based on facts. Foucault completes the archeology approach as tool to test and understand complex social phenomenon through the way it is produced. Further, Foucault also underline four principals of archeologist method that can be summarize as follow: 1. Archaeology is not trying to deﬁne thinking, representation, description or image, themes, pre occupation either implicit or explicit in discourse. Meanwhile, it is trying to explain discourse itself which should be obligate the certain regulations. 1. Archaeology is not trying to deﬁne thinking, representation, description or image, themes, pre occupation either implicit or explicit in discourse. Meanwhile, it is trying to explain discourse itself which should be obligate the certain regulations. 2. Archeology is not trying to discuss continuity and insensible transition in discourse, rather it is discuss the speciﬁcation to show regulation set behind. 3. Archeology is not aiming to discuss enigmatic point in which individual and society is inﬂuence each other. Further, it is also not a sociology, psychology, or anthropology creation, but it is merely focus with the determining regulation types to discursive practical. g g p p 4. Archeology is not trying to restore what being thought, hoped, purposed and desired by human expressed in discourse. It is more concern on systematic description about discourse object. e way Foucault thinking show that archeology approach basically a ﬁeld to investigate discourse, especially power that regulate and produce discourse. Walshaw (2007: 9) dealing with this concern, explain that archeology place discourse as ﬁeld object. In its application, archeology approach should view the observed subject as not merely psychological, sociological, or anthropological subject, rather it is discursive subject. Wherever a discourse and the subject are found, hence the ﬁeld to investigate archeology is found as well. Aerward, in recognizing discourse, it is not enough only by tracking the talk and written. e systematic practical and determined regulation to organize the way people talk and act should be the concern as well. In this case, archeology should be view beyond the surface of the subject. For instance, religious texts are the surface. Individual should be tracking in discursive system, applicable rules, and power, hence the text can be acknowledged and practiced broadly by adherents of such religion. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative 1. Archeology and Genealogy Method in Michel Foucault’ Discourse Analysis Foucault using archaeology concept to diﬀer the approach regarding with history, especially hermeneutic which is generally need understanding in discourse basic or scheme clariﬁcation subjective meaning. Moreover, it is dealing with the diﬀerence between any kinds of history model which has causal or superﬁcial characteristic. 3 3 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 All of these things will be closely related to the analysis of discourse, especially by involving episteme, archives, power, and discourse itself. As for genealogy method is still continuous part of archeology method proposed by Foucault. rough this method, Foucault tries to connect discourse with daily practice which useful for testing and understanding how the subject is formed as discursive or real in the same condition. Foucault (1977: 139-140) explains further: “Genealogy is gray, meticulous and patiently documentary. It operates on a ﬁeld of entangled and confused parchments, on documents that have been scracthed over and recopied many times... genealogy retrieves an indipensable restraint: it must record the singularity of events outside any monotonous ﬁnality; it must 4 Salamet, et al. Leadership Discourses of Keyae in Islamic Boarding School seek them in the most unpromising places, in what we tend to feel is without history–in sentiments, love, conscience, instincts; it must be sensitive to their recurrence, not in order to trace the gradual curve of their evolution but to isolate the diﬀerent scenes where they engaged in diﬀerent roles. Finally genealogy must deﬁne even those instances when they are absent, the moment when they remained unrealized… Genealogy requires patience and a knowledge of details, and it depends on a vast accumulation of source material.” Genealogy basically focuses on daily activities and tries to explain statements in society but more concern on the power. Genealogy introduces power through history of present, how the statements are processed, and concentrate in the use of archaeology strategically to answer any kind of problems that appeared in society. is approach is actually important to reveal power and determine discourse meaning, and the aims which taken for granted by the subject. Walshaw (2007: 14) explains genealogical analyses as a tool to explore the interaction of power and knowledge within the practices and social structures of education. Moreover, it uses as highlight the profound inﬂuence of discourse on shaping everyday life in education. Genealogy analysis makes people possibly to observe relation between the power and knowledge in practical and social structural that will be formed everyday life. At ﬁrst glance, there is no signiﬁcant diﬀerence between archeology and genealogy method in its application, since both of the method should be run together. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 However, Smart (in Mills, 2003: 25) reveals the diﬀerence between archeology and genealogy method in which archaeology is directed in the analysis about rules of the unconscious from formation which organize the emergence of discourse in human science. Meanwhile, genealogy analysis is try to open the emergence of human’s knowledge, conditions of existence, which are closely related to technology or certain powers contained in social practice. erefore, these two approaches or analysis are two sides of one coin. Nevertheless, in its practice, as stated by Mills (2003: 25), the two methodologies can be diﬀerence method. Archeology can give people a snippet, a slice in the existing discourse, while genealogy focuses on the process aspects of the network of discourses. p p Foucault (1980a: 85) also argues as follow: “...if we were to characterise it in two terms, then “archaeology” would be the appropriate methodology of [the] analysis of local discursivities, and “genealogy” would be the tactics whereby, on the basis of the descriptions of these local discursivities, the subjected knowledges which were thus released would be brought into play.” en, based on Foucault’s explanation, archeology can be used as method to analysis local discourse, whereas genealogy can be used as tactic genealogy which based on the local discourses description. 5 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative 2. Analysis and Discussion In the analysis of Foucault’s model discourse analysis, the leadership of Keyae in Annuqayah Islamic Boarding School can be viewed as discourse formed through a long historical journey. is leadership discourse is involving many factors and other discourses in the forming process start from society development of the cultural complexion, the existence of Islam and its inﬂuence on the values and beliefs of society, to the subjective role of cross-generation Keyae who continue to conﬁrm their leadership discourse in the community itself. us, in this case, following the Foucault’s analysis ﬂow, there will be several things that can be reviewed from Keyae leadership as follow: PDF generated from XML JATS4R by Redalyc 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura Leadership discourse not merely seen as language of leadership, or deﬁnitive explanation, yet it is more than that. Leadership discourse means it covering the way people interpret the existence of Keyae, the perspective of people in their behavior and attitude toward Keyae. In the reality, the leadership of Keyae in Annuqayah Islamic Boarding School is acknowledged by Madura society due to the support of several subjects as like Keyae Syarqawi as the foundation of ideological and pedagogical in Annuqayah Islamic boarding school, the generation of relatives who also became Keyae who ran the Islamic Boarding School and continued to preach, the santri of various teaching classes who attended the Annuqayah Islamic Boarding School. ese subjects are united in the form of teaching activity, teaching books, religion doctrine, togetherness and others. Seeing into its history, the leadership of Keyae nowadays, is not separated from the roles of the Keyae. e Keyae has ﬁght in practical religion preaching to the society. In addition, the existence of Keyae Syarqawi and other generations, has successfully make their existence as the main knowledge authority or a person who master the religious matters in its application in all aspect of society. When the authority is successfully obtained and continuously existing and forming in the teaching activity and daily interaction, then the role of Keyae is not merely concern on solving society’s problems based on Islamic values, but it is also as main preference to attitude, behavior, and good life model as thought by Keyae to its society. e authority in the scientiﬁc ﬁeld and the understanding of Islamic teachings are increasingly reinforced by the establishment of religious education institutions in the form of Annuqayah Islamic boarding schools. e presence of Annuqayah Islamic Boarding School is not only reinforces their scientiﬁc legitimacy, but also binds the community's need for education for their children. e presence of an Islamic boarding school educational institution besides being an aﬃrmation of Keyae’s title, which generally in Madurese society is considered as a person who 6 Salamet, et al. Leadership Discourses of Keyae in Islamic Boarding School has scientiﬁc qualiﬁcations and has santri, can also be a place where Keyae build the Islamic discourse he wants. e utterances of Keyae which manifested in the form of texts, documents, and discourse, in further has successfully aﬃrming the leadership discourse of Keyae in society, especially Annuqayah Islamic Boarding School in Madura. 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura is process then called as discursive formation of leadership Keyae in Annuqayah Islamic Boarding School. e reason of successfulness of formation discursive is supported by religious and feudalistic society culture in which religion is assumed as the most important thing in life. Moreover, the Islamic ﬁgure as like Keyae is the one who is worthy to be gloriﬁed (in the high position of society, even above the nobleman position and government oﬃcials). In traditional and feudal society, the statements which based on religion doctrine will be more eased to get acknowledgement from society rather than utterances that contain of ideologies. In this case, when the Keyae in the Anuuqayah Islamic Boarding School successfully acquiring trust and recognition of the community in terms of religious authority or interpretation of religious texts, hence they will also be easier to display their presence as leaders in other aspects of life. e formation of leadership discourse of Keyae in Annuqayah Islamic Boarding School basically has same Keyae leadership as like in other regions as long as the supporting discursive system factors are the same. However, the result of leadership discourse can be diﬀerent, especially because each individual who plays a role in it, is also has a diﬀerent understanding of what he wants to build. But, when discourse begins to form, then the people or individuals, who are bound in it, are actually no longer able to freely speak and believe other statements that do not conform to the existing discourse. When the discourse is formed, it does not stop dialogue and development (production) in itself, whether through conﬂict between statements, internal and external exclusion, which in practice has moved quietly and controls the way of thinking of the subjects. A belief or perspective built on established discourse will be diﬃcult to change, unless there is a certain dispersion and pause (discontinuity of discourse), which usually starts from a subject that is more able to take distance from existing realities, or when conﬂicts occur between diﬀerent statements and not succeeded in being suppressed by the existing power. In other words, discursive formation is not a container or a passive process. ere is a process of interaction, dialogue, conﬂict, and exclusion in it, which all contribute to the level of establishment of a discourse in the community, including the leadership of the Keyae of Annuqayah Islamic Boarding School. 3.2. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative PDF generated from XML JATS4R by Redalyc 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura Episteme and Leadership Archive of Annuqayah Sumenep Islamic Boarding School Keyae Keyae’s leadership discourse is not only formed by a discursive process but also formed through the process in deciding the right and proper 7 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 statements to be distributed or spread to public. Focault explained that discourse works with collecting and debating some statements in order to take and produce a proper statement as a compromise result to become an applicable authentic standard in society. Episteme, in the context of Keyae’s leadership discourse of Annuqayah Islamic Boarding School, refers to a complex set that summarize various relationship between statements, religious teachings, or Islamic scientiﬁc knowledge and life ethic. is leadership function is most visible in certain time, such as in the ﬁrst period of Annuqayah Islamic Boarding School’s Keyae or the period in which Keyae had succeed in establishing education locality and the rules that underlined the production of statements, teachings, and developed scientiﬁc knowledge at that time. Meanwhile, archive refers to the valid rules set in a certain period, especially on the boundaries between what can be made as a statement and what form of statement produced by society. ese boundaries are valid for both society as the consumer and Keyae as the producer. y p Every leadership period has its own episteme and archive that diﬀer from one period and other. However, in Annuqayah Islamic Boarding School, discourse is built by the ﬁrst Keyae and has not undergo the essential transformation, except the institutional development needed to accommodate the need and education. e episteme and archive of the ﬁrst Keyae period in Annuqayah Islamic Boarding School is still limited and narrow due to the Islamic discourse that still not yet complex. e way people thought, act, or expressed their cultural identity is not what it is like nowadays. A harder challenge will be faced by the next generation in which they will not only charged to defend the old leadership discourse but also charged on how to anticipate and accommodate further discourses. Defending a discourse is an eﬀort to build new episteme and archive for society, along with strengthening the existed Keyae’s leadership discourse. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura Building episteme and archive is surely a complex eﬀort since it is not merely a process in denying statements which considered as inappropriate with the religious understanding and interpretation but on how to accommodate various statements and perspectives that newly developed in society. Keyae should be able to formulate statement and documented it in the form of text in religious teaching taught in Islamic Boarding School or Communal Quran Reciting based on the religious ideology, either in the group of Keyae itself or general public. e educational institution of Annuqayah Islamic Boarding School has signiﬁcant role since it becomes the place for Keyae to produce statements, teachings and/or religious interpretative texts taught to santri and society around. Santri will continue the Islamic discourse and wider the discourse itself. Moreover, Annuqayah Islamic Boarding School in its continuity, has become the authoritative institution that have legitimation to validate between proper and improper statements. A proper statement will become the learning source and be documented in the form of book, text or document to spread to public. rough this way, Islamic Boarding School institution has played its role on Keyae’s 8 Salamet, et al. Leadership Discourses of Keyae in Islamic Boarding School leadership and its inﬂuence in society. e process in producing and denying statements by making authentic religious standard is the eﬀort in creating the boundaries for santri and society to issued statements outside the existed ideological narration. In this case, every subject within the episteme and archive of Keyae leadership was slowly formed to have the way of thought, to behave, to run the religious ritual and even to live based on the validity standard that has been created. e unwritten boundaries and rules that appeared from the discursive are were not only interfere the occurrence of other narration that unsuitable with the desire of powerful authority, but also limit the freedom of thought and creativity of the main subjects in the discursive authority circle. In other words, boundaries and rules that managed the statement productivity within Keyae’s leadership discourse are valid for every subject that involved, including Keyae, teacher of Annuqayah Islamic Boarding School, santri, and society around Annuqayah Islamic Boarding School itself. us, what should be done by the next generation is to justify the discursive assumptions that have been built before. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative PDF generated from XML JATS4R by Redalyc 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura Nevertheless, in a certain context, the transformation on Keyae’s leadership generation in Annuqayah Islamic Boarding School is similar to Foucault’s analysis that open the probability on discursive interlude in which some new statements can occur by taking the whole diﬀerent form of the old statement. e existed discourse also can get counterwork within, if it is cannot be anticipated, it will create new Keyae’s leadership discourse in Annuqayah Islamic Boarding School. is new discourse will colorize the new Keyae’s leadership period along with discursive episteme and archive in Annuqayah Islamic Boarding School and society around it. 3.3. Authenticity Statements and Regime of Kyai Annuqayah Sumenep Islamic Boarding School Leadership 3.3. Authenticity Statements and Regime of Kyai Annuqayah Sumenep Islamic Boarding School Leadership Statements that occurred within the discursive room, either in the form of utterance, teachings, da’wah, or daily speech, is the main element to form a discourse. For instance, Keyae’s leadership. e more the leadership is being talked in society, then it will create a discourse on Keyae’s leadership along with shaping a certain community. However, it should be noted that in discursive room, not all people can utter their statements or make their statements accepted and recognized by other people. Some statements delivered by certain parties will be more acceptable and recognizable with authenticity value rather than the statements issued by other subjects. Discourse, through existing episteme and archive, will give a structure based on the uttered statements and the condition in which the statements can be considered as right and proper to then distribute and spread to society. When Keyae delivers the materials on religiosity and character value of life ethic, the utterance or statements issued by Keyae will be considered as authentic statements and the guidance for society to pursue their life. Keyae’s statements have become the discourse since it is already covered 9 9 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 various things as a guidance. Similar to Foucault’s argumentation that discourse could replace the individual role in ﬁltering the statements. is called as statement exclusion that happen in both internal and external discursive environment. e role of Keyae is not only becoming the source of statements production but also the authority in ﬁltering the statements in society. 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura In its practice, Annuqayah Islamic Boarding School Keyae is the legal source for society since they can ask about the laws related to religious things, whether it is obligatory or not, or it is forbid by religion or not. Referring to Foucault, there are four internal procedures for statements exclusion that appeared in discursive room, namely; (1) commentary; (2) the author; (3) discipline; and (4) rarefactions. ese four procedures can be drawn in Annuqayah Islamic Boarding School Keyae’s leadership discourse as; e ﬁrst is commentary. Commentary is a statement issued by a certain subject, such as Keyae, that acceptable by other subjects, (santri or society), to be talked, discussed, and/or practiced in a certain act as a comment to existing statement. Commentary is functioned as a determination and, in some contexts, as an explanation on what has been intended by Keyae for santri and other subjects who received the statements. It then leads to the boundaries for other interpretations on the existing statement. e commentary that has been uttered and distributed will become the ﬁlter for other statements that unsuitable with Keyae’s statements. e second is the author. Keyae is the author when he produces a certain statements even it is a mere material, in the beginning, which not yet collected in the form of text, document, or book. Keyae is considered as the authoritative subject to produce right or authentic statements then if there is a diﬀerentiation between one statement and another, it can be considered as something that connect each other. In other cases, Keyae’s statement can be the law and authentic standard that make Keyae diﬃcult to issue the opposite statement. Keyae, as the author, is presenting a certain boundary for statement that he built. e third is discipline. Keyae’s statement on something that determine his leadership in general environment is oen limit to his expertise. Other Keyae’s statements in his daily interaction can be measured again for its authenticity when his statement has no relation or do not based on the religious teaching. Keyae had limitation that could exclude the statements within his expertise. Nevertheless, in Annuqayah Islamic Boarding School, Keyae’s statements, even for problem which not related with religion, is oen have similar value with his religious statements. e fourth is rarefactions. According to Foucault, rarefactions is a limitation on who and who does not has the right to speak. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura In the context of Annuqayah Islamic Boarding School, those who have right to speak and represent Keyae as the leader of his institution is teachers or Keyae’s relatives. While santri have diﬀerent limit when they appear in society. Santi are becoming the subject that considered good to speak and represent Keyae in public since they received direct teachings and informations from Keyae. 10 Salamet, et al. Leadership Discourses of Keyae in Islamic Boarding School Besides the internal exclusion, the external exclusion is also built. According to Foucault the external exclusion has three elements; (1) taboo; (2) mad and sane; and (3 true and false. ese three elements were worked as below; e ﬁrst is taboo. In a society, it oen ﬁnds a rule or value that limit the discussion on something that considered can damage the moral structure, change the behavior, and even harmful for others. A belief on things that inappropriate or taboo to be talked is the form of external exclusion in society. In Annuqayah Islamic Boarding School Keyae’s leadership discourse, taboo has role as a law that limits the occurrence of improper statements to be uttered. One of the examples is the criticism on Keyae’s leadership and his role in society. is attitude is considered as an abuse toward the noblest ﬁgure in society. is rule can be the external exclusion that eﬀective enough to limit the discourse or statements, also to strengthen the discourse of Keyae’s leadership in society. e second is mad and sane. e term mad and sane, according to Foucault, are pointed to the clinical history and how Western treat people outside the normality boundary (people with mental health disorder). ese people can be categorized as people who had diﬀerent arguments or perspectives with society in general. In the context of Annuqayah Islamic Boarding School Keyae’s leadership discourse, the distinction between normal and abnormal can be found on how society treat people who does not have similar understanding with Keyae in religious interpretation. is distinction appeared along with the discourse on Keyae’s leadership and his role as a leader in spiritual and other aspects in society. erefore, people within the Islamic Boarding School environment who have diﬀerent view toward Keyae will end up as a public gossip material. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative PDF generated from XML JATS4R by Redalyc 3.4. Power relation and production of Annuqayah Islamic Boarding School Keyae’s Leadership Discourse 3.4. Power relation and production of Annuqayah Islamic Boarding School Keyae’s Leadership Discourse e other important point in discourse analysis by Foucault is there is always certain power relation appeared on a discourse. An established discourse is mean to strengthen the authority of a certain parties against the other party by controlling the statements or discourse developed within the society. Discourse can control which one is the proper statement and which one is not. is probably happen when there is a certain party that has power or authority. It means that the ﬁnal destination of a discourse is the society hegemony which dismissed them from conﬂict that could damage the harmony. Authority is not only seen as a physical power or the availability of a certain sources, but also the ability to control the authenticity as the most essential basic for human to act. e ability to control the authenticity is becoming the main power to a discourse that developed in society. Keyae’s leadership discourse in Annuqayah Islamic Boarding School also has certain authorities, including the scientiﬁc authority, religious understanding, leadership characterization and also the Islamic Boarding School institution as an authoritative to produce statements or Islamic discourses for society. Referring to Foucault analysis, the authority within Keyae’s leadership discourse of Annuqayah Islamic Boarding School in Sumenep is not centered on the ﬁgure of Keyae or Islamic Boarding School institution. e authorities within the discursive room are plural and connected one and another. Similar to Foucault’s argument that it is a set of relation spread to whole society classes. Foucault claimed that authority as something that not fully repressive and coercive but productive. Repressed authority will create opposition in the form of new action and thought from society as a way to adapt with repressive condition. In other words, even if Keyae and/or Islamic Boarding School institution along with other ﬁgures around is becoming the authoritative parties, but their authorities are not accepted in absolute way by society. If the teachings are pressing them or making them uncomfortable with their life, then conﬂict will be occurred. Authority is not something acceptable but something that could be managed continuity. If Keyae is not managing his Islamic Boarding School anymore, then the authority that he had can be erased slowly and changed to another authority. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative 3.1. Discursive Formation of Keyae Leadership in Annuqayah Islamic Boarding School in Madura Statements which issued by people who considered as improper to be heard should be guided in order to have the same point of view of the general society in viewing Keyae. e third is true and false. e distinction between true and false is usually involved the institution role or certain party that considered of having authority to deﬁne or decide between the true and false of the statements. In the context of Annuqayah Islamic Boarding School Keyae’s leadership discourse, the authority in deﬁning whether the statement is true or false was under the Islamic Boarding School institution itself, especially the subjects with certain scientiﬁc legitimation within, including Keyae, teachers and/or people that came from the same circle. Keyae and educational institution of Islamic Boarding School are supporting each other. Keyae can manifest various statements as his teaching materials which transmitted to santri, and the educational institution of Islamic Boarding School can conﬁrmed Keyae’s authority through the establishment of educational practices. Both have authorities to support each other in deﬁning which one is the right and proper statement and which one should be spread in society. e statements that appeared and formed an Islamic discourse have strengthen the Keyae’s role and authority as a leader in either educational institution of Islamic Boarding School or society around. 11 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 us, the statements that occurred nowadays has appeared as an authentic aﬃrmation and/or support on Keyae’s leadership discourse and his role. In addition, the absent of conﬂict on Keyae’s statements showed that Keyae’s leadership discourse has an established discourse in society. However, this condition may change if the occurrence of other diﬀerent statements has giving another point of view on how society view on the existence of Keyae. PDF generated from XML JATS4R by Redalyc 3.4. Power relation and production of Annuqayah Islamic Boarding School Keyae’s Leadership Discourse e relation which built between Keyae and educational institution of Islamic boarding school is not as simple as dominative relation. e society’s obedience toward Keyae’s statement is not resulted by the repressive eﬀort 12 Salamet, et al. Leadership Discourses of Keyae in Islamic Boarding School by the Keyae. In its practice, Keyae is not preaching religious teachings in both coercive and repressive way, such as punish people who do not follow his teachings, because it will lead to rejection by society. e society obedience is the accumulation from many factors, start from cultural characteristic of a society, Keyae’s attempt to spread the statements based on religious dalil or postulate. e authority within Annuqayah Islamic Boarding School Keyae can be the basic law toward what was done by society. is can only be happened because of the acceptance and recognition by the society itself. However, the acceptance and recognition by society also becomes a part controlled by the existing discourse. PDF generated from XML JATS4R by Redalyc Project academic non-profit, developed under the open access initiative 4. Conclusion Dealing with the analyisis and discussion section, this article is hoped to give a good conribution to the Madura society in which they are able to know the roles of Keyae besides as the leader of Islamic Boarding School. g Aerwards, based on the analysis on Keyae’s leadership discourse, it can be concluded as below: 1. Keyae’s leadership is a complex discourse since it consists of many factors and individual subject within. Keyae is not the single actor in discursive room, but stand on the authority circle to give authentic statements that should be accepted by society. 2. Keyae’s leadership discourse that developed in society, especially Annuqayah Islamic Boarding School, Sumenep, is the result of a complex process in formulating certain statements. ese statements related to the role of Keyae and culture in society, which accepted and recognized by society, until create its own legitimation on related Keyae as the one who had authority to deliver some statements in society. 3. Keyae’s leadership discourse has become the rules to control the way society thought, act and behave toward Keyae, also the way they understand about Islamic teaching and the application in daily activity. For society within and around Annuqayah Islamic Boarding School, Sumenep, the discursive rules and limitations were working with the way that oen hard to be noticed. For instance, society without any kind of forces from Keyae had strengthen the authenticity of Keyae’s statements and had no courage to issued their own statements because they feel that they had no right to issue a statement. 4. Keyae’s leadership discourse in Annuqayah Islamic Boarding School had been established through a long process, therefore it is hard to transform related to society’s assumption on Keyae or Islamic Boarding School. e discursive transformation could probably happen when there is condition that support the transformation, such as the changing of leadership generation, society’s point of view on new intervention values in society, and others that showed new discursive that is more powerful to be spread in public. 13 Research, Society and Development, 2019, vol. 8, no. 11, ISSN: 2525-3409 / 2525-3409 From the points that has been described above, the researcher suggest that for the future research can analyze or observe the role of Keyae in other regions, since the existence of Keyae in Indonesia is not merely exist in the Madura. References Basyuni, Ison. 1985. “Dakwah Bil Hal Gaya Pesantren”. M. Dawam Raharjo (ed), Pergulatan Dunia Pesantren. Jakarta: P3M. Bertens, Kees. 2001. Filsafat Barat Kontemporer. Jakarta: Gramedia Pustaka Utama. Dhoﬁer, Zamakhsari. 1982. Tradisi Pessantren; Studi Tentang pandangan Hidup Kiai. Jakarta: LP3ES. Dirdjosanjoto, Pradjarta. 1999. Memelihara Umat: Kiai Pesantren-Kiai Langgar di Jawa. Yogyakarta: LKiS. Eﬀendy, Bisri. 1990. Annuqayah: Gerak Transformasi Sosial di Madura. Jakarta: P3M. Foucault, Michel. 1972. e Archaeology of Knowledge. New York: Pantheon Books. Foucault, Michel. 1977. Discipline and Punish; e birth of the Prison. Terj. Alan Sheridan. London-Worcester: Billing and Sons. Foucault, Michel. 1980a. “Prison Talk, Interview by J. Brochier”, in C. Gordon (ed). Power/Knowledge: Selected interview and Other Writings 1972-1977. New York: Pantheon Books. pp. 37-54. Khusyairi, Akhmad. 1989. “Agama, Orientasi Politik, dan Kepemimpinan Lokal di Antara Orang-Orang Madura”, dalam Huub de Jonge (ed), Agama, Kebudayaan dan Ekonomi. Jakarta: Rajawali Press. Madaniy, A. Malik. 1981/1982. Pola Motivasi Berhaji di Kalangan Masyarakat Madura. Jakarta: Proyek Peningkatan Sarana Pendidikan Tinggi Departemen Pendidikan dan Kebudayaan. Mansumoor, Iik Ariﬁn. 1990. Islam in an Indonesia World Ulama of Madura. Yogyakarta: Gadjah Mada Press. Mills, Sara. 2003. Michel Foucault. London dan New York: Routledge. Pribadi, Yanwar. 2013. Religious Networks In Madura; Pesantren, Nahdlatul Ulama and Kiai as the Core of Santri. Vol. 51. No. 1, Al-Jami'ah. Ritzer, George. 2003. Teori Sosial Postmodern. Jogjakarta: Kreasi Wacana. Turmudi, Endang. 2004. Perselingkuhan Kiai dan Kekuasaa. Yogyakarta: LKiS. Ummatin, Khoiro. 2002. Perilaku Politik Kiai. Yogjakarta: Pustaka pelajar. Walshaw, Margaret. 2007. Working with Foucault in education. Rotterdam: Sense Publishers. Percentage contribution of each author in the manuscript Salamet - 40% Arqom Kuswanjono - 30% Ridwan Ahmad Sukri - 30% Salamet - 40% Arqom Kuswanjono - 30% Ridwan Ahmad Sukri - 30% Salamet - 40% Arqom Kuswanjono - 30% Ridwan Ahmad Sukri - 30% 14
https://openalex.org/W3154241601	https://www.frontiersin.org/articles/10.3389/fmicb.2021.647164/pdf	English	null	Rapidly Improving High Light and High Temperature Tolerances of Cyanobacterial Cell Factories Through the Convenient Introduction of an AtpA-C252F Mutation	Frontiers in microbiology	2,021	cc-by	6,576	ORIGINAL RESEARCH published: 08 April 2021 doi: 10.3389/fmicb.2021.647164 Specialty section: Specialty section: This article was submitted to Microbiotechnology, a section of the journal Frontiers in Microbiology Keywords: cyanobacteria, Synechococcus elongatus PCC 7942, ATP synthase, sucrose, high temperature and high light tolerances Received: 29 December 2020 Accepted: 22 February 2021 Published: 08 April 2021 Shanshan Zhang1,2,3, Sini Zheng1,2,4, Jiahui Sun1,2,3, Xuexia Zeng1,2,4, Yangkai Duan1,2, Guodong Luan1,2* and Xuefeng Lu1,2,3,5,6* 1 Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China, 2 Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China, 3 College of Life Science, University of Chinese Academy of Sciences, Beijing, China, 4 College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, China, 5 Dalian National Laboratory for Clean Energy, Dalian, China, 6 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China INTRODUCTION With resource shortages and environmental pollution issues becoming increasingly prominent, photosynthetic biomanufacturing provides important options for the green and sustainable production of biofuels and biochemicals (Melis, 2009; Lu, 2010). Cyanobacteria are potential photosynthetic platforms and have been successfully engineered for production of multiple natural and non-natural products (Oliver and Atsumi, 2014). To put cyanobacterial photosynthetic biomanufacturing technology into practice, high light and high temperature tolerances would be important properties of the cyanobacterial cell factories, aiming to facilitate stable growth and production under stressful conditions during scaled outdoor cultivations (Luan and Lu, 2018). To improve high light and high temperature tolerances of cyanobacterial cells, complex genetic Keywords: cyanobacteria, Synechococcus elongatus PCC 7942, ATP synthase, sucrose, high temperature and high light tolerances Rapidly Improving High Light and High Temperature Tolerances of Cyanobacterial Cell Factories Through the Convenient Introduction of an AtpA-C252F Mutation Shanshan Zhang1,2,3, Sini Zheng1,2,4, Jiahui Sun1,2,3, Xuexia Zeng1,2,4, Yangkai Duan1,2, Guodong Luan1,2* and Xuefeng Lu1,2,3,5,6* Edited by: Tian Zhang, Wuhan University of Technology, China Photosynthetic biomanufacturing is a promising route for green production of biofuels and biochemicals utilizing carbon dioxide and solar energy. Cyanobacteria are important microbial platforms for constructing photosynthetic cell factories. Toward scaled outdoor cultivations in the future, high light and high temperature tolerances of cyanobacterial chassis strains and cell factories would be determinant properties to be optimized. We proposed a convenient strategy for rapidly improving high light and high temperature tolerances of an important cyanobacterial chassis Synechococcus elongatus PCC 7942 and the derived cell factories. Through introduction and isolation of an AtpA-C252F mutation, PCC 7942 mutants with improved high light and high temperature tolerances could be obtained in only 4 days with an antibiotics-free mode. Adopting this strategy, cellular robustness and sucrose synthesizing capacities of a PCC 7942 cell factory were successfully improved. Reviewed by: Lei Chen, Tianjin University, China Jinjin Diao, Washington University in St. Louis, United States Correspondence: Guodong Luan luangd@qibebt.ac.cn Xuefeng Lu lvxf@qibebt.ac.cn Citation: Zhang S, Zheng S, Sun J, Zeng X, Duan Y, Luan G and Lu X (2021) Rapidly Improving High Light and High Temperature Tolerances of Cyanobacterial Cell Factories Through the Convenient Introduction of an AtpA-C252F Mutation. Front. Microbiol. 12:647164. doi: 10.3389/fmicb.2021.647164 April 2021 \| Volume 12 \| Article 647164 Frontiers in Microbiology \| www.frontiersin.org 1 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. engineering strategies have been explored and adopted to modify or to update the native stress-response protective systems or the photosystems. For example, overexpression of speciﬁc heat shock proteins improved high temperature tolerance of cyanobacterial cells (Chaurasia and Apte, 2009; Su et al., 2017), and reduction of light harvesting antenna led to improved tolerances to high light stress (Kirst et al., 2014). However, implementations of these strategies rely on antibiotic resistance selection based on genetic manipulations and require long-term cultivations and passages for isolation of the homozygous mutant, and the entire cycle would usually take several weeks. consisting of transformation, initial screening, and rescreening. As shown in Figure 1, plasmids carrying tailored AtpA fragments would be transformed into PCC 7942 to induce homologous recombination. During the initial screening step, mutant cells obtaining the AtpA-C252 mutagenesis would survive under the selective conditions and form colonies on the agar plates. To exclude false positive results and to conﬁrm the tolerant phenotypes of the mutants, the colonies obtained from the initial screening step would be streaked and cultivated on fresh agar plates under the same selective conditions. The ﬁnal colonies would be collected for further assays and evaluations. Synechococcus elongatus PCC 7942 (PCC 7942 for short) is a widely used cyanobacterial chassis strain and has been engineered to be cell factories for producing dozens of chemicals, including ethanol (Deng and Coleman, 1999), 2,3-butanediol (Oliver et al., 2013), isobutyraldehyde (Atsumi et al., 2009), ethylene (Sakai et al., 1997), acetone (Chwa et al., 2016), 1-butanol (Lan and Liao, 2011), isopropanol (Kusakabe et al., 2013), 2-methyl-1-butanol (Shen and Liao, 2012), 1,2-propanediol (Li and Liao, 2013), sucrose (Ducat et al., 2012), hexose sugars (Niederholtmeyer et al., 2010), free fatty acid (Ruﬃng and Jones, 2012), and 3- hydroxypropionic acid (Lan et al., 2015). Moreover, PCC 7942 is a critical platform to explore mechanisms and strategies for optimizing stabilities and eﬃciency of photosynthetic carbon ﬁxation (Su et al., 2017; Ungerer et al., 2018; Yu et al., 2018). Rapid Isolation of PCC 7942 Mutants Carrying the AtpA-C252F Mutation y g p Among the four AtpA-C252 mutations endowing PCC 7942 with improved tolerances to high light and high temperature (Lou et al., 2018), we selected AtpA-C252F for proof-of-concept of the proposed strategy in this work. A plasmid carrying an AtpA-C252F gene fragment (termed as pSS18 in Figure 1) was constructed and transformed into PCC 7942 cells, and another plasmid carrying wild-type (WT) AtpA was used as a control. PCC 7942 tolerant transformants appeared on BG11 agar plates after only 48 h of cultivation in the selective conditions with high light and high temperature. More than 1,000 transformants were obtained on the screening plate of PCC 7942 (transformed with pSS18, AtpA-C252F), whereas no transformants appeared on PCC 7942 transformed with pSS3 (AtpA WT). Phenotypes of the transformants were further checked by re-screening under the same conditions, and 23 randomly selected transformants (from the initial screening step) all survived in the rescreening step. We further collected the 23 transformants (after the two-round selection) for AtpA sequence analysis, and the Sanger sequencing results revealed all of the strains carrying the AtpA-C252F mutation, indicating that the tolerances to high light and high temperature were endowed by the targeted mutagenesis. In addition, the sequencing results showed that all the mutants carrying the AtpA-C252F mutation existed as homozygous (as shown in Supplementary Figure 1), indicating that the signiﬁcant growth advantages caused by the AtpA-C252F facilitated eﬀective segregation of the mutated chromosomes. Previously, we have demonstrated that speciﬁc point mutations in FOF1 ATP synthase α subunit (AtpA), converting the 252nd amino acid from cysteine to any of the four conjugated amino acids (tyrosine/phenylalanine/tryptophan/histidine), could endow PCC 7942 cells with high temperature and high light resistances (Lou et al., 2018). Inspired by this, we proposed a convenient strategy for rapidly improving high light and high temperature tolerances of PCC 7942 derived cell factories by targeted mutagenesis of AtpA-C252. Compared with previously reported approaches, this new strategy has two potential advantages. First, the direct stress selection (with high light and high temperature) process would eliminate the dependence on introducing and screening antibiotics markers in the recombinant strains. Second, the growth advantages of the desired transformants would reduce the time required for cultivation and segregation. For proof-of-concept, we successfully isolated the PCC 7942 mutant strains with improved high light and high temperature tolerances that could be obtained in only 4 days in an antibiotics-free mode. Rapid Isolation of PCC 7942 Mutants Carrying the AtpA-C252F Mutation In addition, cellular robustness and sucrose synthesizing capacities of a previously constructed PCC 7942 cell factory were signiﬁcantly optimized, increasing the sucrose productivities by nearly onefold. High light and high temperature tolerances of the isolated mutants were further evaluated by liquid cultivation in column photobioreactors. As shown in Figure 2A, the growth of the PCC 7942 mutants carrying the AtpA-C252F mutation was similar to that of the control under normal conditions (30◦C and 50 µmol photons/m2/s). While in a stressful environment (44◦C and 400 µmol photons/m2/s), the mutant strain exhibited signiﬁcantly improved adaptabilities compared with the WT control and maintained rapid growth (Figure 2B). WT cells of PCC 7942 could not survive or grow facing the environmental stress, whereas OD730 of the AtpA-C252F mutant cells reached up to 7 with a signiﬁcantly improved growth rate compared with when cultivated in normal conditions (30◦C and 50 µmol photons/m2/s). The results indicated that Citation: Thus, the improvement of the eﬃciency and eﬀects on engineering PCC 7942 high light and high temperature tolerances will be of broad and important biotechnological signiﬁcance. Overview To facilitate rapid mutagenesis of AtpA-C252 and eﬀective isolation of the mutants, we designed a three-step procedure, April 2021 \| Volume 12 \| Article 647164 Frontiers in Microbiology \| www.frontiersin.org 2 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. FIGURE 1 \| Procedures for rapid isolation of high light and high temperature tolerant PCC 7942 mutants by introduction of the AtpA-C252F mutation. FIGURE 1 \| Procedures for rapid isolation of high light and high temperature tolerant PCC 7942 mutants by introduction of the AtpA-C252F mutation. FL130 was transformed with the pSS18 plasmid (AtpA- C252F) to conﬁrm the feasibility and eﬀectiveness of our strategy when adopted on cell factories. Following the described procedures, we successfully isolated high light and high temperature tolerant transformants of FL130 (Figure 3A). The ﬁnal isolated and veriﬁed FL130 mutant (AtpA-C252F) was termed as SZ41, which was able to grow under the conditions of 45◦C with 300 µmol photons/m2/s illumination (Figure 3B). the mutant strain of PCC 7942 obtained through the rapid isolation process displayed signiﬁcantly improved capacities to tolerate and utilize strong illuminations even when cultivated at high temperatures. AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory When cultivated under normal conditions of 30◦C and 100 µmol photons/m2/s, growths FL130 and SZ41 show minor diﬀerences (Figure 3C, FL130-NTL and SZ41-NTL), and the sucrose production reached up to 1,835 and 2,025 mg/L in 8 days, respectively (Figure 3D, FL130-NTL and SZ41-NTL). The slight increase of total carbon ﬁxation (the sum of biomass and sucrose) in SZ41 might result from an increased oxygen evolution rate (Figure 4A), indicating that the introduction of AtpA-C252F brought in beneﬁts on the overall eﬃciency or We further adopted this strategy to engineer a sucrose synthesizing cyanobacterial cell factory. Previously, we have introduced an Escherichia coli sourced sucrose permease CscB into PCC 7942 to facilitate the secretory production of sucrose under salts stress and overexpressed the native sucrose-phosphate synthase Sps to enhance sucrose synthesis (Duan et al., 2016; Qiao et al., 2018). The ﬁnal strain April 2021 \| Volume 12 \| Article 647164 Frontiers in Microbiology \| www.frontiersin.org 3 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. FIGURE 2 \| Effect of the AtpA-C252F mutation on cellular growth and tolerances of PCC 7942. Growth of the PCC 7942 strain with or without the AtpA-C252F mutation in liquid BG11 medium at 30◦C with 50 µmol photons/m2/s illumination (A) and at 44◦C with 400 µmol photons/m2/s illumination (B). Error bars indicate standard deviations (n = 3). FIGURE 3 \| Effects of the AtpA-C252F mutation on growth, tolerance, and sucrose synthesis of a PCC 7942 derived cell factory. (A) Tolerant transformants (with the AtpA-C252F mutation) of FL130 on BG11 agar plates after 2 days of cultivation under high light and high temperature conditions (45◦C with 300 µmol photons/m2/s illumination). (B) FL130 and the derived SZ41 strain (FL130-AtpA-C252F) grown on BG11 plates at 45◦C with 300 µmol photons/m2/s illumination. (C) Cell growth of FL130 and SZ41 under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions as measured by OD730. (D) Extracellular sucrose production of FL130 and SZ41. NaCl (150 mM) and IPTG (0.1 mM) were added to the BG11 medium (C D) Error bars indicate standard deviations (n ≥3) Statistical analysis was performed by using Student’s t test (p 0 05) FIGURE 2 \| Effect of the AtpA-C252F mutation on cellular growth and tolerances of PCC 7942. AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory Growth of the PCC 7942 strain with or without the AtpA-C252F mutation in liquid BG11 medium at 30◦C with 50 µmol photons/m2/s illumination (A) and at 44◦C with 400 µmol photons/m2/s illumination (B). Error bars indicate standard deviations (n = 3). FIGURE 2 \| Effect of the AtpA-C252F mutation on cellular growth and tolerances of PCC 7942. Growth of the PCC 7942 strain with or without the AtpA-C252F mutation in liquid BG11 medium at 30◦C with 50 µmol photons/m2/s illumination (A) and at 44◦C with 400 µmol photons/m2/s illumination (B). Error bars indicate standard deviations (n = 3). FIGURE 3 \| Effects of the AtpA-C252F mutation on growth, tolerance, and sucrose synthesis of a PCC 7942 derived cell factory. (A) Tolerant transformants (with the AtpA-C252F mutation) of FL130 on BG11 agar plates after 2 days of cultivation under high light and high temperature conditions (45◦C with 300 µmol photons/m2/s illumination). (B) FL130 and the derived SZ41 strain (FL130-AtpA-C252F) grown on BG11 plates at 45◦C with 300 µmol photons/m2/s illumination. (C) Cell growth of FL130 and SZ41 under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions as measured by OD730. (D) Extracellular sucrose production of FL130 and SZ41. NaCl (150 mM) and IPTG (0.1 mM) were added to the BG11 medium (C,D). Error bars indicate standard deviations (n ≥3). Statistical analysis was performed by using Student’s t-test (p < 0.05). FIGURE 3 \| Effects of the AtpA-C252F mutation on growth, tolerance, and sucrose synthesis of a PCC 7942 derived cell factory. (A) Tolerant transformants (with the AtpA-C252F mutation) of FL130 on BG11 agar plates after 2 days of cultivation under high light and high temperature conditions (45◦C with 300 µmol photons/m2/s illumination). (B) FL130 and the derived SZ41 strain (FL130-AtpA-C252F) grown on BG11 plates at 45◦C with 300 µmol photons/m2/s illumination. (C) Cell growth of FL130 and SZ41 under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions as measured by OD730. (D) Extracellular sucrose production of FL130 and SZ41. NaCl (150 mM) and IPTG (0.1 mM) were added to the BG11 medium (C,D). Error bars indicate standard deviations (n ≥3). Statistical analysis was performed by using Student’s t-test (p < 0.05). stability of cellular photosynthesis in PCC 7942 under these conditions (30◦C, 100 µmol photons/m2/s, 150 mM NaCl). AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory the role of carbon pool for synthesis of desired metabolites; thus, the enhanced glycogen synthesis activities and glycogen contents could be an additional advantage of the strategy developed in this work. SZ41 and FL130, respectively, under the high light and high temperature conditions (Figure 3D). The diﬀerent performances on cell growths and sucrose production between the two strains under the stressful conditions are in accordance with the photosynthesis activities diﬀerences revealed from the oxygen evolution rates. With enhanced illumination strengths (and increased temperatures), photosynthesis activities of FL130 and SZ41 were both signiﬁcantly elevated compared with these under normal conditions, and the oxygen evolution rate of SZ41 would be 40% higher than that of FL130 (Figure 4A). We also evaluated the eﬀects of this strategy on engineering an ethanologenic cell factory derived from PCC 7942. A previously optimized ethanologenic pathway consisting of the pyruvate decarboxylase from Zymomonas mobilis and a type II alcohol dehydrogenase from Synechocystis sp. PCC 6803 was introduced into PCC 7942 to generate the ZN44 strain, producing about 0.22 g/L ethanol in 2 days (Supplementary Figure 2). Introduction of the AtpA-C252F mutation into ZN44 signiﬁcantly improved the growth of the ethanologenic cell factory (ZN45) under high light conditions (OD730 of 7.2 in ZN45 compared with OD730 of 5.1 in ZN44 after 2 days of cultivation). However, the ethanol production was just slightly improved from 0.22 to 0.25 g/L, indicating that as for the PCC 7942 derived ethanologenic cell factory, the activities of the ethanol synthesis pathways rather than the total were holding control over the ethanol production capacities, and the increase of carbon ﬁxation could not be eﬀectively rewired into ethanol synthesis, which could be solved through further metabolic engineering. g It is noteworthy that the speciﬁc sucrose production on per cell levels was on similar levels between the two strains (Figure 4B) whether in normal or stressful conditions, indicating that the improved accumulation of sucrose in the SZ41 strain resulted from the optimized cell growth and enhanced carbon ﬁxation caused by the AtpA-C252F mutation, rather than a distribution of a larger portion of biomass to sucrose synthesis. After 4 days of high light and high temperature cultivation, bleaching phenotypes would be observed for both the SZ41 and FL130 cells, and the sucrose production would be terminated. AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory When stressful high light and high temperature conditions (40◦C and 400 µmol photons/m2/s) were adopted, SZ41 exhibited signiﬁcantly improved growth advantages over FL130. After 4 days of cultivation supplemented with 150 mM NaCl, the cell density of the SZ41 culture broth reached about 3, whereas that of the FL130 was lower than 2.0 (Figure 3C). The bleaching phenotypes might be caused by the synergy eﬀects of high light and high temperature stress and salts stress on the Synechococcus cells, and during this process, 1.8 and 0.98 g/L of extracellular sucrose were synthesized by April 2021 \| Volume 12 \| Article 647164 Frontiers in Microbiology \| www.frontiersin.org 4 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. FIGURE 4 \| Effects of the AtpA-C252F mutation on carbohydrate production of a PCC 7,942 derived cell factory. (A) Oxygen evolution rate of the FL130 and SZ41 strains under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions. (B) Speciﬁc sucrose productivities of the FL130 and SZ41 strains under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions. (C) Glycogen accumulation of the FL130 and SZ41 strains under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions during the initial 3 days of cultivation. Error bars indicate standard deviations (n ≥3). Statistical analysis was performed by using Student’s t-test (p < 0.01). FIGURE 4 \| Effects of the AtpA-C252F mutation on carbohydrate production of a PCC 7,942 derived cell factory. (A) Oxygen evolution rate of the FL130 and SZ41 strains under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions. (B) Speciﬁc sucrose productivities of the FL130 and SZ41 strains under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions. (C) Glycogen accumulation of the FL130 and SZ41 strains under normal (NTL, 30◦C with 100 µmol photons/m2/s illumination) and stressful (HTL, 40◦C with 400 µmol photons/m2/s illumination) conditions during the initial 3 days of cultivation. Error bars indicate standard deviations (n ≥3). Statistical analysis was performed by using Student’s t-test (p < 0.01). Frontiers in Microbiology \| www.frontiersin.org AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory Xudong Xu, Institute of Hydrobiology, Chinese Academy of Sciences PCC 7942-C252F Mutant strain of PCC 7942 harboring a AtpA with C252F point mutation Lou et al., 2018 FL130 Mutant strain of PCC 7942 harboring NS1::Ptrc-sps and NS3::Ptrc-cscB, Spr Cmr Duan et al., 2016 SZ41 Mutant strain derived from FL130, carrying atpA with the C252F point mutation This study ZN44 Mutant strain of PCC 7942 harboring NS1::PrbcL-pdcZM-slr1192, Spr This study ZN45 Mutant strain derived from ZN44, carrying AtpA with the C252F point mutation This study Plasmids pSS3 Carrying the wild-type PCC 7942 atpA gene This study pSS18 Carrying the PCC 7942 atpA mutant gene with the C252F mutation This study pZN45 Carrying the NS1UP-Spr-PrbcL-pdcZM-slr1192-NS1DOWN cassette This study aPtrc, trc promoter; cscB, proton/sucrose exporter gene from Escherichia coli; sps, encoding a natively fused protein of sucrose-phosphate synthase SPS; NS1 and NS3, different neutral sites in the genome of PCC 7942; Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Spr, spectinomycin resistance. TABLE 2 \| Primers used in this study. Primer Sequence (5′ →3′) Purpose AtpA-UP500-F-NdeI AAATTCATATGGGATGCAGCCCTATTCCGAA Amplifying the atpA gene AtpA-Down500-R-EcoRI AACCTGAATTCCGGGATTTGCTCCAAACCAC Amplifying the atpA gene AtpA-before-F GATTTCGATCAGTTTGGCCGCC Checking AtpA genotypes of Synechococcus elongatus PCC 7942 mutants AtpA-after-R TCGCGAATCGCCTTGAGGTTTG Checking AtpA genotypes of Synechococcus elongatus PCC 7942 mutants pSN44-NS1-r TTTGTTCGCCCAGCTTCTGTATGGCTCGAGCTTCT GGAGCAGGAAGATGT Amplifying the backbone of pSN44 pSN44-NS1-f TGCTCAGCCATAGTAAAAATTAGTCCCTGCTCGTC ACGCTTTCAGGCACC Amplifying the backbone of pSN44 pSN44-slr1192-r CTAATTTTTACTATGGCTGA Amplifying the Spr-PrbcL-pdcZM-slr1192 DNA fragment pSN44-sp-f GCATGCCCGTTCCATACAGA Amplifying the Spr-Prbcl-pdcZM-slr1192 DNA fragment different neutral sites in the genome of PCC 7942; Ap , ampicillin resistance; Cm , chloramphenicol resistance; Sp , spectinomycin resistance. TABLE 2 \| Primers used in this study. Primer Sequence (5′ →3′) Purpose AtpA-UP500-F-NdeI AAATTCATATGGGATGCAGCCCTATTCCGAA Amplifying the atpA gene AtpA-Down500-R-EcoRI AACCTGAATTCCGGGATTTGCTCCAAACCAC Amplifying the atpA gene AtpA-before-F GATTTCGATCAGTTTGGCCGCC Checking AtpA genotypes of Synechococcus elongatus PCC 7942 mutants AtpA-after-R TCGCGAATCGCCTTGAGGTTTG Checking AtpA genotypes of Synechococcus elongatus PCC 7942 mutants pSN44-NS1-r TTTGTTCGCCCAGCTTCTGTATGGCTCGAGCTTCT GGAGCAGGAAGATGT Amplifying the backbone of pSN44 pSN44-NS1-f TGCTCAGCCATAGTAAAAATTAGTCCCTGCTCGTC ACGCTTTCAGGCACC Amplifying the backbone of pSN44 pSN44-slr1192-r CTAATTTTTACTATGGCTGA Amplifying the Spr-PrbcL-pdcZM-slr1192 DNA fragment pSN44-sp-f GCATGCCCGTTCCATACAGA Amplifying the Spr-Prbcl-pdcZM-slr1192 DNA fragment transplantation to photosynthetic cell factories could be expected through similar procedures developed in this manuscript, aiming to optimize the growth or tolerance properties. In addition, to some gene deﬁciency-related growth advantages (meaning beneﬁts caused by loss of speciﬁc gene functions) (Joseph et al., 2014; Kirst et al., 2014), this strategy could be expected to work and facilitate the rapid isolation of the gene-deﬁcient mutants. AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory However, it is still noteworthy that the application of this strategy to other targets might be limited by the signiﬁcant degrees of the growth advantages endowed by speciﬁc mutations, which might not be as eﬀective as that from AtpA- C252 (stress tolerances coupled with growth advantages). To this end, systematic optimization of selective pressure strengths and methods would be required in order to achieve the ideal eﬀects of strain improvements. mutation. Adopting this strategy, cellular robustness and sucrose synthesizing capacities of the PCC 7942 derived cell factory were signiﬁcantly improved. AtpA-C252F Mutagenesis Rapidly Improves High Light and High Temperature Tolerances and Sucrose Production Rates of a PCC 7942 Cell Factory However, it is noteworthy that the AtpA-C252F mutation carrying the SZ41 strain under the stressful conditions could synthesize similar concentrations of sucrose utilizing half of the cultivation term as that obtained under normal conditions (1.8 g/L in 4 days versus 2.0 g/L in 8 days). In addition to sucrose, the SZ41 strain also accumulated higher intracellular glycogen contents than the FL130 under high light and high temperature conditions, conﬁrming the improved capacities for carbohydrates synthesis caused by the AtpA-C252F mutations (Figure 4C). Both sucrose and glycogen biosyntheses depend on the precursor glucose-1-phosphate, and it has been reported that the metabolic ﬂux through glucose-1-phosphate signiﬁcantly determines the ﬂexibility of the intracellular carbon distribution of cyanobacteria via a dynamic balance between diﬀerent metabolic branches (Luan et al., 2019). Furthermore, it has been reported that S. elongatus UTEX 2973, the fast-growing strain carrying AtpA-C252Y single nucleotide polymorphism (SNP) compared with PCC 7942, would overaccumulate glycogen to buﬀer the enhanced carbon ﬂux from the CBB cycle (Song et al., 2016; Tan et al., 2018). Glycogen storage serves as the main carbon sink mechanism of cyanobacterial cells and could play Compared with the recently reported fast-growing and robust cyanobacteria S. elongatus UTEX 2973, PCC 7942 still possesses the advantages of clear genetic background and more convenient genetic manipulation, and dozens of photosynthetic cell factories have been engineered based on this typical model strain. The strategy developed in this work provided a convenient approach to update the related PCC 7942 derived cell factories, which would avoid repeated implementations of the complex metabolic engineering with a new chassis. In addition, the success in this work provided a novel targeted mutagenesis and selective isolation manipulation paradigm for cyanobacterial phenotypes improvements. In recent years, many SNPs have been identiﬁed to be related with cellular growth or survival beneﬁts, e.g., acid tolerances (Uchiyama et al., 2015), alcohol tolerances (Hirokawa et al., 2018), high light and high temperature tolerances (Lou et al., 2018), and growth rates (Ungerer et al., 2018). Regarding such mutations endowing cells with speciﬁc growth advantages, rapid and convenient April 2021 \| Volume 12 \| Article 647164 Frontiers in Microbiology \| www.frontiersin.org 5 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. TABLE 1 \| Cyanobacterial strains and plasmids used in this study. Strain Genotypesa References PCC 7942 Synechococcus elongatus PCC 7942 wild-type Gifted by Prof. Chemicals and Reagents All reagents were purchased from Sigma-Aldrich (United States). Taq and pfu DNA polymerases for PCR were purchased from Transgene Biotech (Beijing, China). T4 DNA ligase and restriction enzymes were purchased from Fermentas (Canada) or New England Biolabs (Japan). The kits for molecular cloning were from Omega Bio-tek (Norcross, GA, United States). Oligonucleotides were synthesized, and DNA sequencing was performed by Tsingke (Qingdao, China). Frontiers in Microbiology \| www.frontiersin.org Construction of Plasmids and Strains Escherichia coli DH5α (TaKaRa, Dalian, China) was used as a host for cloning. The strains constructed and used in the present study are listed in Table 1. All the constructed plasmids employed pUC19 as backbone. To construct pSS3 and pSS18, AtpA-encoding gene containing 252C/252F was cloned from the chromosome of PCC 7942 and PCC 7942-C252F via PCR using the primers AtpA-UP500-F-NdeI and AtpA-Down500-R-EcoRI listed in Table 2. PCR products were puriﬁed using the E.Z.N.A High light and high temperature tolerances are important properties for cyanobacterial cell factories, aiming to put the photosynthetic biomanufacturing technology into practice. We developed a rapid and convenient strategy for improving cellular tolerances to high light and high temperature in an important cyanobacterial chassis PCC 7942 derived cell factory by antibiotics-free introduction of an AtpA-C252F April 2021 \| Volume 12 \| Article 647164 6 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. Cycle Pure Kit (Omega Bio-Tek, Norcross, GA, United States), and then the AtpA/AtpA-C252F fragments were digested with NdeI and EcoRI and inserted into pUC19 for constructing the target plasmids pSS3 and pSS18. To construct the ZN44 strain, a cassette containing the spectinomycin resistance gene, the PrbcL promoter sequence, the PdcZM gene, and the slr1192 gene was ampliﬁed from a previously constructed pZG25 plasmid (Gao et al., 2012) and ligated with the backbone of another previously constructed plasmid pFL20n (Duan et al., 2016) containing the upstream and downstream sequences of neutral site I (NS1). The ﬁnal obtained plasmid was termed as pZN44. All the resulting plasmids were then veriﬁed by Sanger gene sequencing and are listed in Table 1. Germany). The sensor was calibrated with air-saturated water and de-oxygenated water as 100% and 0% O2 levels. The culture broths of FL130 or SZ41 were transferred to the respiration vials and placed in the corresponding culture conditions. The oxygen concentration in the respiration vials was continuously monitored by the oxygen microsensor connected to the oxygen logger software. The ﬁnal photosynthetic oxygen evolution rate was calculated according to the Chla content, which was determined spectrophotometrically at OD665 and OD720 in methanol extracts and calculated with the formula: Chla (mg/L) = 12.9447 × (A665 −A720). Transformation of the PCC 7942 Strains Transformation of the PCC 7942 Strains Transformation of PCC 7942 derived cell factory FL130 was performed according to a previously reported method with optimization and quantitation (Lou et al., 2018). Brieﬂy, 2 ml WT or FL130 broth with an OD730 of 1.0 was centrifuged at 6,000 × g for 5 min and then re-suspended in 250 µl of fresh BG11 medium. Next, 200 ng plasmid was added and incubated in the dark at 150 rpm and 30◦C for 6–24 h. Then, 5 µl of the mixtures (about 4 × 106 cells) was plated on solid BG11 plates with 1.5% agar after transformation and incubated at 45◦C and 300 µmol photons/m2/s for 2 days. The obtained colonies would be picked, streaked on fresh BG11 plates, and incubated for another 24 h under the same conditions. Genotypes of the ﬁnal survived transformants would be checked, and the atpA gene would be ampliﬁed for Sanger sequencing. To determine the amount of extracellular sucrose in PCC 7942 culture broth, 1 ml of cyanobacterial culture was centrifuged, and sucrose in the supernatant was measured using the sucrose/D-glucose assay kit (Megazyme). The glycogen contents in PCC 7942 cells were measured as previously introduced with modiﬁcations (Chi et al., 2019). After the glycogen precipitants were hydrolyzed to glucose by treatment with amyloglucosidase at 60◦C for 2 h, glucose in 100 mM sodium acetate (pH 4.5) solution was measured using the sucrose/D-glucose assay kit (Megazyme). FUNDING This work was supported by the National Natural Science Foundation of China (grant numbers 31525002, 31872624, 31770092, 32070084, and 31700048), Strategic Priority Research Program of the Chinese Academy of Sciences (Transformational Technologies for Clean Energy and Demonstration, XDA21010211), and Shandong Taishan Scholarship (to XL). This work was supported by the National Natural Science Foundation of China (grant numbers 31525002, 31872624, 31770092, 32070084, and 31700048), Strategic Priority Research Program of the Chinese Academy of Sciences (Transformational Technologies for Clean Energy and Demonstration, XDA21010211), and Shandong Taishan Scholarship (to XL). Cultivations of Cyanobacterial Strains Cultivations of Cyanobacterial Strains For phenotypes evaluations, PCC 7942 WT and the derived strains would be cultivated in BG11 medium with column photobioreactors (3 cm diameter), and the temperature and illumination strengths would be set as required. The light would be provided by incandescent lamps (NVC, NDL433SI-28W). Then, 3% (volume to volume) CO2 air would be bubbled for carbon source supplementations. For liquid cultivation of the WT PCC 7942 and the derived mutant carrying the AtpA-C252F mutation, the normal conditions were set as 30◦C and 50 µmol photons/m2/s, and the respective stressful conditions were set as 44◦C and 400 µmol photons/m2/s. For sucrose production, 150 mM NaCl and 0.1 mM isopropyl-D-1-thiogalactopyranoside (IPTG) would be added to the culture medium. During this process, normal conditions were set as 30◦C and 100 µmol photons/m2/s, whereas stressful conditions were set as 40◦C and 400 µmol photons/m2/s. The growth of each cyanobacterial strain was monitored by measuring the optical density at 730 nm (OD730). Dry cell weights (DCWs) were determined following the methods previously described (Wang et al., 2019). As for the cultivation of ethanologenic strains, the conditions were also set as 30◦C and 400 µmol photons/m2/s, and the ethanol production would be evaluated as previously described (Wang et al., 2019). 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SUPPLEMENTARY MATERIAL Oxygen Evolution Rates Determination Oxygen concentration was measured using ﬁber-based and contactless oxygen microsensors (PyroScience, Aachen, The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.647164/full#supplementary-material Oxygen concentration was measured using ﬁber-based and contactless oxygen microsensors (PyroScience, Aachen, April 2021 \| Volume 12 \| Article 647164 Frontiers in Microbiology \| www.frontiersin.org 7 AtpA-C252F Facilitates Rapid Phenotype Improvement Zhang et al. REFERENCES Genomic analysis of parallel-evolved cyanobacterium Synechocystis sp. PCC 6803 under acid stress. Photosynth Res. 125, 243–254. doi: 10.1007/s11120- 015-0111-3 Kusakabe, T., Tatsuke, T., Tsuruno, K., Hirokawa, Y., Atsumi, S., Liao, J. C., et al. (2013). Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light. Metab. 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Microbiol. 84, e1218–e1222. doi: 10.1128/AEM.01222-18 Copyright © 2021 Zhang, Zheng, Sun, Zeng, Duan, Luan and Lu. 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. REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Appl. Environ. Microbiol. 84, e1218–e1222. doi: 10.1128/AEM.012 Lu, X. (2010). A perspective: photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria. Biotechnol. Adv. 28, 742–746. doi: 10. 1016/j.biotechadv.2010.05.021 Luan, G., and Lu, X. (2018). Tailoring cyanobacterial cell factory for improved industrial properties. Biotechnol. 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https://openalex.org/W4296953383	https://link.springer.com/content/pdf/10.1007/s10549-022-06726-w.pdf	English	null	Clinical significance of the neutrophil-to-lymphocyte ratio in oligometastatic breast cancer	Breast cancer research and treatment	2,022	cc-by	5,976	Abstract Purpose This study investigated the clinical impact of pretreatment neutrophil-to-lymphocyte ratio (NLR) on survival in patients with oligometastatic breast cancer. Patients and methods We collected data from 397 patients who underwent primary breast surgery from 2004 to 2015 and developed recurrence during the follow-up. We reviewed the images and clinical information and defined OMD according to the European Society for Medical Oncology advanced breast cancer guidelines. The NLR was calculated using pretreat- ment data of primary breast cancer. The cutoff value of the NLR was determined by receiver operating characteristic curve with Youden Index.i Results Among 397 patients, 131 had OMD at recurrence. The low-NLR group included patients of significantly older age at primary cancer than those in the high-NLR group. A low NLR indicated a better overall survival (p = 0.023) after adjusting for relevant factors, including estrogen receptor status, surgical resection of metastatic disease, metastatic organ number, disease-free interval, and liver metastasis than did the high-NLR group. We developed prognostic models for OMD using six independent prognostic factors, including the NLR. The number of factors was associated with overall survival; patients with all six favorable factors showed a good overall survival of 90.9% at 8 years and those with four or more factors showed 70.4%. Conclusions The NLR was an independent prognostic factor for overall survival in OMD. The number of favorable prog- nostic factors was associated with overall survival. A prognostic model, including the NLR, will help identify patients with a favorable prognosis. Keywords Oligometastatic breast cancer · Neutrophil-to-lymphocyte ratio · NLR · Oligometastatic disease · Breast cancer Keywords Oligometastatic breast cancer · Neutrophil-to-lymphocyte ratio · NLR · Oligometastat CLINICAL TRIAL CLINICAL TRIAL Clinical significance of the neutrophil‑to‑lymphocyte ratio in oligometastatic breast cancer Yuka Inoue1 · Makoto Fujishima2 · Makiko Ono3 · Jun Masuda4 · Yukinori Ozaki4 · Tetsuyo Maeda1 · Natsue Uehiro1 · Yoko Takahashi1 · Takayuki Kobayashi4 · Takehiko Sakai1 · Tomo Osako5 · Takayuki Ueno1 · Shinji Ohno6 Yuka Inoue1 · Makoto Fujishima2 · Makiko Ono3 · Jun Masuda4 · Yukinori Ozaki4 · Tetsuyo Maeda1 · Natsue Uehiro1 · Yoko Takahashi1 · Takayuki Kobayashi4 · Takehiko Sakai1 · Tomo Osako5 · Takayuki Ueno1 · Shinji Ohno6 Received: 7 July 2022 / Accepted: 22 August 2022 / Published online: 24 September 2022 © The Author(s) 2022 Extended author information available on the last page of the article * Takayuki Ueno takayuki.ueno@jfcr.or.jp Extended author information available on the last page of the article Breast Cancer Research and Treatment (2022) 196:341–348 https://doi.org/10.1007/s10549-022-06726-w Breast Cancer Research and Treatment (2022) 196:341–348 https://doi.org/10.1007/s10549-022-06726-w CLINICAL TRIAL * Takayuki Ueno takayuki.ueno@jfcr.or.jp Introduction between locally confined and systemically metastatic disease in 1995 [9]. Although patients with OMD are considered as potentially curable [6], there are no clear diagnostic crite- ria or treatment guidelines for OMD, and it is unclear who will benefit from treatment of curative intent. The European Society for Medical Oncology (ESMO) advanced breast can- cer (ABC) guidelines define OMD as low volume metastatic disease with a limited number and size of metastatic lesions (up to five lesions and not necessarily in the same organ) [10]. Conversely, German experts define it as a limited num- ber of metastases in one body organ [11]. Because metastatic breast cancer (MBC) is an incurable disease, systemic therapy for palliation is the standard of care for affected patients [1, 2]. Approximately 30%–40% of patients with MBC will develop widespread metastases [3–5]. Although the advancement of systemic therapy for MBC has led to improved overall survival (OS), the median OS ranges widely from 8 months to 4 years [6–8]. However, in clinical practice, some patients achieve the status of no evident disease or long-term survival with controlled MBC.i Hellman and Weichselbaum first proposed the concept of oligometastatic disease (OMD) as a distinct cancer state Recent reports have shown that the neutrophil-to-lym- phocyte ratio (NLR) correlates with survival in patients with cancer, including breast cancer [12, 13]. Although inflammatory cells and mediators in the tumor microen- vironment play an important role in cancer progression, which may be reflected by systemic immune status [14], (0121 3456789) 3 3456789) 3 Breast Cancer Research and Treatment (2022) 196:341–348 342 significantly older age at primary breast cancer than those in the high-NLR group (p = 0.0026). There were no sig- nificant differences in clinical stage or subtype between the two groups. More patients received endocrine therapy as adjuvant therapy in the high-NLR group than in the low- NLR group (p = 0.0447). the clinical significance of NLR in oligometastatic breast cancer requires elucidation. This study investigated the clini- cal impact of pretreatment NLR on the survival of patients with oligometastatic breast cancer to help identify patients who will benefit from treatment of curative intent. Table 2 shows the clinical characteristics of patients at recurrence and the treatment for OMD by the NLR groups. The low-NLR group included patients of significantly older age at recurrence than those in the high-NLR group (p = 0.0037). Patients and methods We analyzed the data from 397 patients who underwent primary breast surgery from 2004 to 2015 and developed recurrence in sites other than central nervous system during follow-up in our institution. Their (neo)adjuvant therapy was administered based on the guidelines of the Japanese Breast Cancer Society [15]. We reviewed the images and clini- cal information and defined OMD according to the ESMO ABC guidelines [10]. The NLR was defined as the absolute blood neutrophil count divided by the absolute lymphocyte count in the peripheral blood and was calculated using pre- treatment data of the patients at the time of primary breast cancer. The cutoff value of the NLR was determined by receiver operating characteristics curve analysis using the Youden index. OS was defined as the period from the day of diagnosis of breast cancer recurrence until the day of death from any cause. JMP software, version 8.0.0 (SAS Institute, Cary, NC), was used for all statistical analyses. The results are expressed as the mean ± standard deviation or number. Group differences in continuous variables were assessed by the Mann–Whitney test. Group differences in categorical variables were assessed by the chi-squared test. Survival curves were plotted by the Kaplan–Meier method and com- pared by the log-rank test. Survival data were evaluated using a multivariate Cox proportional hazards model. Dif- ferences were considered significant at two-sided p < 0.05. A prognostic model for OMD was developed using favorable prognostic factors on multivariate analysis. Harrell's C-index was used to evaluate the prognostic strength of models [16]. Figure 1A shows the OS of patients with OMD by the NLR groups. Those in the low NLR had better OS (p = 0.023) after adjusting for estrogen receptor (ER) status, age at recurrence, (neo)adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis (Table 3). In addition, no (neo)adjuvant chemotherapy (p = 0.0044), surgical resection (p = 0.0416), single metastatic organ (p = 0.05), DFI > 2 years (p = 0.007), and no liver metastasis (p = 0.02) were independent favorable prognostic factors (Table 3). In the whole population of patients with recur- rent breast cancer, the NLR was prognostic (p = 0.0052) while it was not in patients with recurrent breast cancer without OMD (p = 0.134) (Supplementary Fig. 2A and B). Thus, the prognostic significance of NLR was more prominent for patients with OMD than for those without. Introduction There were no significant differences in disease-free interval (DFI), number of metastatic organs, number of metastatic lesions, presence/absence of distant metastasis, or site of metastasis between the two groups. Moreover, there were no significant differences in the 1st- line therapy for OMD after recurrence between the two groups (Table 2). Patients and methods To select patients with favorable prognosis likely to benefit from treatment of curative intent, prognosis was compared according to the number of favorable prognostic factors, including no (neo)adjuvant chemotherapy, surgical resection, single metastatic organ, DFI > 2 years, no liver metastasis, and low NLR. The number of prognostic fac- tors was associated with OS (p < 0.001) (Fig. 1B). The OS at 8 years was calculated based on the number of included factors (Table 4). Patients with all six of the favorable fac- tors showed an excellent 8-year OS of 90.9% (p = 0.001; hazard ratio [HR] = 9.14), while patients with five or more factors showed an OS of 75.6% (p = 0.0025) and those with four or more factors showed an OS of 70.4% (p < 0.001) (Supplementary Fig. 3). The concordance of each model was evaluated using Harrell's C-index, with a C-index of 0.737 for the model with NLR, which was higher than for the model without NLR (a C-index of 0.72). For the pre- diction of 5-year OS, we constructed a nomogram using these predictive factors (Fig. 2). Results Among 397 cases with recurrent breast cancer, 131 cases (33%) had OMD. The median follow-up from recurrence was 59 months (range 6–151 months). The cutoff value of the NLR was 2.52 by the Youden index using data from patients with OMD, and 93 patients (71%) were classified in the low-NLR group. The absolute counts of neutrophil and lymphocyte were plotted on a graph (Supplemen- tary Fig. 1). Table 1 shows the background characteris- tics of patients at primary breast cancer according to the NLR groups. The low-NLR group included patients of 1 3 3 Breast Cancer Research and Treatment (2022) 196:341–348 Breast Cancer Research and Treatment (2022) 196:341–348 343 Table 1 The background characteristics of patients with primary breast cancer according to NLR group NLR Neutrophil-to-lymphocyte ratio, ER estrogen receptor, PR progesterone receptor, HER2 human epi- dermal growth factor receptor type2, Statistically significant P values are shown in bold. High-NLR n = 38 Low-NLR n = 93 P value Age at primary breast cancer (median, range) 47 (29–73) 55 (27–86) 0.0026 cStage 0 0 (0) 3 (3) 0.40 I 6 (16) 18 (19) II 18 (47) 49 (53) III 14 (37) 23 (25) ER − 6 (16) 31 (33) 0.054 + 32 (84) 62 (67) PR − 18 (47) 50 (54) 0.56 + 20 (53) 43 (46) HER2 − 31 (82) 72 (77) 0.65 + 7 (18) 23 (21) Subtype HR+HER2− 28 (74) 55 (59) 0.16 HR+HER2+ 5 (13) 8 (9) HR−HER2+ 2 (5) 12 (13) HR−HER2− 3 (8) 18 (19) Adjuvant endocrine therapy No 8 (21) 37 (40) 0.0447 Yes 30 (79) 56 (60) (Neo)adjuvant chemotherapy No 7 (18) 24 (26) 0.50 Yes 31 (82) 69 (74) (Neo)adjuvant anti-HER2 therapy No 35 (92) 78 (84) 0.27 Yes 3 (8) 15 (16) (%) (%) NLR Neutrophil-to-lymphocyte ratio, ER estrogen receptor, PR progesterone receptor, HER2 human epi- dermal growth factor receptor type2, Statistically significant P values are shown in bold. Results High-NLR n = 38 Low-NLR n = 93 P value Age at primary breast cancer (median, range) 47 (29–73) 55 (27–86) 0.0026 cStage 0 0 (0) 3 (3) 0.40 I 6 (16) 18 (19) II 18 (47) 49 (53) III 14 (37) 23 (25) ER − 6 (16) 31 (33) 0.054 + 32 (84) 62 (67) PR − 18 (47) 50 (54) 0.56 + 20 (53) 43 (46) HER2 − 31 (82) 72 (77) 0.65 + 7 (18) 23 (21) Subtype HR+HER2− 28 (74) 55 (59) 0.16 HR+HER2+ 5 (13) 8 (9) HR−HER2+ 2 (5) 12 (13) HR−HER2− 3 (8) 18 (19) Adjuvant endocrine therapy No 8 (21) 37 (40) 0.0447 Yes 30 (79) 56 (60) (Neo)adjuvant chemotherapy No 7 (18) 24 (26) 0.50 Yes 31 (82) 69 (74) (Neo)adjuvant anti-HER2 therapy No 35 (92) 78 (84) 0.27 Yes 3 (8) 15 (16) (%) (%) Table 1 The background characteristics of patients with primary breast cancer according to NLR group NLR Neutrophil-to-lymphocyte ratio, ER estrogen receptor, PR progesterone receptor, HER2 human epi- dermal growth factor receptor type2, Statistically significant P values are shown in bold. Some studies have suggested that local treatment for metastatic lesions, such as surgery and radiotherapy, improves the survival of patients with OMD [18] The SABR-COMET trial, a phase 2 randomized trial, dem- onstrated that stereotactic ablative radiotherapy improved the prognosis of patients with OMD from different pri- mary cancers, including breast cancer [19]. The SABR- COMET-3 trial, a phase 3 trial of the same concept, including breast cancer patients with OMD, is currently underway [20]. Some studies have examined outcomes after surgical resection of lung, liver, and brain metastases and suggested good long-term disease control and sur- vival for selected patients [21–24]. There are also some case–control studies suggesting a survival benefit from surgical resection of metastatic lesions in patients with breast OMD [25–30]. However, these studies are retro- spective, limited by number of patients, and conducted in Discussion This study showed that a low NLR was an independent favorable prognostic factor for breast OMD. Although the association between the NLR and prognosis in breast cancer has been reported in multiple studies [12, 13], to the best of our knowledge, this is the first report to show the prognos- tic significance of the NLR in OMD. A recent systematic review of breast OMD demonstrated that comparing to those without, patients with solitary metastasis, > 24-month inter- val between primary tumor and OMD, no or limited involved axillary lymph nodes at primary diagnosis, and hormone- receptor positivity were associated with better prognosis [17]. However, the NLR was not considered in that study. Our study results may enable clinicians to better predict the prognosis of patients with OMD by considering the NLR in the prognostic model. 1 3 Breast Cancer Research and Treatment (2022) 196:341–348 344 OMD oligometastatic disease, DFI disease-free interval, RT radiotherapy, Statistically significant P values are shown in bold. A breakdown of the anti-HER2 therapy is shown in supplementary Table 1 High-NLR n = 38 Low-NLR n = 93 P value Age at recurrence(median, range) 49 (30–78) 57 (31–86) 0.0037 DFI(month, median, range) 30 (6–97) 30 (2–98) 0.60 Number of metastatic organs 1 36 (95) 83 (89) 0.57 2 2 (5) 9 (10) 3 0 (0) 1 (1) Number of metastatic lesions 1 20 (53) 57 (62) 0.73 2 7 (18) 16 (17) 3 7 (18) 13 (14) 4 3 (8) 3 (3) 5 1 (3) 3 (3) Distant metastasis 1.00 Yes 25 (66) 62 (67) No 13 (34) 31 (33) Site of metastasis Local 4 (11) 13 (14) 0.77 Region LN 11 (29) 23 (25) 0.66 Bone 12 (32) 27 (29) 0.83 Liver 7 (18) 16 (17) 1.00 Lung/pleura 5 (13) 19 (20) 0.46 Distant LN 1 (3) 6 (6) 0.67 Surgery for OMD No 29 (76) 63 (71) 0.66 Yes 9 (24) 26 (29) RT for OMD No 32 (84) 75 (81) 0.80 Yes 6 (16) 18 (19) 1st line Chemotherapy 11 (30) 44 (47) 0.079 Endocrine therapy 24 (65) 45 (48) 0.11 Anti-HER2 therapy 5 (13) 18 (19) 0.61 (%) (%) Table 2 The clinical characteristics at recurrence and the treatment for OMD according to NLR groups with favorable prognosis would help to indicate those who would benefit from intensive treatment of curative intent, including surgery. Discussion OMD oli- gometastatic disease, DFI disease-free survival, NLR neutrophil-to- lymphocyte ratio Table 3 Multivariate analysis for the overall survival ER estrogen receptor, CI confidence interval, DFI disease-free interval, NLR neutrophil-to-lymphocyte ratio, Statistically significant P values are shown in bold. Variable Multivariate analysis HR (95%CI) P value ER Positive vs. negative 0.91 (0.52–1.62) 0.74 Age at recurrence < 50 vs. 50- 0.59 (0.33–1.08) 0.09 (neo) adjuvant chemotherapy No vs. yes 0.38 (0.17–0.75) 0.0044 Surgical resection Yes vs. no 0.53 (0.27–0.98) 0.0416 Number of metastatic organs 1 vs. 2 or more 0.45 (0.23–1.00) 0.05 DFI (year) 2- vs. < 2 0.40 (0.23–0.70) 0.007 Liver metastasis No vs. yes 0.50 (0.29–0.89) 0.02 NLR Low vs. High 0.52 (0.30–0.91) 0.023 NLR-low(n=93) NLR-high(n=38) Time after recurrence (year) Overall survival 0 1 0 0 20 40 60 100 80 p = 0.09 (log-rank test) p = 0.023 (adjusted) 5 Time after recurrence (year) Overall survival 0 20 40 60 100 80 0 10 6 (n=11) 5 (n=10) 4 (n=41) 3 (n=41) 2 (n=26) 1 (n=2) p<0.001 5 A B Fig. 1 A Kaplan–Meier survival curves for overall survival in patients with OMD by NLR. A low NLR indicated better survival (p = 0.023) after adjusting for estrogen receptor status, age at recur- rence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis, than did a high NLR. adjusted for estrogen receptor status, age at recurrence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis. B Kaplan–Meier survival curves for overall survival in patients with OMD by the number of prognostic factors for OMD. Survival curves were drawn according to the number of the favorable prognostic factors, including no (neo)adjuvant chemotherapy, surgical resection, single metastatic organ, DFI > 2 years, no liver metastasis, and low NLR. The number of the factors was associated with post-recurrence survival. OMD oli- gometastatic disease, DFI disease-free survival, NLR neutrophil-to- lymphocyte ratio Time after recurrence (year) Overall survival 0 20 40 60 100 80 0 10 6 (n=11) 5 (n=10) 4 (n=41) 3 (n=41) 2 (n=26) 1 (n=2) p<0.001 5 B NLR-low(n=93) NLR-high(n=38) Time after recurrence (year) Overall survival 0 1 0 0 20 40 60 100 80 p = 0.09 (log-rank test) p = 0.023 (adjusted) 5 A B Fig. 1 A Kaplan–Meier survival curves for overall survival in patients with OMD by NLR. Discussion Limitations One of the major limitations in this study was its small number of patients, which resulted partly from it being a Table 4 The association the number of the prognostic factors and overall survival OS overall survival Number of favorable prog- nostic factors OS at 8 years (%) HR P value 6 90.9 9.14 0.001 ≥ 5 75.6 3.68 0.0025 ≥ 4 70.4 3.91 < 0.001 Table 4 The association the number of the prognostic factors and overall survival OS overall survival Number of favorable prog- nostic factors OS at 8 years (%) HR P value 6 90.9 9.14 0.001 ≥ 5 75.6 3.68 0.0025 ≥ 4 70.4 3.91 < 0.001 single institutional study. Therefore, the survival analysis of this population needs to be interpreted with caution. We are planning a multicenter study with a larger population to confirm the results of this study. Another limitation is that the NLR at the time of recurrence could not be calcu- lated because white blood cell fractions were not measured in all patients at recurrence. However, our result suggested that the NLR at primary cancer impacted survival even after recurrence, indicating the importance of the primary immune status throughout the disease course. The differ- ence in the proportion of patients given adjuvant endo- crine therapy between the two groups is another limitation (Table 1). Because female hormones have been reported to affect T-cell proliferation and neutrophil counts [31, 32], adjuvant endocrine therapy may have affected the sys- temic immune status. To reduce such a bias, we included ER status, which was associated with administration of adjuvant endocrine therapy, in the multivariate analysis, single institutional study. Therefore, the survival analysis of this population needs to be interpreted with caution. We are planning a multicenter study with a larger population to confirm the results of this study. Another limitation is that the NLR at the time of recurrence could not be calcu- lated because white blood cell fractions were not measured in all patients at recurrence. However, our result suggested that the NLR at primary cancer impacted survival even after recurrence, indicating the importance of the primary immune status throughout the disease course. The differ- ence in the proportion of patients given adjuvant endo- crine therapy between the two groups is another limitation (Table 1). Discussion Our results suggest that the addition of the NLR to conventional prognostic factors would be use- ful in such a prognostic model. highly selective cohorts; thus, a selection bias cannot be avoided. Indeed, the Korean case–control study showed better survival in patients with surgical resection of pul- monary metastases than in patients without surgery, but in the multivariate analysis, surgical resection did not remain an independent prognostic factor [26]. Therefore, it is unclear whether surgery itself contributes to the improved prognosis of patients with OMD. However, these stud- ies do not exclude the possibility that surgical resection of metastatic lesions may provide some survival benefit in highly selected patients with favorable prognosis. A refined prognostic model that can select these patients Most studies that examined prognostic factors in OMD focused on tumor-related factors, such as the number of metastatic lesions, metastatic organs, and tumor subtypes. However, it is now clear that host-related factors also affect patient prognosis. In this study, we showed that the NLR at primary diagnosis indicated the survival in patients with 3 3 Breast Cancer Research and Treatment (2022) 196:341–348 345 NLR-low(n=93) NLR-high(n=38) Time after recurrence (year) Overall survival 0 1 0 0 20 40 60 100 80 p = 0.09 (log-rank test) p = 0.023 (adjusted) 5 Time after recurrence (year) Overall survival 0 20 40 60 100 80 0 10 6 (n=11) 5 (n=10) 4 (n=41) 3 (n=41) 2 (n=26) 1 (n=2) p<0.001 5 A B Fig. 1 A Kaplan–Meier survival curves for overall survival in patients with OMD by NLR. A low NLR indicated better survival (p = 0.023) after adjusting for estrogen receptor status, age at recur- rence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis, than did a high NLR. adjusted for estrogen receptor status, age at recurrence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis. B Kaplan–Meier survival curves for overall survival in patients with OMD by the number of prognostic factors for OMD. Survival curves were drawn according to the number of the favorable prognostic factors, including no (neo)adjuvant chemotherapy, surgical resection, single metastatic organ, DFI > 2 years, no liver metastasis, and low NLR. The number of the factors was associated with post-recurrence survival. Discussion A low NLR indicated better survival (p = 0.023) after adjusting for estrogen receptor status, age at recur- rence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis, than did a high NLR. adjusted for estrogen receptor status, age at recurrence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis. B Kaplan–Meier Fig. 1 A Kaplan–Meier survival curves for overall survival in patients with OMD by NLR. A low NLR indicated better survival (p = 0.023) after adjusting for estrogen receptor status, age at recur- rence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis, than did a high NLR. *adjusted for estrogen receptor status, age at recurrence, (neo) adjuvant chemotherapy, surgical resection of metastatic disease, metastatic organ number, DFI, and liver metastasis. B Kaplan–Meier survival curves for overall survival in patients with OMD by the number of prognostic factors for OMD. Survival curves were drawn according to the number of the favorable prognostic factors, including no (neo)adjuvant chemotherapy, surgical resection, single metastatic organ, DFI > 2 years, no liver metastasis, and low NLR. The number of the factors was associated with post-recurrence survival. OMD oli- gometastatic disease, DFI disease-free survival, NLR neutrophil-to- lymphocyte ratio ER estrogen receptor, CI confidence interval, DFI disease-free interval, NLR neutrophil-to-lymphocyte ratio, Statistically significant P values are shown in bold. Variable Multivariate analysis HR (95%CI) P value ER Positive vs. negative 0.91 (0.52–1.62) 0.74 Age at recurrence < 50 vs. 50- 0.59 (0.33–1.08) 0.09 (neo) adjuvant chemotherapy No vs. yes 0.38 (0.17–0.75) 0.0044 Surgical resection Yes vs. no 0.53 (0.27–0.98) 0.0416 Number of metastatic organs 1 vs. 2 or more 0.45 (0.23–1.00) 0.05 DFI (year) 2- vs. < 2 0.40 (0.23–0.70) 0.007 Liver metastasis No vs. yes 0.50 (0.29–0.89) 0.02 NLR Low vs. High 0.52 (0.30–0.91) 0.023 Table 3 Multivariate analysis for the overall survival Table 3 Multivariate analysis for the overall survival ER estrogen receptor, CI confidence interval, DFI disease-free interval, NLR neutrophil-to-lymphocyte ratio, Statistically significant P values are shown in bold. breast OMD, probably because it may reflect the host anti- cancer immune status. Discussion Because female hormones have been reported to affect T-cell proliferation and neutrophil counts [31, 32], adjuvant endocrine therapy may have affected the sys- temic immune status. To reduce such a bias, we included ER status, which was associated with administration of adjuvant endocrine therapy, in the multivariate analysis, Table 4 The association the number of the prognostic factors and overall survival breast OMD, probably because it may reflect the host anti- cancer immune status. References 1. Senkus E et al (2015) Primary breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 26(Suppl 5):v8-30 1. Senkus E et al (2015) Primary breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 26(Suppl 5):v8-30 Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. pp 2. Kwapisz D (2019) Oligometastatic breast cancer. Breast Cancer 26(2):138–146 pp 2. Kwapisz D (2019) Oligometastatic breast cancer. Breast Cancer 26(2):138–146 3. Reyes DK, Pienta KJ (2015) The biology and treatment of oli- gometastatic cancer. Oncotarget 6(11):8491–8524 3. Reyes DK, Pienta KJ (2015) The biology and treatment of oli- gometastatic cancer. Oncotarget 6(11):8491–8524 Limitations One of the major limitations in this study was its small number of patients, which resulted partly from it being a 1 3 346 Breast Cancer Research and Treatment (2022) 196:341–348 Breast Cancer Research and Treatment (2022) 196:341–348 Fig. 2 Nomogram for predic- tion of 5-year overall survival. A nomogram was constructed using the prognostic factors included in our model to predict 5-year overall survival. DFI disease-free survival, NLR neutrophil-to-lymphocyte ratio <2 2- 1 yes no yes no no yes high low 0 50 100 150 200 250 300 350 400 450 500 550 0.02 0.05 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.94 DFI Number of metastatic organs Liver metastasis (neo) adjuvant chemotherapy Surgical resection NLR Total points 5 year-overall survival 0 10 20 30 40 50 60 70 80 90 100 Points 2- which showed that the NLR was prognostic independent of ER status (Table 3). Ethical approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the institu- tional Ethics Committee. Fig. 2 Nomogram for predic- tion of 5-year overall survival. A nomogram was constructed using the prognostic factors included in our model to predict 5-year overall survival. DFI disease-free survival, NLR neutrophil-to-lymphocyte ratio <2 2- 1 yes no yes no no yes high low 0 50 100 150 200 250 300 350 400 450 500 550 0.02 0.05 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.94 DFI Number of metastatic organs Liver metastasis (neo) adjuvant chemotherapy Surgical resection NLR Total points 5 year-overall survival 0 10 20 30 40 50 60 70 80 90 100 Points 2- 5 year-overall survival Ethical approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the institu- tional Ethics Committee. which showed that the NLR was prognostic independent of ER status (Table 3). Ethical approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the institu- tional Ethics Committee. which showed that the NLR was prognostic independent of ER status (Table 3). Consent for publication All authors agree to publish this article. Conclusion Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The NLR was an independent prognostic factor for OS in OMD. The number of favorable prognostic factors was associated with survival. We developed a new prognostic model for OMD using the NLR, which will help to decide treatment strategy for patients with OMD. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s10549-022-06726-w. Author contributions YI, MF, and MO conceived and designed the study. YI and MF carried out the analysis. SO advised on the study design. TO contributed to the pathological assessment. JM, YO, TM, NU, YT, TK, and TS contributed to data acquisition. All authors con- tributed to the interpretation of the findings. TU drafted the initial manuscript, and all authors contributed to the revisions of the manu- script. The authors read and approved the final manuscript. Fig. 2 Nomogram for predic- tion of 5-year overall survival. A nomogram was constructed using the prognostic factors included in our model to predict 5-year overall survival. DFI disease-free survival, NLR neutrophil-to-lymphocyte ratio Declarations 4. 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BMC Cancer 20(1):380 1 Breast Surgical Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 4 Breast Medical Oncology, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 3 Medical Oncology, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan Yuka Inoue1 · Makoto Fujishima2 · Makiko Ono3 · Jun Masuda4 · Yukinori Ozaki4 · Tetsuyo Maeda1 · Natsue Uehiro1 · Yoko Takahashi1 · Takayuki Kobayashi4 · Takehiko Sakai1 · Tomo Osako5 · Takayuki Ueno1 · Shinji Ohno6 Yuka Inoue1 · Makoto Fujishima2 · Makiko Ono3 · Jun Masuda4 · Yukinori Ozaki4 · Tetsuyo Maeda1 · Natsue Uehiro1 · Yoko Takahashi1 · Takayuki Kobayashi4 · Takehiko Sakai1 · Tomo Osako5 · Takayuki Ueno1 · Shinji Ohno6 Natsue Uehiro natsue.uehiro@jfcr.or.jp Yoko Takahashi yo.takahashi@jfcr.or.jp Takayuki Kobayashi takayuki.kobayashi@jfcr.or.jp Takehiko Sakai takehiko.sakai@jfcr.or.jp Tomo Osako tomo.osako@jfcr.or.jp Shinji Ohno shinji.ohno@jfcr.or.jp Yuka Inoue yuka.inoue@jfcr.or.jp Makoto Fujishima fujishima@kuma-h.or.jp Makiko Ono makiko.ono@jfcr.or.jp Jun Masuda jun.masuda@jfcr.or.jp Yukinori Ozaki yukinori.ozaki@jfcr.or.jp Tetsuyo Maeda tetsuyo.maeda@jfcr.or.jp 1 3 1 3 1 3 348 Breast Cancer Research and Treatment (2022) 196:341–348 1 Breast Surgical Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 2 Department of Surgery, Kuma Hospital, Shinkokai Medical Corporation, 8‑2‑35, Shimoyamate‑dori, Chuo‑ku, Kobe‑shi, Hyogo‑ken 650‑0011, Japan 3 Medical Oncology, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 4 Breast Medical Oncology, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 1 Breast Surgical Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 2 Department of Surgery, Kuma Hospital, Shinkokai Medical Corporation, 8‑2‑35, Shimoyamate‑dori, Chuo‑ku, Kobe‑shi, Hyogo‑ken 650‑0011, Japan 5 Division of Pathology, The Cancer Institute of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 3 Medical Oncology, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 6 Breast Oncology Center, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3‑8‑31 Ariake, Koto‑ku, Tokyo 135‑8550, Japan 1 3 3
https://openalex.org/W4253036086	https://refubium.fu-berlin.de/bitstream/fub188/38379/1/2021_Iqbal_etal.pdf	English	null	Predictors of parental mediation in teenagers’ internet use: A cross-sectional study of female caregivers in Lahore, Pakistan	Research Square (Research Square)	2,020	cc-by	9,250	Open Access Predictors of parental mediation in teenagers’ internet use: a cross-sectional study of female caregivers in Lahore, Pakistan Sarosh Iqbal1, Rubeena Zakar2 and Florian Fischer3,4* Sarosh Iqbal1, Rubeena Zakar2 and Florian Fischer3,4* Abstract Background: The internet has become the most widely used medium among teenagers, who spend much of their time online, which raises parental concerns. Notwithstanding teens’ increased internet use and exposure to online risks, little is yet known about parental internet mediation in local settings. The present research aimed to assess the various dimensions of parental mediation to regulate teens’ use of the internet and their predictors. Methods: A cross-sectional survey was conducted in the district of Lahore, Pakistan, among mothers/female caregivers of teens (aged 13–19 years). Only women were interviewed because they are more frequently engaged as primary caregivers than fathers or male caregivers. Furthermore, only qualified and working mothers from the top two professions among women, i.e. academia and medicine, were interviewed. A stratified random sampling technique was adopted, and 347 mothers were interviewed using face-to-face interviews at 11 universities and 11 hospitals/medical colleges. Data were entered and analysed using descriptive, bivariate and multivariate logistic regression analyses. Results: The findings highlighted that more than 65% of respondents applied highly active mediation of internet safety, around 60% used highly active co-use mediation and more than 56% applied restrictive mediation. In addition, 36% of respondents monitored and 15.3% technically mediated to regulate their teens’ use of the internet. The results of the multivariate logistic regression revealed that the majority of respondents were more inclined to adopt active internet safety mediation if they had teens aged 16–19 years, with medium internet addiction, possessed good digital skills, felt confident about their teens’ coping appraisal to perform online protection, and considered their teens to have high self-esteem and resilience. (Continued on next page) (Continued on next page) * Correspondence: florian.fischer1@charite.de 3Institute of Public Health, Charité – Universitätsmedizin Berlin, Berlin, Germany y 4Institute of Gerontological Health Services and Nursing Research, Ravensburg-Weingarten University of Applied Sciences, Weingarten, Germany Full list of author information is available at the end of the article Iqbal et al. BMC Public Health (2021) 21:317 https://doi.org/10.1186/s12889-021-10349-z Iqbal et al. BMC Public Health (2021) 21:317 https://doi.org/10.1186/s12889-021-10349-z Background The internet has become the most widely used medium among the young generation in today’s media- and technology-rich environment, particularly among teen- agers, commonly known as ‘teens’. Even at the beginning of the widespread usage of the internet, teens used it for more hours than adults [1]. Nowadays, teens have been born and raised in a digital era and, hence, are also recognised as ‘digital natives’ [2]. Online virtual environ- ments stimulate teens’ self-presentation and identity ex- periments, particularly through the sharing of their self- created content, posts and pictures online [3]. For this reason, they are considered more digitally literate than their parents – leading to a generation gap [4–6]. The increased use of the internet among teens, their con- cerns about online identity and privacy, and their strong association with peers, alongside reduced communica- tion with parents, enhance their susceptibility to online risks [7]. Previous studies on television and video games have categorised PM into three dimensions: instructive or ac- tive, restrictive and co-use mediation [16–18]. Further- more, with the evolution of the internet and digital devices, for example, smartphones and tablets, different researchers have strengthened and refined the concept of PM over a period of time. Livingstone et al. [19] re- cently recognised that digital devices and the internet, being more technologically complex, personalised and portable than previous technology, were difficult for par- ents to manage. Hence, five dimensions of parental internet mediation were developed, keeping in view the specific attributes of the internet. These are: 1) active co-use or instructive mediation, where parents encour- age, share and discuss mutually; 2) active mediation of internet safety, where parents guide teens towards safer online practices; 3) restrictive mediation, where parents set rules and regulations; 4) monitoring, where parents check the record available afterwards; and 5) technical mediation, where parents use software or control mech- anisms to restrict, filter or monitor online activities [20]. Teenage years are coupled with developmental changes. Teens mostly devote their time online to using self-selected devices for recreational and social activities without any parental supervision [8]. These unsupervised activities have a long-lasting impact on them; therefore, parents apply multiple dimensions of mediation, particu- larly to promote positive outcomes amongst teens. Inter- net use has mounted over the past two decades with free web browsing, social networking, online shopping, gaming and instant messaging. (Continued from previous page) (Continued from previous page) Conclusions: This research found that parental internet mediation is a multifaceted concept used to regulate teens’ online activity and enhance a resilient approach to reduce the risks associated with use of the internet. The researchers recommend developing parental guidelines, e-safety resource material, local support networks and community programmes to educate parents, teachers and teens in order to raise awareness and promote resilient pathways amongst teens. Keywords: Teen, Children, Online, Internet addiction, Risk, Resilience Keywords: Teen, Children, Online, Internet addiction, Risk, Resilience acknowledges that parents actively manage and regu- late their children’s internet use [11], while mitigating its negative effects amongst teens [12]. The notion of PM originated primarily in media studies, especially in the areas of television and video games, to compre- hend the effects of media content on teens’ or chil- dren’s behaviour [13]. Researchers have demonstrated that young audiences adopt certain behaviours that are presented on television and in video games unless parents mediate [14]. Hence, parental involvement en- courages the potential for positive outcomes, while also effectively neutralising the negative effects of the internet [15]. © 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 14 Iqbal et al. BMC Public Health (2021) 21:317 Background Furthermore, the introduction of smartphones and multiple ‘apps’ has also fuelled internet use [9]. The internet provides numerous benefits in the areas of information, edutainment and socialisation; none- theless, it also exposes users to a unique set of online risks, such as privacy invasion, cyberbullying and exposure to violent, hateful or inappropriate material or contacts [5]. Moreover, the effects of online risks, such as pornography, on teens and the adverse impact on youths’ self-esteem is a matter for concern [10]. Therefore, high sensitivity and concern among parents about their teens’ risks related to online addiction and victimisation are needed in order to protect teens from the negative aspects of internet use and to avoid harm [9]. Previous research suggests that parental preferences for applying these various dimensions of PM are subject to multiple predictors, such as the teens’ online addiction and parents’ own characteristics, including education, in- come and digital skills [21]. Moreover, parents’ beliefs about risk and response appraisal, as well as their effect on teens, also determine the various dimensions of PM. Giving due importance to parental beliefs and inputs, the theoretical foundations of this research lie in Protec- tion Motivation Theory (PMT) to aid in understanding Given this context, the concept of parental medi- ation (PM) has emerged. Parental internet mediation Iqbal et al. BMC Public Health (2021) 21:317 Iqbal et al. BMC Public Health (2021) 21:317 Page 3 of 14 PM-related predictors. The PMT postulates that one’s intention to adopt protective behaviour is linked to how individuals process threats and cope with adverse cir- cumstances [22]. Under the ambit of this research, PMT suggests that parents’ own perceptions of threat and coping appraisal could be the predictors of PM. Severity indicates the seriousness of an online risk in threat ap- praisal, while susceptibility refers to vulnerability to- wards these risks. Furthermore, threat appraisal also considers the teens’ (excessive) internet use. Parents, for instance, who found online addiction among their teens, perceived the online risks to be more severe and be- lieved their teens to be more susceptible to them applied mediation. Under coping appraisal, response efficacy de- notes effectiveness in preventing risks, while self-efficacy indicates the individual’s ability to achieve optimal on- line safety behaviour. Parents mediate more often, for example, if they believe that their involvement enables teens to manage the online risks effectively and adopt online protection behaviour. Research setting and participants A cross-sectional survey was conducted in Lahore, a dis- trict in Pakistan. Lahore District is the capital of Punjab province, with 11 million inhabitants. It is the second largest and most populated urban district in Pakistan [31]. Almost one quarter of the population is below 19 years of age [32]. Parents or primary caregivers of teens aged 13–19 years were included in the study. Only mothers or fe- male caregivers were interviewed, because they are more frequently involved as primary caregivers than fathers or male caregivers [33]. Furthermore, the study focused only on qualified and working mothers from the top two professions chosen by women, i.e. academia and medi- cine. This focus was chosen because, with higher educa- tion, serving professionals and those belonging to the middle class or having high socio-economic status are more aware of internet-related benefits and risks and are, therefore, more likely to apply mediation [34–36]. Learning from previous studies [34–36], this research narrowed its focus only to the mothers belonging to middle to high socio-economic status due to several facts. Firstly, teens from the middle to high social class use the internet more often on a range of devices com- pared to the low social class [37]. Secondly, parents from the middle to high social class and education have more awareness about online risks, thus, report more online harm to their children/teens and mediate more often [37]. Thirdly, literature also recommends that PM de- pends upon the family’s socio-economic status, where parents from middle to high status raise their children in digitally rich environments and home ecologies, which determine their quantity and quality of internet use, as well as parents’ confidence in mediation [38]. Conse- quently, associate and assistant professors and lecturers from academic universities were interviewed, while lady doctors and head nurses were interviewed from hospitals and attached medical colleges in the district of Lahore. Parental internet mediation is the concern of all par- ents and societies with the widespread use of digital technologies, regardless of background or culture. How- ever, although there are multiple PM-related studies available for western societies and cultures [26], there is a dearth of comparable literature for eastern societies and cultures, such as Pakistan. The latter ranks in the top 10 among countries within the Asian region regard- ing digital growth [27]. There are currently more than 44 million internet users in the country [28]. Research setting and participants Among them, a majority of young people and teens surf the internet for a minimum of 2 h a day, largely gaining ac- cess on tablets and smartphones [29]. There is a sub- stantial cultural difference between eastern (Asian) and western parenting practices [30]. Moreover, the notion of parental internet mediation is quite new in the devel- oping country of Pakistan. Notwithstanding the teens’ increased internet use or addiction and their exposure to online risks, little is yet known about parental internet mediation or the factors influencing it in local settings. Background Coping appraisal also high- lights parents’ own digital skills, which help them to evaluate their teens’ responses and self-efficacy. Hence, taken altogether, PMT proposes that PM could be con- sidered as self-protective behaviour against adversity and online risks [23, 24]. This adversity could be overcome through the teens’ higher self-esteem and resilience. Re- silience is defined here as a strength-based and positive outcome in the face of online risks or challenges [25]. teens’ internet addiction, parents’ own digital skills, parents’ assessments of threat and coping appraisals, as well as the effects of PM among teens, particularly in nurturing self- esteem and resilience. Sampling strategy The Cochran formula of n ¼ z2 α=2∙p∙ð1 −pÞ d2 was used to cal- culate the sample size [39], assuming z = 95% signifi- cance level, α = 95% confidence interval, p = 18% population proportion using the internet [28], d = 5% ab- solute precision and 1.5 design effect. This formula led to a sample size of 340, which was also selected by con- sidering the minimum number required for the central Given the context above, this research is an attempt to fill the gap in the existing literature and seeks to understand the varied dimensions of PM to regulate the teens’ use of the internet and their predictors in the district of Lahore (Pakistan), as illustrated in Fig. 1. These predictors include socio-demographic and teen-related characteristics, the Iqbal et al. BMC Public Health (2021) 21:317 Page 4 of 14 Fig. 1 Theoretical framework limit theorem, a key principle of statistics for ensuring the cost-effectiveness of population surveys [40]. filter, block or track websites and their use of software to limit or protect teens from viruses or spam mails. Lastly, the respondents’ active mediation of internet safety was assessed in terms of helping or suggesting safety measures to their teens or having supported their teens in the past when they were bothered online. A mean score was used to dichotomise all 5 PM dimen- sions into high and low mediation after computing all the variables above. Development of the questionnaire A closed-ended and precoded interview schedule was prepared to address the research objectives (Supplemen- tary Appendix 1). This interview schedule was pretested before finalisation and going into the field. Pretesting helped to determine the effectiveness of the interview schedule, particularly related to the language, wording, order, format of questions and analytical approach. Based on the findings of the pretesting, the interview schedule was finalised. Furthermore, the validity and re- liability of the interview schedule were also measured using Cronbach’s alpha, which was found to be satisfactory. Independent variables h d d The independent variables consisted of socio-demographic characteristics and other predictors, as mentioned in the theoretical framework. Socio-demographic variables in- cluded the respondents’ age in years (31–40, 41–50, 51– 60), marital status (married, divorced, separated, widowed), monthly income in PKR (up to 50,000; > 50,000–100,000; > 100,000–150,000; > 150,000–200,000; > 200,000), the teens’ age in years (13–15, 16–17, 18–19) and gender (boy, girl). Other predictors included internet addiction, digital skills, threat and coping appraisal, and the teens’ self- esteem and resilience. Outcome variable Parental internet mediation was the outcome variable. It was assessed in five dimensions, measured through selecting multiple situations on closed-ended responses (yes/no), which is consistent with previous similar stud- ies [20]. Firstly, the respondents’ active co-use mediation was assessed, such as talking about the teens’ online ac- tivities, sharing activities together, sitting alongside or staying nearby during internet use, or encouraging teens to learn and explore things online. Secondly, restrictive mediation was measured by asking whether parents ei- ther restrict their teens or give permission for them to own social media profiles, share personal information, use instant messaging, watch and download music/films/ videos or upload videos/photos online. Thirdly, the re- spondents’ monitoring was assessed in terms of checking social media profiles, friends lists and account messages, and visiting websites after the teens’ internet use. Fourthly, the respondents’ technical mediation was mea- sured by their use of parental control mechanisms to The respondents’ perception of the teens’ addictive be- haviour was measured through selecting five situations (yes/no), consistent with the literature [41], in which, due to the internet, teens: 1) do not eat or sleep; 2) spend less time with family and friends; 3) are caught surfing when not interested; 4) feel bothered when not online; and 5) unsuccessfully tried to spend less time on the internet. The categories of high, medium and low internet addiction were constructed after computing the index. Another key variable was the respondents’ own under- standing of multiple digital skills [42], such as comparing or bookmarking websites, changing filters or privacy set- tings, blocking messages or unwanted pop-ups/adverts or spam/junk mails, and deleting the record of websites Iqbal et al. BMC Public Health (2021) 21:317 Page 5 of 14 visited. A closed-ended response on multiple situations was recorded (yes/no). This variable was dichotomised into good or weak digital skills after computing the index. celebrations/festivals and their treatment, and always getting support from friends and family during difficult times. The variable was dichotomised into high vs low resilience after computing an index. Details of the above indices are provided in Supplementary Appendix 2. g g g An important predictor is the respondents’ threat ap- praisal, which was measured in terms of the severity of the threat and the teens’ susceptibility to potential online risks. Initially, female caregivers were asked about how serious they considered the online risks to teens (serious vs. Outcome variable not serious), such as online threats, receiving hate- based or sexual remarks, someone pretending to be a teen, publishing personal information, videos, pictures or negative comments about teens with bad intentions, or being infected with a computer virus [43]. The re- spondents were subsequently asked how likely they felt it was that the online risks mentioned above, excluding the virus, might happen to teens (likely vs. not likely) [43]. The variables were dichotomised after computing the indices above. Data collection The survey was conducted during April and May 2018 using two-stage stratified random sampling, during the first stage of which 11 universities and 11 hospitals/med- ical colleges were randomly selected, based on district- specific lists of academic universities and hospitals/med- ical colleges. These lists were obtained from the Punjab Higher Education Department for academic universities and the Pakistan Medical and Dental Council for hospi- tals/medical colleges. In the second stage, eligible re- spondents, i.e. mothers/female caregivers who had teens in the age range of 13 to 19 years, were randomly selected for the survey. The last-birthday method was applied when one eligible respondent had more than one teen [48]. Thus, a total of 347 respondents (mothers/female caregivers) were interviewed, as illus- trated in Fig. 2, which presents the break-down of the stratified sampling. The respondents’ coping appraisal was also measured in terms of the two elements of the teens’ response- efficacy and self-efficacy to ensure online safety. The re- spondents’ perception of their teens’ response-efficacy was measured against a 6-item list (agree vs. disagree): use of a nickname to conceal their identity and personal information, providing inaccurate information for priv- acy protection, limiting access to only friends/family, avoiding contact with online strangers, being aware of whom to talk to for online safety advice, and believing that they could receive help from parents and teachers in the form of good advice [43]. Similarly, the respon- dents’ opinion about their teens’ response-efficacy was assessed against the same 6-item list (likely vs. not likely) [43]. Dichotomous variables were constructed after com- puting the indices above. Data entry and statistical analysis EpiData software was used for data entry. After this was complete, the data were exported and analysed in SPSS version 21 to present univariate descriptive, bivariable and multivariate logistic regression analyses. Reliability and internal consistency of all measures were calculated using Cronbach’s alpha, which was found to be accept- able between 0.72 and 0.86. Cross-tabulations were cal- culated, along with chi-square as a test of association, where p-values < 0.05 showed statistical significance. Odds ratios (OR) with 95% confidence intervals (CI) were calculated in bivariable logistics regression. The predictors showing significance with a p-value < 0.05 during bivariable analysis were further included in the regression modelling. Thus, five bivariate logistic regres- sion models, with each of the PM dimensions, were cal- culated to identify the unadjusted effect of predictors on the outcome variables. Adjusted odds ratios (AOR) with 95% CI were also determined during the multivariate analysis. p g In order to determine the respondents’ opinions of their teens’ self-esteem, Rosenberg’s [44] 10-item scale was used (yes/no), which is widely recognised as a reli- able and valid instrument to measure self-esteem in multiple settings including Pakistan [45, 46]. This scale includes both positive qualities (feels satisfied, worthy, capable, has a positive attitude and good behaviour) and negative qualities (feels useless, bad, a failure, nothing to be proud of and wants to earn more respect). It was dichotomised into high vs. low self-esteem after compu- tation of the index. Furthermore, the research measured the respondents’ opinions of their teens’ resilience using the 12-item child and youth resilience measure, which was explicitly developed for parents/caregivers [47]. A closed-ended response to multiple situations was re- corded (yes/no). These items about the teens’ resilient behaviour include living with likeable people, learning useful things, completing all tasks, considering education and institutions important, being aware of how to fix things and seeking help, liking community-related Results Socio-demographic characteristics The respondents’ mean age was 47 years (SD + 5.44), with the majority (61.7%) belonging to the age group 41–50 years. Most women were married (93.4%), and had a monthly income between 51,000 and 100,000 Pakistani rupees (PKR) (45.8%). The mean age of the teens was 16 years (SD + 2.05) (Table 1). Iqbal et al. BMC Public Health (2021) 21:317 Page 6 of 14 Fig. 2 Two-stage stratified random sampling for survey Fig. 2 Two-stage stratified random sampling for survey Multivariate logistic regression analysis Characteristics of parental internet mediation and its influencing factors Table 3 shows the multivariate logistic regression ana- lysis presenting five models, one for each of the parental internet mediation dimensions and its predictors. The findings of Model I revealed that the respondents were more likely to apply active co-use mediation if they had female teens (AOR = 1.89, 95% CI: 1.03–3.51), had good digital skills (AOR = 2.53, 95% CI: 1.35–4.76), found the online risks for their teens serious (AOR = 3.42, 95% CI: 1.28–9.11) and judged their teen’s self-esteem to be high (AOR = 2.35, 95% CI: 1.27–4.32). On the other hand, ap- plying active co-use mediation was less likely for the re- spondents whose children had medium internet addiction (AOR = 0.37, 95% CI: 0.14–1.02) compared to no addiction and who felt confident about their teens’ response-efficacy to perform online protection (AOR = 0.34, 95% CI: 0.12–1.03). Figure 3 shows that more than 65% of the respondents applied highly active mediation of internet safety, around 60% applied highly active co-use mediation and more than 56% adopted restrictive mediation to regulate their teens’ use of the internet. About 36% of the respondents monitored their teens and only 15.3% applied technical mediation. Table 1 highlights the predictors of parental internet mediation. The results show that most respondents (65.1%) had good digital skills. A large number reported high internet addiction amongst their teens (58.8%); only 21.3% confirmed no internet addiction among their teens. The findings reveal that 89.6 and 59.9% of the re- spondents reported severe online risks and susceptibility of teens, respectively. A large proportion was informed about response efficacy (61.4%) and self-efficacy (55.0%) to adopt online safety measures against risks. Lastly, the results indicate that respondents found high self-esteem (59.4%) and resilience (71.5%) among their teens. The results of Model II reveal that the respondents with higher incomes were generally less likely to apply restrictive mediation. Furthermore, those respondents with teens of older ages were less likely to apply restrict- ive mediation. High internet addiction – compared to no addiction – was a significant predictor for applying restrictive mediation (AOR = 2.85, 95% CI: 1.16–7.02), whereas parents were less likely to use this dimension of mediation for medium addiction (AOR = 0.32, 95% CI: 0.13–0.83). Bivariate logistic regression The results of the bivariate logistic regression (Table 2) indicate that active mediation of internet safety was sig- nificantly associated with the teens’ age, internet addic- tion, digital skills, severity of online risks, self-esteem and resilience. Furthermore, 8 out of the overall 12 pre- dictors included in the bivariate logistic regression model showed significant associations with the active mediation of internet safety. Particularly good digital skills among the respondents (OR = 4.05, 95% CI: 2.52– 6.49) and judging the severity of online risks as serious (OR = 2.99, 95% CI: 1.48–6.06) showed strong associa- tions with active mediation of internet safety. Technical mediation was only significantly associated with suscep- tibility to online risks. Model III demonstrates that the adjusted odds of moni- toring were lower for the respondents having teens be- tween 18 and 19 years of age (AOR = 0.39, 95% CI: 0.15– 0.99) compared to 13 and 15 years. The use of monitoring as a mediation dimension was more likely in cases of high internet addiction (AOR = 4.79, 95% CI: 2.33–9.86), for re- spondents with good digital skills (AOR = 2.02, 95% CI: 1.11–3.68), and for those caregivers who felt confident about their teens’ response-efficacy (AOR = 3.07, 95% CI: 1.06–8.89) and attributed high self-esteem to their chil- dren (AOR = 1.81, 95% CI: 1.02–3.19). Iqbal et al. BMC Public Health (2021) 21:317 Page 7 of 14 Iqbal et al. BMC Public Health Model IV was not very clear because most of the pre- dictors for technical mediation were not significant. However, caregivers who judged their teens’ susceptibil- ity to online risks as likely were also more likely to use technical mediation (AOR = 2.54, 95% CI: 1.07–5.99). The findings of Model V reveal a positive association between the use of active mediation of internet safety and good digital skills (AOR = 1.94, 95% CI: 1.03–3.67), self-efficacy to perform online protection (AOR = 4.59, 95% CI: 1.52–13.8), and considering high self-esteem (AOR = 2.06, 95% CI: 1.20–3.53) and resilience (AOR = 2.65, 95% CI: 1.46–4.82) among teens. Bivariate logistic regression The respondents were less likely to apply this kind of mediation when their teens were older than 13–15 years (16–17 years: AOR = 0.31, 95% CI: 0.12–0.80; 18–19 years: AOR = 0.17, 95% CI: 0.05–0.54), had a medium addiction to the internet (AOR = 0.23, 95% CI: 0.08–0.67) compared to no addiction, and if caregivers felt confident about their teens’ response-efficacy (AOR = 0.23, 95% CI: 0.07–0.73). Discussion This study aimed to assess the various dimensions of parental internet mediation to regulate teens’ internet use and its predictors. Given the research objectives, the findings are based on parents’ perceptions and assess- ment about their teens’ online engagements and other dimensions, corresponding to previous studies [36, 37, 49, 50]. The research provides local evidence for Pakistan and, thus, adds value by filling some of the gaps in the existing literature. The teens’ use of the internet on various portable de- vices, such as smartphones, laptops and tablets, increases their probability of internet addiction [51]. Moreover, the popularity of multiple apps and social networking sites, with the provision of instant notifications, also pro- voked the teens’ online addiction. Bivariate logistic regression The analysis found a medium to high level of internet addiction among teens, h h b f h d h Table 1 Socio-demographic characteristics of respondents (caregivers of teenagers) (n = 347) Characteristics n % Respondents’ age Median (range) 47.0 (31–60) M (SD) 47.13 (5.44) 31–40 years 48 13.8 41–50 years 214 61.7 51–60 years 85 24.5 Respondents’ marital status Married 324 93.4 Separated 7 2.0 Divorced 7 2.0 Widowed 9 2.6 Respondents’ monthly income (in PKR) Up to 50,000 45 13.0 > 50,000–100,000 159 45.8 > 100,000–150,000 70 20.2 > 150,000–200,000 34 9.8 > 200,000 39 11.2 Age of teens Median (range) 16.0 (13–19) M (SD) 16.0 (2.1) 13–15 years 145 41.8 16–17 years 97 28.0 18–19 years 105 30.3 Sex of teens Teen boys 183 52.7 Teen girls 164 47.3 Respondents’ digital skills Good skills 226 65.1 Weak skills 121 34.9 Experience of teens’ internet addiction No addiction 74 21.3 Medium addiction 69 19.9 High addiction 204 58.8 Threat appraisal Severity of online risks Serious 311 89.6 Not serious 36 10.4 Susceptibility to online risks Likely 208 59.9 Not likely 139 40.1 Coping appraisal Response efficacy to perform online protection Agree 213 61.4 Table 1 Socio-demographic characteristics of respondents (caregivers of teenagers) (n = 347) (Continued) Characteristics n % Disagree 134 38.6 Self-efficacy to perform online protection Likely 191 55.0 Not likely 156 45.0 Teens’ self-esteem High self-esteem 206 59.4 Low self-esteem 141 40.6 Teens’ resilience High resilience 248 71.5 Low resilience 99 28.5 Model IV was not very clear because most of the pre- dictors for technical mediation were not significant. However, caregivers who judged their teens’ susceptibil- ity to online risks as likely were also more likely to use technical mediation (AOR = 2.54, 95% CI: 1.07–5.99). This study aimed to assess the various dimensions of parental internet mediation to regulate teens’ internet use and its predictors. Given the research objectives, the findings are based on parents’ perceptions and assess- ment about their teens’ online engagements and other dimensions, corresponding to previous studies [36, 37, 49, 50]. The research provides local evidence for Pakistan and, thus, adds value by filling some of the gaps in the existing literature. 21.3 19.9 58.8 89.6 10.4 The teens’ use of the internet on various portable de- vices, such as smartphones, laptops and tablets, increases their probability of internet addiction [51]. Bivariate logistic regression Moreover, the popularity of multiple apps and social networking sites, with the provision of instant notifications, also pro- voked the teens’ online addiction. The analysis found a medium to high level of internet addiction among teens, where the absence of smartphones and the internet Iqbal et al. BMC Public Health (2021) 21:317 Page 8 of 14 Fig. 3 Parental perception of applying five dimensions of internet mediation reported high self-esteem amongst their teens were more likely to apply active co-use, internet safety medi- ation and monitoring. These findings somewhat corrob- orate previous research [58]. Lastly, the findings highlight that the respondents applied active internet safety mediation when they found high resilience among their teens. This research conceptualises resilience as a positive outcome of parental internet mediation for coping with risks. The results also suggested that par- ental guidance and certain individual traits, including improved coping skills and high self-esteem, are es- sential for nurturing resilience [25]. caused anxiety. These results are similar to those of pre- vious research conducted in Europe, Singapore and Pakistan [41, 52, 53]. The teens’ online activities are individualised and pri- vatised, due to the variety of portable devices available and the multiple locations (homes, schools/colleges) where they are used. Hence, it is challenging for parents to oversee their teens [19]. Therefore, parental internet mediation is highly significant in guiding, supervising and regulating teens’ use of the internet. The results showed that parents in the district of Lahore applied mixed forms of mediation, most commonly the active mediation of internet safety, active co-use, restrictive and monitoring mediation; only a few parents adopted technical or software-related mediation. These findings are consistent with previous studies [20, 33, 54]. How- ever, the results were slightly inconsistent compared to preceding research, which could be attributed to cultural variations. Parents in the United States, for example, preferred active co-use mediation [55], whilst they ap- plied both active and restrictive mediation in Europe [56]. Our findings demonstrated that parents in Pakistan generally preferred to apply a combination of mediation according to their own priorities and values as well as their teens’ needs and competences [20]. This has also been argued in other studies, where a mixed approach was found to be the most effective [17, 57]. Bivariate logistic regression Summarising the above, this research highlights the parental perception and infers that parental internet me- diation influences critical thinking and resilience in teens to minimise online risks and maximise opportunities. Since resilience and risks go hand in hand, the findings support the recommendation that parental internet me- diation enables teens to cope with adverse situations as a strength-based approach. Limitations No causal relationships can be drawn due to the cross- sectional study design. One of the key limitations could be regarding the data obtained, highlighting only par- ents’ perception, particularly about their teens’ dimen- sions. However, this methodological approach has been widely used and adopted in various studies. A varied analytical approach was adopted, after performing neces- sary statistical tests to assure reliability and avoid any er- rors or outliers, due to skewed data for some variables, which may have had an undue influence on the statis- tical analysis [59, 60]. Another limitation was faced dur- ing the fieldwork regarding interviewing eligible respondents at their workplaces. Therefore, the re- searchers initially contacted the relevant authorities at each institution sampled, briefed them on the research objective and requested a permission letter from them This research also featured the role of parental threat appraisal and coping appraisal in determining their pref- erence for various mediation strategies. This study dem- onstrates that parents found online risks to be serious and judged it likely that their teens would be susceptible. Furthermore, a majority of respondents also endorsed their teens’ self- and response-efficacy to ensure online safety in the face of risks. Similar results are also evident in past studies, highlighting the significance of parental threat and coping appraisal to mediation [23]. Further- more, the results also indicate that those parents who Iqbal et al. Limitations BMC Public Health (2021) 21:317 Page 9 of 14 Table 2 Bivariate logistics regression of parental internet mediation with various socio-demographic and PM predictors Characteristics Active co-use Restrictive Monitoring Technical Active internet safety OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value Respondents’ age 31–40 years 1 1 1 1 1 41–50 years 0.47 0.23–0.99 0.04 1.07 0.57–2.01 0.83 2.21 1.07–4.58 0.03 1.46 0.58–3.69 0.42 1.09 0.56–2.13 0.78 51–60 years 0.24 0.11–0.53 < 0.01 0.83 0.41–1.70 0.62 1.74 0.77–3.91 0.17 0.93 0.31–2.74 0.90 0.64 0.31–1.35 0.25 Respondents’ monthly income (in PKR) Up to 50,000 1 1 1 1 1 > 50,000–100,000 2.15 1.09–4.23 0.02 0.67 0.33–1.34 0.25 1.45 0.71–2.98 0.31 0.51 0.21–1.30 0.16 1.96 1.00–3.85 0.05 > 100,000–150,000 1.10 0.51–2.33 0.80 0.84 0.38–1.85 0.67 1.12 0.49–2.55 0.77 0.77 0.27–2.13 0.61 2.23 1.02–4.85 0.04 > 150,000–200,000 0.47 0.19–1.19 0.11 0.44 0.17–1.11 0.08 1.52 0.59–3.92 0.38 1.92 0.66–5.57 0.23 2.65 1.01–6.94 0.04 > 200,000 0.83 0.35–1.96 0.67 0.28 0.11–0.69 0.01 2.11 0.85–5.19 0.10 1.38 0.47–4.03 0.54 1.37 0.57–3.26 0.47 Age of teens 13–15 years 1 1 1 1 1 16–17 years 0.34 0.19–0.58 < 0.01 0.79 0.47–1.33 0.38 1.26 0.74–2.15 0.39 0.67 0.33–1.38 0.28 0.59 0.34–1.03 0.06 18–19 years 0.25 0.15–0.44 < 0.01 1.07 0.64–1.79 0.77 1.11 0.65–1.88 0.69 0.62 0.30–1.26 0.18 0.51 0.29–0.87 0.01 Sex of teens Male 1 1 1 1 1 Female 1.80 1.16–2.79 0.01 0.86 0.56–1.32 0.49 0.90 0.58–1.39 0.64 0.83 0.46–1.50 0.54 1.41 0.90–2.21 0.13 Experience of teens’ internet addiction No addiction 1 1 1 1 1 Medium addiction 0.28 0.14–0.57 < 0.01 0.54 0.26–1.07 0.07 2.28 1.06–4.90 0.03 1.42 0.62–3.21 0.40 0.46 0.23–0.91 0.02 High addiction 0.56 0.31–1.01 0.05 3.07 1.77–5.32 < 0.01 3.18 1.67–6.07 < 0.01 0.63 0.30–1.30 0.21 1.12 0.63–1.99 0.68 Respondents’ digital skills Weak skills 1 1 1 1 1 Good skills 3.41 2.12–5.51 < 0.01 1.58 1.01–2.47 0.04 1.94 1.20–3.15 0.01 0.95 0.51–1.75 0.87 4.05 2.52–6.49 < 0.01 Threat appraisal Severity of online risks Not serious 1 1 1 1 1 Serious 3.87 1.84–9.16 < 0.01 1.32 0.66–2.64 0.43 1.52 0.71–3.27 0.28 1.13 0.42–3.05 0.81 2.99 1.48–6.06 < 0.01 Susceptibility to online risks Not likely 1 1 1 1 1 Likely 1.56 1.01–2.41 0.04 0.91 0.59–1.41 0.67 1.06 0.67–1.65 0.81 2.06 1.07–3.95 0.03 1.29 0.83–2.03 0.26 Page 10 of 14 Iqbal et al. BMC Public Health (2021) 21:317 Iqbal et al. Limitations BMC Public Health Table 2 Bivariate logistics regression of parental internet mediation with various socio-demographic and PM predictors (Continued) Characteristics Active co-use Restrictive Monitoring Technical Active internet safety OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value Coping appraisal Response-efficacy to perform online protection Disagree 1 1 1 1 1 Agree 0.33 0.14–0.73 0.01 0.94 0.43–2.05 0.88 2.17 0.96–4.89 0.06 1.57 0.57–4.34 0.38 0.43 0.19–0.96 0.04 Self-efficacy to perform online protection Not likely 1 1 1 1 1 Likely 0.98 0.45–2.14 0.97 1.74 0.81–3.73 0.15 1.01 0.46–2.21 0.97 0.68 0.26–1.83 0.45 1.68 0.77–3.68 0.19 Teens’ self-esteem Low self-esteem 1 1 1 1 1 High self-esteem 2.01 1.29–3.12 < 0.01 1.64 1.06–2.53 0.02 2.09 1.31–3.34 < 0.01 1.15 0.63–2.11 0.64 2.34 1.49–3.69 < 0.01 Teens’ resilience Low resilience 1 1 1 1 1 High resilience 1.42 0.88–2.27 0.14 1.16 0.73–1.85 0.53 1.74 1.04–2.90 0.03 1.27 0.64–2.49 0.48 2.59 1.60–4.19 < 0.01 Page 11 of 14 Iqbal et al. Limitations BMC Public Health (2021) 21:317 Table 3 Multivariate logistics regression of parental internet mediation with various socio-demographic and PM predictors Characteristics Model I Active co-use Model II Restrictive Model III Monitoring Model IV Technical Model V Active internet safety AOR 95% CI p-value AOR 95% CI p-value AOR 95% CI p-value AOR 95% CI p-value AOR 95% CI p-value Respondents’ age 31–40 years 1 1 1 1 1 41–50 years 0.93 0.37–2.35 0.88 1.07 0.39–2.91 0.89 1.87 0.73–4.83 0.19 2.31 0.57–9.31 0.24 2.73 0.91–8.23 0.07 51–60 years 0.59 0.20–1.78 0.36 0.99 0.31–3.18 0.99 1.19 0.38–3.67 0.75 1.47 0.28–7.53 0.64 1.64 0.47–5.79 0.44 Respondents’ monthly income (in PKR) Up to 50,000 1 1 1 1 1 > 50,000–100,000 2.22 0.89–5.51 0.10 0.35 0.13–0.92 0.03 0.95 0.40–2.25 0.91 0.55 0.17–1.74 0.31 1.57 0.63–3.90 0.32 > 100,000–150,000 0.94 0.35–2.57 0.83 0.63 0.21–1.86 0.40 0.66 0.25–1.73 0.39 1.08 0.31–3.75 0.89 2.55 0.87–7.47 0.08 > 150,000–200,000 0.38 0.11–1.29 0.09 0.16 0.04–0.57 < 0.01 0.82 0.26–2.59 0.73 4.03 0.98–16.5 0.05 2.21 0.61–8.02 0.23 > 200,000 0.93 0.28–3.08 0.96 0.14 0.04–0.51 < 0.01 1.58 0.52–4.83 0.42 3.28 0.78–13.7 0.10 1.57 0.46–5.36 0.47 Age of teens 13–15 years 1 1 1 1 1 16–17 years 0.60 0.24–1.47 0.26 0.26 0.10–0.65 < 0.01 0.54 0.24–1.21 0.13 0.31 0.10–0.92 0.03 0.31 0.12–0.80 0.01 18–19 years 0.42 0.15–1.18 0.09 0.23 0.07–0.68 0.01 0.39 0.15–0.99 0.04 0.33 0.09–1.12 0.07 0.17 0.05–0.54 < 0.01 Sex of teens Male 1 1 1 1 1 Female 1.89 1.03–3.51 0.04 1.30 0.70–2.42 0.41 1.11 0.63–1.96 0.71 0.85 0.39–1.86 0.69 1.56 0.83–2.96 0.17 Experience of teens’ internet addiction No addiction 1 1 1 1 1 High addiction 1.28 0.49–3.28 0.61 2.85 1.16–7.02 0.02 4.79 2.33–9.86 < 0.01 0.74 0.46–4.79 0.63 1.28 0.47–3.49 0.62 Medium addiction 0.37 0.14–1.02 0.05 0.32 0.13–0.83 0.01 1.48 0.57–3.81 0.08 1.49 0.42–2.54 0.50 0.23 0.08–0.67 < 0.01 Respondents’ digital skills Weak skills 1 1 1 1 1 Good skills 2.53 1.35–4.76 < 0.01 1.59 0.84–3.01 0.15 2.02 1.11–3.68 0.02 0.54 0.24–1.25 1.51 1.94 1.03–3.67 < 0.01 Threat appraisal Severity of online risks Not serious 1 1 1 1 1 Serious 3.42 1.28–9.11 0.01 1.50 0.58–3.83 0.39 1.29 0.53–3.13 0.57 0.97 0.24–3.94 0.96 2.24 0.86–5.78 0.09 Susceptibility to online risks Not likely 1 1 1 1 1 Likely 1.08 0.58–2.00 0.80 0.87 0.47–1.64 0.68 0.88 0.50–1.56 0.67 2.54 1.07–5.99 0.03 0.91 0.47–1.73 0.77 Page 12 of 14 Iqbal et al. Ethics approval and consent to participate The Advanced Studies and Review Board, and Doctoral Programme Coordination Committee, University of the Punjab Lahore (Pakistan), provided ethical approval for this study. Written informed consent was obtained from respondents before starting the interviews. Respondents were informed about their rights for volunteer participation. Moreover, the researcher also ensured the confidentiality and anonymity of participants, and their responses. Abbreviations This research highlighted the significance of parental internet mediation and multiple predictors. The research reiterated that the role of parents is critical in regulating teens’ use of internet, keeping in view their day-to-day activities and personal dimensions, such as self-esteem and resilience. Parents regulate and guide their teens about the appropriate use of the internet, enabling them to critically analyse the situation and adopt optimised online behaviour while in the face of risks. The local evi- dence generated through this research facilitates the par- ents, researchers and educators to realise the increasing online engagement of teens and the role and benefits of parental internet mediation. The findings also drew the attention of researchers and policymakers to develop, adapt and implement e-safety guidelines and resource material to ensure the online safety of young genera- tions. There is a need for organised awareness creating programmes at schools/colleges and community levels for both teens and parents regarding cyber risks and safety behaviours. Practitioners working with and for both parents and teens can benefit from the insights of this research, particularly in understanding how PM fa- cilitates the resilience among teens. AOR: Adjusted odds ratio; CI: Confidence interval; OR: Odds ratio; PKR: Pakistani rupee; PM: Parental mediation; PMT: Protection Motivation Theory; SD: Standard deviation; SPSS: Statistical Package for the Social Sciences Acknowledgements We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité – Universitätsmedizin Berlin. Authors’ contributions The study was conceptualised by SI and RZ. SI conducted data collection and analysis, RZ supervised this process. SA drafted the manuscript, RZ and FF revised it critically for important intellectual content. All authors reviewed the final version of the manuscript. Received: 7 January 2020 Accepted: 27 January 2021 Received: 7 January 2020 Accepted: 27 January 2021 Availability of data and materials Data are available from authors upon reasonable request. Supplementary Information to support access. Thus, eligible respondents were approached and an appointment was scheduled for the interview. However, this might also have enforced a se- lection bias as people with no or very low activity in internet mediation may not have participated. pp y The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-10349-z. The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-10349-z. The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-10349-z. Additional file 1: Supplementary Appendix 1: Questionnaire Additional file 2: Supplementary Appendix 2: Operationalization of variables – Details of the scales/indexes Competing interests p g The authors declare that no competing interest exist. FF serves on the Editorial Board of BMC Public Health as Associate Editor. Consent for publication Not applicable. The researchers conclude that parental internet medi- ation is a multidimensional concept which is directed to- wards not only regulating teens’ use of the internet but also augmenting their abilities to create resilient path- ways to prevent online risks. Therefore, it is necessary to implement PM guidelines, e-safety resource material and local support networks to raise community awareness and promote positive outcomes among teens. Based on parents’ perceptions, these findings also support the sug- gestion of launching government-supported initiatives and updating the curriculum module to raise awareness among parents, teachers, professionals and communities about potential online risks, online protection tools and safer internet best practices in order to cultivate a safe environment for children, teens and the young gener- ation. It also highlights the social responsibility of inter- net service providers to block offensive and hate-filled websites/pages. Lastly, the research emphasises the need to initiate community-based programmes to educate parents, teachers and teens about online safety tools and mechanisms. Limitations BMC Public Health (2021) 21:317 Iqbal et al. BMC Public Health Table 3 Multivariate logistics regression of parental internet mediation with various socio-demographic and PM predictors (Continued) Characteristics Model I Active co-use Model II Restrictive Model III Monitoring Model IV Technical Model V Active internet safety AOR 95% CI p-value AOR 95% CI p-value AOR 95% CI p-value AOR 95% CI p-value AOR 95% CI p-value Coping appraisal Response-efficacy to perform online protection Disagree 1 1 1 1 1 Agree 0.34 0.12–1.03 0.05 1.28 0.42–3.88 0.66 3.07 1.06–8.89 0.03 0.75 0.17–3.41 0.71 0.23 0.07–0.73 0.01 Self-efficacy to perform online protection Not likely 1 1 1 1 1 Likely 1.86 0.64–5.39 0.25 2.49 0.85–7.33 0.09 0.70 0.26–1.91 0.48 2.48 0.57–10.8 0.23 4.59 1.52–13.8 < 0.01 Teens’ self-esteem Low self-esteem 1 1 1 1 1 High self-esteem 2.35 1.27–4.32 < 0.01 1.40 0.77–2.55 0.26 1.81 1.02–3.19 0.04 1.22 0.55–2.67 0.63 2.06 1.20–3.53 < 0.01 Teens’ resilience Low resilience 1 1 1 1 1 High resilience 1.13 0.56–2.25 0.73 1.11 0.56–2.18 0.77 1.31 0.68–2.53 0.41 1.17 0.47–2.89 0.73 2.65 1.46–4.82 < 0.01 Iqbal et al. BMC Public Health (2021) 21:317 Page 13 of 14 Funding g This research received no supporting funds from any funding agency in the public, commercial, or not-for-profit sector. Open Access funding enabled and organized by Projekt DEAL. Author details 1 1Institute of Social and Cultural Studies, University of the Punjab, Lahore, Pakistan. 2Department of Public Health, Institute of Social and Cultural Studies, University of the Punjab, Lahore, Pakistan. 3Institute of Public Health, Charité – Universitätsmedizin Berlin, Berlin, Germany. 4Institute of Gerontological Health Services and Nursing Research, Ravensburg-Weingarten University of Applied Sciences, Weingarten, Germany. Received: 7 January 2020 Accepted: 27 January 2021 References 1. Kraut R, Patterson M, Lundmark V, Kiesler S, Mukhopadhyay T, Scherlis W. Internet paradox: a social technology that reduces social involvement and psychological well-being? Am Psychol. 1998;53(9):1017–32. 2. Prensky M. 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https://openalex.org/W4385550962	https://zenodo.org/records/8216917/files/BDJ_article_109082.pdf	English	null	Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the Islands of São Miguel, Terceira and Pico (Azores)	Biodiversity data jurnal	2,023	cc-by	14,042	Biodiversity Data Journal 11: e109082 doi: 10.3897/BDJ.11.e109082 Data Paper Biodiversity Data Journal 11: e109082 doi: 10.3897/BDJ.11.e109082 Data Paper Biodiversity Data Journal 11: e109082 doi: 10.3897/BDJ.11.e109082 Data Paper Data Paper Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the Islands of São Miguel, Terceira and Pico (Azores) Lurdes Borges Silva , Patrícia Madeira , Diogo Pavão , Rui B Elias , Monica Moura , Luís Silva ‡,§,\| ‡,§,\| ‡,§,\|,¶ #,¤ ‡,§,\|,¶ ‡,§,\|,¶ ‡ CIBIO, Research Center in Biodiversity and Genetic Resources, InBIO Associate Laboratory, Campus Ponta Delgada- Faculty of Sciences and Technology, University of the Azores, Rua da Mãe de Deus, 9500-321, Ponta Delgada, Portugal § BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairão, 4485-661, Vairão, Portugal \| UNESCO Chair – Land within Sea: Biodiversity & Sustainability in Atlantic Islands. University of the Azores, Rua da Mãe de Deus, 9500-321, Ponta Delgada, Portugal ¶ Faculty of Sciences and Technology, University of the Azores, Rua da Mãe de Deus, 9500-321, Ponta Delgada, Portugal # cE3c - Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute & Azorean Biodiversity Group, University of the Azores, 9700-042, Angra do Heroísmo, Portugal ¤ Faculty of Agricultural Sciences and Environmental, University of the Azores, Rua Capitão João d’Ávila – Pico da Urze, 9700‑042, Angra do Heroísmo, Portugal Corresponding author: Lurdes Borges Silva (lurdes.cb.silva@uac.pt) Academic editor: Paulo Borges Received: 05 Jul 2023 \| Accepted: 27 Jul 2023 \| Published: 03 Aug 2023 Citation: Borges Silva L, Madeira P, Pavão D, Elias RB, Moura M, Silva L (2023) Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the Islands of São Miguel, Terceira and Pico (Azores). Biodiversity Data Journal 11: e109082. https://doi.org/10.3897/BDJ.11.e109082 © Borges Silva L et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. New information We provide an inventory of indigenous and non-indigenous vascular plant taxa from 90 forest stands. A total of 105 taxa were identified and registered, belonging to 60 families, 91 genera, 101 species and four subspecies. A total of 35% of the taxa were endemic, 27% native and 38% non-indigenous, including 19% of invasive taxa. Endangered and vulnerable taxa were registered, including Elaphoglossum hirtum (Sw.) C.Chr., Lactuca watsoniana Trel. and others which were considered by the authors a priority for conservation (e.g. Arceuthobium azoricum Wiens & Hawksw., Bellis azorica Hochst. ex Seub., Sanicula azorica Guthnick ex Seub., Platanthera micrantha (Hochst. ex Seub.) Schltr.). Our records provide detailed and updated knowledge of Azorean Forest flora and highlight the role of natural forests as indigenous plant diversity hotspots and exotic woodland as a source of invasive taxa within the Archipelago. Borges Silva L et al Borges Silva L et al 2 forest, exotic woodland and production forest. The aim of this report is to further expand knowledge on biodiversity trends enclosed in the different forest types present in the Azores, by providing a list of the occurrences of the 105 different vascular plant taxa together with a brief characterisation of their origin and life-form. forest, exotic woodland and production forest. The aim of this report is to further expand knowledge on biodiversity trends enclosed in the different forest types present in the Azores, by providing a list of the occurrences of the 105 different vascular plant taxa together with a brief characterisation of their origin and life-form. Keywords Azores, islands, Plantae, endemic, native, exotic woodland, natural forest, production forest, Magnoliophyta, Magnoliopsida, Liliopsida, Lycopodiophyta, Pinophyta, Pteridophyta, occurrences Background The data presented here originated from field expeditions carried out between 2017 and 2018, within the framework of Forest-Eco project: "Towards an Ecological and Economic valorisation of the Azorean Forest". The project aimed to quantify the ecological value of the Azorean forests, including carbon accumulation and to design and propose measures that could further enhance forest sustainability. For that, 90 forest plots were sampled on three Azores islands - São Miguel, Terceira and Pico - equally distributed into natural 2 Introduction Studies on ecology and the distribution of organisms in a gradient of forest types, particularly with an emphasis on flora and species attributes, have been the focal point in many projects on forests ecosystems across the world (Rahbek 1995, Kessler 2000, Pavón et al. 2000, Grytnes 2003, Olthoff et al. 2016, Vinod et al. 2022) and also in the Azores Islands (Marcelino et al. 2013, Marcelino et al. 2014, Elias et al. 2016, Borges Silva L et al. 2022b). Forests and woodlands constitute a striking and structuring element of the Azorean landscape. Occupying about 30% of the insular terrestrial territory (DRRF 2014), they harbour enormous terrestrial biodiversity, making them, in essence, critical habitats for plant species and for providing a wide range of ecosystem services (Matos et al. 2019, Borges Silva L et al. 2022b). The flora of the Azores comprises about 4000 vascular plant taxa (Tracheobionta) (Silva et al. 2008, Silva et al. 2022, Borges Silva L et al. 2022a), including cultivated taxa; but excluding another 1397 taxa without a confirmed presence in the Azores. Distributed within Vascular plant taxa occurrences in exotic woodland and in natural and production ... 3 several groups, namely Magnoliophyta, Pinophyta, Pteridophyta and Lycopodiophyta (Silva et al. 2008, Silva et al. 2010), approximately 133 species are considered as native and 101 endemic, distributed over 60 families, the vast majority of which only contain a single endemic taxon (Silva et al. 2008, Silva et al. 2022, Borges Silva L et al. 2022a). The families with the highest numbers of endemic taxa correspond to Asteraceae (13), Dryopteridaceae (8), Poaceae (12) and Apiaceae (7) (Silva et al. 2009, Silva et al. 2010, Moura et al. 2013, Borges Silva et al. 2016, Moura et al. 2018, Moura et al. 2019, Vieira et al. 2020, Elias et al. 2022). The remaining taxa consist of exotic plants, distributed over 200 families, 2901 considered as cultivated or at least imported, 322 as casual escapes and 578 as naturalized, of which 140 are invasive, occupying large extensions (Silva et al. 2008, Silva et al. 2022, Borges Silva L et al. 2022a). As in many archipelagos, the Azorean primary forest was largely cleared and replaced by secondary forest and grassland (Matos et al. 2019, Pavão et al. 2021). Introduction Currently, land use in the Azores is dominated by pastures and agriculture (60%), planted or alien dominated forests (22%), with natural forests and vegetation representing 10% of the territory (DRRF 2014). The Azorean production forest is dominated by a reduced number of species, including Cryptomeria japonica D. Don (12,856 ha), Eucalyptus globulus Labill. (3786 ha), Pinus pinaster Aiton (874 ha) and by non-productive exotic woodland occupying more than 30% of the forest areas, where Pittosporum undulatum Vent. is the dominant woody species, occupying 23,939 ha (Lourenço et al. 2011, DRRF 2014, Borges Silva et al. 2014, Borges Silva et al. 2017, Borges Silva et al. 2018). Non-indigenous invasive species, such as P. undulatum, Hedychium gardnerianum Sheppard ex Ker-Gawl., Leycesteria formosa Wall., Clethra arborea Aiton, Gunnera tinctoria (Molina) Mirb. and tree ferns, such as Sphaeropteris cooperi (F.Muell.) R.M.Tryon, Sphaeropteris medullaris Bernh and Dicksonia antarctica Labill., currently threaten the conservation of endemic Azorean species and natural forests (Silva and Smith 2006, Silva et al. 2009, Costa et al. 2013, Borges Silva et al. 2014, Borges Silva et al. 2017, Silva and Beech 2017, Borges Silva et al. 2018). The publication of updated species lists and suitable floristic data, makes available relevant information for the evaluation of the conservation status of species and ecosystems (Simões et al. 2021, Büttner et al. 2022). This paper aims to publish a dataset of vascular plant occurrences in 90 Azorean forests, highlighting the importance of natural forests as indigenous plant diversity hotspots and of exotic woodland as a source of invasive taxa. General description Purpose: The purpose of this paper was to publish a dataset of vascular plant occurrences in three forest types (NF-Natural Forest, EW-Exotic Woodland and PF-Production Forest) Borges Silva L et al 4 4 on three islands of the Azores Archipelago (São Miguel, Terceira and Pico), already published in GBIF as a Darwin Core Archive. on three islands of the Azores Archipelago (São Miguel, Terceira and Pico), already published in GBIF as a Darwin Core Archive. Project description Title: Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the islands of São Miguel, Terceira, and Pico (Azores). Personnel: Lurdes Borges Silva, Patrícia Madeira, Diogo Pavão, Rui Bento Elias, Mónica Moura and Luís Silva. Study area description: The Azores Archipelago is situated in the North Atlantic Ocean, between North America and Europe, about 1500 km west of mainland Portugal, roughly at 38°44'52''N, 31°32'16''W and 38°55'27''N, 25°0'36''W (Fig. 1). The Archipelago is formed by nine main islands and some small islets, all of them of volcanic origin. The islands are divided into three main groups: the western group (Corvo and Flores), the central group (Faial, Pico, Graciosa, São Jorge and Terceira) and the eastern group (São Miguel and Santa Maria). The climate in the Azores is temperate oceanic, with regular and abundant rainfall, high levels of relative humidity and persistent winds, mainly during winter and autumn (Azevedo et al. 2004). Figure 1. The Azores Archipelago with its location in the middle Atlantic (right panel) and Azorean Islands namely São Miguel, Terceira and Pico islands (left panel). The Azores Archipelago with its location in the middle Atlantic (right panel) and Azorean Islands namely São Miguel, Terceira and Pico islands (left panel). The landscape of the islands is composed by a mosaic of habitats, ranging from herbaceous to arboreal and from natural to anthropogenic (Silva et al. 2008, Soares et al. 2021). The original landscape was strongly altered by replacing pristine and native forest areas with exotic tree plantations, crops, pastures and urban areas (Silva et al. 2008). During the last decades of the 20 century, the reduction of native forest area was significant, with the clearing of large fragments, at mid- and high altitude, for pasture ( Gaspar et al. 2008). Pittosporum undulatum, Acacia melanoxylon R.Br. or Eucalyptus th The landscape of the islands is composed by a mosaic of habitats, ranging from herbaceous to arboreal and from natural to anthropogenic (Silva et al. 2008, Soares et al. 2021). The original landscape was strongly altered by replacing pristine and native forest areas with exotic tree plantations, crops, pastures and urban areas (Silva et al. 2008). During the last decades of the 20 century, the reduction of native forest area was significant, with the clearing of large fragments, at mid- and high altitude, for pasture ( Gaspar et al. 2008). Pittosporum undulatum, Acacia melanoxylon R.Br. Project description or Eucalyptus th Vascular plant taxa occurrences in exotic woodland and in natural and production ... 5 globulus dominate most forest patches located in low- to mid-elevation areas. At higher altitudes, Cryptomeria japonica dominates, along with the remaining stands of natural forests, particularly above 600 m a.s.l. (Elias et al. 2016, Borges Silva et al. 2017, Borges Silva et al. 2018, Dutra Silva et al. 2019). The natural vegetation includes diverse communities, namely coastal vegetation, coastal and inland wetlands, meadows, peat bogs and several types of native forests and scrubs. However, forests are the dominant natural vegetation type. In fact, before human settlement, laurel forests could have covered around 75% of Azorean islands (Elias et al. 2016). Currently, the native laurel forest comprises about 5% of the total surface of the Archipelago and has remained only at higher elevations and in inaccessible areas of the islands (Elias et al. 2016). This research comprised three islands contributing with the largest forest areas: São Miguel Island with 745 km , the highest elevation being 1105 m a.s.l. with an estimated age of 0.79 MY (millions of years) (Sibrant et al. 2015), Terceira Island with 400 km , a maximum elevation of 1023 m a.s.l. and 0.39 MY (Hildenbrand et al. 2014) and Pico Island with an area of 447 km , mostly occupied by a volcanoes reaching an altitude of 2351 m a.s.l. and an approximate age of 0.27 MY (Demand et al. 1982) (Fig. 1). 2 2 2 Design description: A total of 90 forests patches were randomly sampled, with 30 quadrats plots (100 m , divided into four subplots), per island. Surveys took place in spring and summer of 2017 (São Miguel and Terceira Islands) and 2018 (São Miguel and Pico Islands), for a period of 8 months (4 months per year), corresponding to a total of 240 days. Study areas were delimited using a geographic information system (GIS; QGis 3.28) to map and select forest stands, based on the data provided by the Azorean Forest Inventory (DRRF 2007) (see Borges Silva L et al. (2022b) , Figure 2). Project description 2 Funding: This work was funded by: i) Project FOREST-ECO2-Towards an Ecological and economic valorization of the Azorean Forest ACORES-01-0145-FEDER-000014-Azores 2020 PO, 2016–2019; ii) FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE; iii) by PO Azores Project “Portal da Biodiversidade dos Açores” - M1.1.A/INFRAEST CIENT/001/2022; and iv) by National Funds through FCT-Foundation for Science and Technology under the UID/BIA/50027/2019 and POCI-01-0145-FEDER-006821. Sampling methods Description: Three types of vegetation were included (Borges Silva L et al. 2022) • Natural forest, corresponding to submontane and montane cloud forests (Elias et al. 2016). Its distribution in the Azores evolved in unique conditions, due to a pronounced isolation, relatively homogeneous climatic conditions and a limited number of native woody species, but high plant biodiversity and a high number of endemic species, which are dominated by Ilex azorica Gand., Juniperus brevifolia (Hochst. ex Seub.) Antoine, Laurus azorica (Seub.) Franco, Morella faya (Aiton) 6 6 Borges Silva L et al Wilbur and Picconia azorica (Tutin) Knobl (Dias et al. 2007, Elias and Dias 2008, Silva et al. 2010, Pavão et al. 2022, Pavão et al. 2023a, Pavão et al. 2023b). Wilbur and Picconia azorica (Tutin) Knobl (Dias et al. 2007, Elias and Dias 2008, Silva et al. 2010, Pavão et al. 2022, Pavão et al. 2023a, Pavão et al. 2023b). Exotic woodland, located at lower to mid-elevations and dominated by P. undulatum covering more than 30% of the forest area, which expanded from sea level up to 600 m (Borges Silva et al. 2017, Dutra Silva L et al. 2017a, Dutra Silva et al. 2017b, Borges Silva et al. 2018, Dutra Silva et al. 2019). • Exotic woodland, located at lower to mid-elevations and dominated by P. undulatum covering more than 30% of the forest area, which expanded from sea level up to 600 m (Borges Silva et al. 2017, Dutra Silva L et al. 2017a, Dutra Silva et al. 2017b, Borges Silva et al. 2018, Dutra Silva et al. 2019). • Production forest, dominated by C. japonica, occupying 60% of the area dedicated to this type of forest, the most economically important forestry species in the Azores and with an important impact on the landscape (DRRF 2007, Lourenço et al. 2011, DRRF 2014). • Production forest, dominated by C. japonica, occupying 60% of the area dedicated to this type of forest, the most economically important forestry species in the Azores and with an important impact on the landscape (DRRF 2007, Lourenço et al. 2011, DRRF 2014). Sampling description: A total of 90 forest stands were randomly sampled, 30 in each of the three selected islands São Miguel, Terceira and Pico (10 NF, 10 EW and 10 PF) (Table 1). Sampling methods At each forest, we delimited a 10 × 10 m (100 m ) plot and recorded the vascular plant taxa. To quantify the cover-abundance of each taxon, we used the Braun Blanquet scale modified by Van der Maarel E (1979). 2 Table 1. List of the 90 sampled forests in São Miguel (SMG, n = 30), Terceira (TER, n = 30) and Pico (PIC, n = 30) Islands. Information about Forest Type (Natural - Natural Forest, Exotic-Pitt - Exotic Woodland dominated by Pittosporum undulatum, Production-Cryp - Production Forest dominated by Cryptomeria japonica), Location ID (Code), Locality, decimal geographical coordinates (datum WGS84) and elevation in metres is provided. List of the 90 sampled forests in São Miguel (SMG, n = 30), Terceira (TER, n = 30) and Pico (PIC, n = 30) Islands. Information about Forest Type (Natural - Natural Forest, Exotic-Pitt - Exotic Woodland dominated by Pittosporum undulatum, Production-Cryp - Production Forest dominated by Cryptomeria japonica), Location ID (Code), Locality, decimal geographical coordinates (datum WGS84) and elevation in metres is provided. List of the 90 sampled forests in São Miguel (SMG, n = 30), Terceira (TER, n = 30) and Pico (PIC, n = 30) Islands. Information about Forest Type (Natural - Natural Forest, Exotic-Pitt - Exotic Woodland dominated by Pittosporum undulatum, Production-Cryp - Production Forest dominated by Cryptomeria japonica), Location ID (Code), Locality, decimal geographical coordinates (datum WGS84) and elevation in metres is provided. Island Forest type Location ID Locality Latitude Longitude Elevation (m) SMG Natural SMLO- NF01 Lombadas 37.78824 -25.46862 654 SMG Natural SMTR- NF02 Tronqueira 37.79914 -25.18354 646 SMG Natural SMVO- NF03 Lomba do Carro 37.78122 -25.27603 568 SMG Natural SMER- NF04 Sete Cidades (Estrada-Sul Vista do Rei) 37.83654 -25.78826 640 SMG Natural SMLF- NF05 Lagoa do Fogo 37.76259 -25.46632 653 SMG Natural SMVA- NF06 Pico da Vela 37.76174 -25.46140 855 SMG Natural SMMT- NF07 Tronqueira 37.79804 -25.18430 677 SMG Natural SMME- NF08 Monte Escuro 37.77866 -25.43709 753 SMG Natural SMTR- NF09 Tronqueira 37.79701 -25.18442 754 Vascular plant taxa occurrences in exotic woodland and in natural and production ... 7 Island Forest type Location ID Locality Latitude Longitude Elevation (m) SMG Natural SMLM- NF10 Lomba do Botão 37.77383 -25.27546 455 SMG Exotic-Pitt. SMCM- EW01 Ferraria (Pico das Camarinhas) 37.85843 -25.84877 205 SMG Exotic-Pitt. SMCB- EW02 Lagoa (Chã do Rego d`Água) 37.77367 -25.56959 240 SMG Exotic-Pitt. SMMF- EW03 Mosteiros (Pico de Mafra) 37.89435 -25.80720 200 SMG Exotic-Pitt. Sampling methods SMLS- EW04 Lagoa Santiago 37.85266 -25.77467 502 SMG Exotic-Pitt. SMPP- EW05 Pinhal da Paz 37.79015 -25.63265 394 SMG Exotic-Pitt. SMFA- EW06 Fenais da Ajuda (Lomba de Cima) 37.82921 -25.31140 405 SMG Exotic-Pitt. SMFT- EW07 Faial da Terra 37.75355 -25.21646 508 SMG Exotic-Pitt. SMPB- EW08 Pico Bartolomeu 37.80110 -25.15830 607 SMG Exotic-Pitt. SMFN- EW09 Furnas Norte (Caminho Norte/Sul) 37.78404 -25.29760 315 SMG Exotic-Pitt. SMNG- EW10 Lagoa do Congro 37.75813 -25.40702 607 SMG Production-Cryp. SMAL- PF01 Caldeira do Alferes 37.87024 -25.80575 483 SMG Production-Cryp. SMEM- PF02 Lagoa das Empadadas 37.82668 -25.74983 845 SMG Production-Cryp. SMCT- PF03 Castelo Branco 37.74732 -25.35377 638 SMG Production-Cryp. SMFN- PF04 Furnas Norte (Caminho Norte/Sul) 37.77344 -25.36415 654 SMG Production-Cryp. SMAG- PF05 Água de Pau 37.73415 -25.49549 432 SMG Production-Cryp. SMPG- PF06 Planalto dos Graminhais 37.80362 -25.26304 850 SMG Production-Cryp. SMTR- PF07 Tronqueira 37.79797 -25.17647 683 SMG Production-Cryp. SMSB- PF08 Lagoa São Brás 37.79404 -25.41236 716 SMG Production-Cryp. SMFT- PF09 Faial da Terra 37.77265 -25.18795 470 Borges Silva L et al Borges Silva L et al 8 8 Island Forest type Location ID Locality Latitude Longitude Elevation (m) SMG Production-Cryp. SMGI- PF10 Ginetes 37.87205 -25.82743 289 TER Natural TETB- NF01 Reserva Natural da Terra Brava 38.73293 -27.20936 692 TER Natural TEBL- NF02 Reserva Natural da Terra Brava 38.73942 -27.21194 710 TER Natural TETR- NF03 Reserva Natural da Terra Brava 38.73086 -27.19350 679 TER Natural TETA- NF04 Reserva Natural da Terra Brava 38.73008 -27.19260 692 TER Natural TEIN-NF05 Caldeira de Santa Bárbara 38.73461 -27.30828 889 TER Natural TEIS-NF06 Caldeira de Santa Bárbara 38.73390 -27.30933 904 TER Natural TEMO- NF07 Morro Assombrado 38.75619 -27.22497 591 TER Natural TEMA- NF08 Morro Assombrado 38.75763 -27.22706 550 TER Natural TELM- NF09 Lomba 38.73911 -27.29289 725 TER Natural TELO- NF10 Lomba 38.73844 -27.29036 700 TER Exotic-Pitt. TEMD- EW01 Monte Brasil (Ponta de São Diogo) 38.64105 -27.22841 212 TER Exotic-Pitt. TESE- EW02 Serreta (Pico do Carneiro) 38.76431 -27.35234 492 TER Exotic-Pitt. TESC- EW03 Biscoitos 38.79195 -27.24367 139 TER Exotic-Pitt. TEPT- EW04 Pico do Teles 38.73238 -27.36180 443 TER Exotic-Pitt. TEFE- EW05 Feteira 38.66165 -27.15121 279 TER Exotic-Pitt. TEMG- EW06 Caparica (Caminho dos Caneleiros) 38.77158 -27.26219 386 TER Exotic-Pitt. TELJ- EW07 Vila das Lajes 38.75774 -27.10938 129 TER Exotic-Pitt. TECU- EW08 Serra do Cume 38.72935 -27.09827 264 TER Exotic-Pitt. TELC- EW09 São Brás (Ladeira do Cardoso) 38.74693 -27.13439 265 TER Exotic-Pitt. Sampling methods TEAG- EW10 Agualva 38.79077 -27.19684 197 Vascular plant taxa occurrences in exotic woodland and in natural and production ... 9 Island Forest type Location ID Locality Latitude Longitude Elevation (m) TER Production-Cryp. TETE- PF01 Terra Chã 38.69952 -27.23730 517 TER Production-Cryp. TESS- PF02 Serra de Santa Bárbara 38.71403 -27.32952 600 TER Production-Cryp. TEGN- PF03 Gruta de Natal 38.73102 -27.28399 661 TER Production-Cryp. TEGR- PF04 Gruta de Natal 38.73999 -27.26335 593 TER Production-Cryp. TEBI-PF05 Biscoitos 38.76861 -27.25169 443 TER Production-Cryp. TECB- PF06 São Bento (Caminho do Cabrito) 38.70378 -27.17663 468 TER Production-Cryp. TEEC- PF07 Algar do Carvão (Caminho) 38.72588 -27.24099 579 TER Production-Cryp. TEMH- PF08 Malha Grande 38.75945 -27.26404 498 TER Production-Cryp. TERF- PF09 Reserva Florestal Parcial (Serreta e Serra de Santa Bárbara) 38.76519 -27.32107 555 TER Production-Cryp. TEMS- PF10 Mato da Serreta 38.74679 -27.33689 800 PIC Natural PISG- NF01 Saída das Lages 38.43333 -28.30689 419 PIC Natural PIPR-NF02 Mistério da Prainha 38.48544 -28.27356 516 PIC Natural PIBU-NF03 Trilho dos Burros 38.47972 -28.27231 621 PIC Natural PIAC-NF04 Planalto da Achada 38.46914 -28.31014 682 PIC Natural PICD- NF05 Caiado 38.45589 -28.25708 808 PIC Natural PICA-NF06 Caveiro 38.43753 -28.20108 905 PIC Natural PICT-NF07 Caveiro 38.43606 -28.20761 940 PIC Natural PIAF-NF08 Caminho do Arrife 38.45067 -28.30986 580 PIC Natural PICS-NF09 Cabeçinhos 38.44350 -28.31883 530 PIC Natural PICX-NF10 Cabeço do Teixo 38.48775 -28.34708 850 PIC Exotic-Pitt. PIPH- EW01 Prainha 38.46781 -28.21922 296 PIC Exotic-Pitt. PISR- EW02 São Roque 38.51144 -28.33072 282 PIC Exotic-Pitt. PISL- EW03 Santa Luzia 38.52550 -28.39169 344 Borges Silva L et al Borges Silva L et al 10 Island Forest type Location ID Locality Latitude Longitude Elevation (m) PIC Exotic-Pitt. PIAM- EW04 Santo Amaro 38.45053 -28.18022 229 PIC Exotic-Pitt. PIPE- EW05 Piedade 38.43203 -28.07394 200 PIC Exotic-Pitt. PIRI-EW06 Ribeiras 38.41289 -28.14211 358 PIC Exotic-Pitt. PILG- EW07 Lajes do Pico 38.42542 -28.27253 288 PIC Exotic-Pitt. PICB- EW08 Candelária 38.47233 -28.50047 124 PIC Exotic-Pitt. PIBD- EW09 Bandeiras 38.52964 -28.46250 202 PIC Exotic-Pitt. PIJO- EW10 São João 38.42733 -28.33086 320 PIC Production-Cryp. PIFR-PF01 Farrobo 38.51358 -28.43758 534 PIC Production-Cryp. PIBU-PF02 Trilho dos Burros 38.47986 -28.27272 627 PIC Production-Cryp. PIAR- PF003 São Miguel Arcanjo 38.49628 -28.29019 377 PIC Production-Cryp. PIAF- PF004 Caminho do Arrife 38.45461 -28.30689 604 PIC Production-Cryp. PIJO- PF005 São João 38.44089 -28.32081 514 PIC Production-Cryp. PICE- PF006 São Caetano 38.43192 -28.36583 434 PIC Production-Cryp. PIIR- PF007 Ribeirinhas 38.43044 -28.09308 362 PIC Production-Cryp. PIAM- PF008 Santo Amaro 38.44797 -28.15064 292 PIC Production-Cryp. Sampling methods PIPH- PF009 Prainha 38.46158 -28.21608 374 PIC Production-Cryp. PICI- PF010 Caminho do Caveiro (Lagoa do Caiado) 38.45689 -28.25922 804 Analysis Colonisation status. We determined the indigenous and non-indigenous plants globally and specifically for the groups Magnoliopsida, Liliopsida, Pinophyta, Pteridophyta and Lycopodiophyta. The contribution of each family was evaluated by calculating the number of genera for indigenous and non-indigenous taxa within each family and the number of infrageneric taxa per genus. Vascular plant taxa occurrences in exotic woodland and in natural and production ... 11 Biogeography of non-indigenous plants. The distribution of non-indigenous taxa was classified by region, according to Pielou (1992). Taxa present in more than two biogeographic regions were considered as subcosmopolitan. Life forms. The classification of life forms followed Franco (1971), Franco (1984), Franco and Afonso (1994) and Franco and Afonso (1998) and the frequency of each life form was then calculated. Life forms were based on the Raunkjaer (1936) main criterion (height of perennating buds): phanerophytes-perennating buds on aerial shoots (nanophanerophytes < 2 m in height, microphanerophytes 2-8 m, mesophanerophytes 8-30 m, megaphanerophytes > 30 m); chamaephytes - perennating buds very close to the ground; hemicryptophytes - perennating buds at ground level; cryptophytes - perennating buds below ground level (geophytes) and therophytes - annual species (Silva and Smith 2004). Useful species. The number of taxa in the following categories was calculated: ornamental, forestry, cultivated (aromatic, animal fodder, hedge-plants), crops (human food) and ruderal. The percentages of taxa considered as plant invaders and as ecological threats by Portuguese legislation were also calculated DLR 15 from 2 April 2012 (DLR15 2012) and DL 92 from 10 July 2019 (DL92 2019). Quality control: Specimens representing most of the inventoried species, were collected in the field, following standard herbarium techniques and then deposited in the Herbarium Ruy Telles Palhinha, University of the Azores (AZB). All sampled individuals were sorted by trained taxonomists. Taxonomic nomenclature obtained from:Seubert and Hochstetter (1843), Seubert (1844), Dröuet (1866), Trelease (1897), Cedercreutz (1941), Tutin et al. (1964), Palhinha (1966), Tutin et al. (1968), Franco (1971), Tutin et al. (1972), Sjögren (1973), Tutin et al. (1976), Tutin et al. (1980), Fernandes and Fernandes (1980), Fernandes and Fernandes (1983), Franco (1984), Fernandes and Fernandes (1987), Valdés et al. (1987), Franco and Afonso (1994), Press and Short (1994), Franco and Afonso (1998), Schaefer (2002), Schaefer (2003), Schaefer (2005), Schaefer (2005a), Silva et al. (2010), Vieira et al. (2020) Taxonomic nomenclature obtained from:Seubert and Hochstetter (1843), Seubert (1844), Dröuet (1866), Trelease (1897), Cedercreutz (1941), Tutin et al. Analysis (1964), Palhinha (1966), Tutin et al. (1968), Franco (1971), Tutin et al. (1972), Sjögren (1973), Tutin et al. (1976), Tutin et al. (1980), Fernandes and Fernandes (1980), Fernandes and Fernandes (1983), Franco (1984), Fernandes and Fernandes (1987), Valdés et al. (1987), Franco and Afonso (1994), Press and Short (1994), Franco and Afonso (1998), Schaefer (2002), Schaefer (2003), Schaefer (2005), Schaefer (2005a), Silva et al. (2010), Vieira et al. (2020) In terms of species colonisation status, we followed Silva et al. (2010) categories: Azorean endemic species, i.e. species (or subspecies) occurring only in the Azores, as a result of either speciation events (neo-endemics) or extinct of the mainland populations (paleo- endemics); Macaronesian endemic species, i.e. species only known from the Macaronesian archipelagos (the Azores, Madeira, Canaries and Cape Verde); Native species, i.e. species which arrived by long-distance dispersal to the Azores and which also occur naturally elsewhere. Regarding Introduced species, that occur in the archipelago as a result of human activities, we distinguished two groups, Naturalised, with self-supporting populations and Casual, occasionally escaped from cultivation. The biogeographic and historic criteria used to classify taxa as non-indigenous were adapted from Silva et al. (2000): (i) classified as such by several authors from the 19 century; (ii) first record in the last 100 years; (iii) distribution restricted to a reduced number of islands; (iv) record of a recent (last 100 years) extension of the distribution in the Azores; th Borges Silva L et al 12 (v) absence in other Macaronesian islands; (vi) disjunct distribution; (vii) anthropochoric taxa - only casual in the native vegetation. These criteria were applied after exclusion of endemic taxa. Database queries allowed the determination of the taxa number in each category, globally and specifically for the Magnoliopsida, Liliopsida, Pinophyta, Pteridophyta and Lycopodiophyta groups (Silva and Smith 2004). Geographic coverage Description: São Miguel, Terceira and Pico Islands, in the Azores Archipelago (Portugal). Coordinates: São Miguel: 37°55'45.6''N and 37°42'22.8''N Latitude; 25°53'28.2''W and 25°0'27.6''W Longitude Terceira: 38°38'16.8''N and 38°48'50.4''N Latitude; 27°23'38.4''W and 27°0'54''W Longitude Pico: 38°34'53''N and 38°21'48''N Latitude; 28°33'40''W and 28°0'14.9''W Longitude. Taxonomic coverage Description: For the three forest types and for the three Islands, the dataset includes 105 vascular plant taxa, represented by 101 species and four subspecies, mostly including indigenous plants (35% endemic and 27% native) and 38% of non-indigenous plants (Fig. 2 and Fig. 3). Magnoliopsida were the highest proportion and Pinophyta the lowest (Fig. 2 and Fig. 3). The 105 taxa were distributed by 60 families belonging to 91 genera (Fig. 2). In general, each family and genus contributed only with a small number of indigenous (1-6) or non-indigenous taxa (1-3) (Fig. 2). Regarding Magnoliopsida, NF showed the highest numbers of endemic and native taxa, while EW and PF showed the highest values of invasive taxa, EW also showing a high number of naturalised and casual species (Fig. 2). For Liliopsida, again NF showed the highest values of endemism, despite also including naturalised and invasive taxa (Fig. 2). For Pteridophyta, all three forests showed similar values of endemic, native and invasive taxa (Fig. 2). In the Pteridophyta, for the three forest types, 10 families included one or two genera, one family included three genera and only two families participated with more than two species, namely the Dryopteridaceae and Hymenophyllaceae (Table 2). We found two Lycophytes, one a Macaronesian endemism, Huperzia suberecta (Lowe) Tardieu. The Pinophyta only included two Cupressaceae, with one endemic taxon, Juniperus brevifolia (Hochst. ex Seub.) Antoine subsp. brevifolia (Table 2). For the three forest types, the Liliopsida included 11 families and 27 genera, most contributing with only one taxon. The Cyperaceae and Poaceae included the highest numbers of indigenous taxa (Table 2). For the Magnoliopsida, we found 35 families and 83 genera. In general, the families including more indigenous taxa were the Asteraceae, Ericaceae, Lauraceae and Rosaceae (Table 2). Vascular plant taxa occurrences in exotic woodland and in natural and production ... 13 13 Figure 2. Categorisation of 105 Azorean vascular plant taxa, observed at 90 forests in the Azores, per forest type (Natural Forest, Exotic Woodland and Production Forest) and according to the five major groups of vascular plants (MA-Magnoliopsida, LI-Liliopsida, PI-Pinophyta, PT- Pteridophyta and LY-Lycopodiophyta,). Number of taxa in each category: END-Endemic (taxa only occurring in the Azores); NAT-Native (colonised the Azores without human intervention, also occurring in other regions); INV-Invasive; NTZ-Naturalised (with self-supporting populations); CA-casual (occasionally escaped from cultivation) and CU-Cultivated (Silva et al. 2010). Figure 2. Categorisation of 105 Azorean vascular plant taxa, observed at 90 forests in the Azores, per forest type (Natural Forest, Exotic Woodland and Production Forest) and according to the five major groups of vascular plants (MA-Magnoliopsida, LI-Liliopsida, PI-Pinophyta, PT- Pteridophyta and LY-Lycopodiophyta,). Number of taxa in each category: END-Endemic (taxa only occurring in the Azores); NAT-Native (colonised the Azores without human intervention, also occurring in other regions); INV-Invasive; NTZ-Naturalised (with self-supporting populations); CA-casual (occasionally escaped from cultivation) and CU-Cultivated (Silva et al. 2010). Categorisation of 105 Azorean vascular plant taxa, observed at 90 forests in the Azores, per forest type (Natural Forest, Exotic Woodland and Production Forest) and according to the five major groups of vascular plants (MA-Magnoliopsida, LI-Liliopsida, PI-Pinophyta, PT- Pteridophyta and LY-Lycopodiophyta,). Number of taxa in each category: END-Endemic (taxa only occurring in the Azores); NAT-Native (colonised the Azores without human intervention, also occurring in other regions); INV-Invasive; NTZ-Naturalised (with self-supporting populations); CA-casual (occasionally escaped from cultivation) and CU-Cultivated (Silva et al. 2010). Figure 3. Categorisation of 105 Azorean vascular plant taxa, observed at 90 forests in the Azores, per Island (São Miguel, Terceira and Pico) and according to the five major groups of vascular plants (MA-Magnoliopsida; LI-Liliopsida; PI-Pinophyta; PT- Pteridophyta; and LY- Lycopodiophyta). Number of taxa in each category: END-Endemic (taxa only occurring in the Azores); NAT-Native (colonised Azores without human intervention, also occurring in other regions); INV-Invasive; NTZ-Naturalised (with self-supporting populations); CA-casual (occasionally escaped from cultivation) and CU-Cultivated (Silva et al. 2010). Figure 3. Categorisation of 105 Azorean vascular plant taxa, observed at 90 forests in the Azores, per Island (São Miguel, Terceira and Pico) and according to the five major groups of vascular plants (MA-Magnoliopsida; LI-Liliopsida; PI-Pinophyta; PT- Pteridophyta; and LY- Lycopodiophyta). Number of taxa in each category: END-Endemic (taxa only occurring in the Azores); NAT-Native (colonised Azores without human intervention, also occurring in other regions); INV-Invasive; NTZ-Naturalised (with self-supporting populations); CA-casual (occasionally escaped from cultivation) and CU-Cultivated (Silva et al. 2010). 14 Borges Silva L et al Number of genera and of infrageneric taxa per family, for indigenous and non-indigenous vascular plants, in three forest types (Natural Forest, Exotic Woodland and Production Forest), on three Azores Islands (São Miguel, Terceira and Pico). Numbers within brackets represent number of genera shared between indigenous and non-indigenous taxa. Figure 2. Family Indigenous Non-indigenous Family Indigenous Non-indigenous Genera Taxa Genera Taxa Genera Taxa Genera Taxa Magnoliopsida Liliopsida Adoxaceae 1 1 Amaryllidaceae 1 1 Apiaceae 1 1 Araceae 2 2 Apocynaceae 1 1 Asparagaceae 1 1 Aquifoliaceae 1 1 Commelinaceae 1 1 Araliaceae 1 1 Cyperaceae 1 4 Asteraceae 4 5 3 3 Iridaceae 1 1 Brassicaceae 1 1 Juncaceae 2(1) 2 1(1) 1 Caprifoliaceae 1 1 Orchidaceae 1 2 Clethraceae 1 1 Poaceae 3(1) 3 2(1) 2 Ericaceae 3 3 Smilacaceae 1 1 Euphorbiaceae 1 1 Zingiberaceae 1 1 Fabaceae 1 1 Pinophyta Geraniaceae 1 1 Cupressaceae 1 1 1 1 Hydrangeaceae 1 1 Pteridophyta Hypericaceae 1 1 Aspleniaceae 1 2 Lamiaceae 1 1 Athyriaceae 2 2 1 1 Lauraceae 1(1) 1 3(1) 3 Blechnaceae 2 2 1 1 Myricaceae 1 1 Culcitataceae 1 1 Myrsinaceae 1 1 Cyatheaceae 1 1 Myrtaceae 2 2 Dennstaedtiaceae 1 1 Oleaceae 1 1 Dryopteridaceae 3 6 1 1 Onagraceae 1 1 Hymenophyllaceae 2 3 Pittosporaceae 1 1 Osmundaceae 1 1 Plantaginaceae 1 1 Polypodiaceae 1 1 Platanaceae 1 1 Pteridaceae 1 1 1 1 Primulaceae 1 1 Lycopodiophyta Vascular plant taxa occurrences in exotic woodland and in natural and production ... Family Indigenous Non-indigenous Family Indigenous Non-indigenous Genera Taxa Genera Taxa Genera Taxa Genera Taxa Rhamnaceae 1 1 Lycopodiaceae 1 1 Rosaceae 4 (1) 4 1(1) 1 Selaginellaceae 1 1 Rubiaceae 1 1 Santalaceae 1 1 Scrophulariaceae 1 1 Solanaceae 2 2 Thymelaeaceae 1 1 Ulmaceae 1 1 Urticaceae 2 2 The frequency of indigenous vascular plant taxa showed the highest values for NF (55% endemic and 38% native) and the lowest for EW (23% endemic and 25% native) and PF (17% endemic and 18% native) (Table 3). Pico Island displayed the highest number of indigenous taxa in the NF and the lowest number in PF (Table 3). Vascular plant taxa occurrences in exotic woodland and in natural and production ... Figure 2. 15 15 Family Indigenous Non-indigenous Family Indigenous Non-indigenous Genera Taxa Genera Taxa Genera Taxa Genera Taxa Rhamnaceae 1 1 Lycopodiaceae 1 1 Rosaceae 4 (1) 4 1(1) 1 Selaginellaceae 1 1 Rubiaceae 1 1 Santalaceae 1 1 Scrophulariaceae 1 1 Solanaceae 2 2 Thymelaeaceae 1 1 Ulmaceae 1 1 Urticaceae 2 2 The frequency of indigenous vascular plant taxa showed the highest values for NF (55% endemic and 38% native) and the lowest for EW (23% endemic and 25% native) and PF (17% endemic and 18% native) (Table 3). Pico Island displayed the highest number of indigenous taxa in the NF and the lowest number in PF (Table 3). Scientific Name CS Forest type Island Conservation NF EW PF SMG TER PIC DLR15 H B UICN Others Arceuthobium azoricum Wiens & Hawksw. END X X X X X T100\|P Asplenium adiantum-nigrum L. NAT X X Asplenium scolopendrium L. NAT X X Athyrium filix-femina (L.) Roth NAT X X X X X X LC Bellis azorica Hochst. ex Seub. END X X X X T100\|P Callitriche stagnalis Scop. NAT X X LC Calluna vulgaris (L.) Hull NAT X X LC Table 3. Conservation status and occurrence of indigenous vascular plant taxa per forest type (NF-Natural Forest, EW-Exotic Woodland and PF-Production Forest), in each Island (SMG-São Miguel; TER- Terceira and PIC-Pico). Based on 90 forests sampled in the Azores. Colonisation status (CS) follows Silva et al. (2010): NAT-Native and END-Endemic. Conservation status according to IUCN (threatened species: VU = Vulnerable, EN = Endangered; LC = Least Concern, NT = Near Threatened) and DLR no. 15/2012/A (Legal regime for nature conservation and protection of the Azores Autonomous Region biodiversity): H (Habitats Directive - Natura 2000 Network); B (Berne Convention); T100 (100 priority threatened species for management in the European biogeographical region of Macaronesia); CITES; R4 (protected by regional interest); P (priority for conservation) and (priority European species). Conservation status and occurrence of indigenous vascular plant taxa per forest type (NF-Natural Forest, EW-Exotic Woodland and PF-Production Forest), in each Island (SMG-São Miguel; TER- Terceira and PIC-Pico). Based on 90 forests sampled in the Azores. Colonisation status (CS) follows Silva et al. (2010): NAT-Native and END-Endemic. Conservation status according to IUCN (threatened species: VU = Vulnerable, EN = Endangered; LC = Least Concern, NT = Near Threatened) and DLR no. Figure 2. 15/2012/A (Legal regime for nature conservation and protection of the Azores Autonomous Region biodiversity): H (Habitats Directive - Natura 2000 Network); B (Berne Convention); T100 (100 priority threatened species for management in the European biogeographical region of Macaronesia); CITES; R4 (protected by regional interest); P (priority for conservation) and (priority European species). Scientific Name CS Forest type Island Conservation NF EW PF SMG TER PIC DLR15 H B UICN Others Arceuthobium azoricum Wiens & Hawksw. END X X X X X T100\|P Asplenium adiantum-nigrum L. NAT X X Asplenium scolopendrium L. NAT X X Athyrium filix-femina (L.) Roth NAT X X X X X X LC Bellis azorica Hochst. ex Seub. END X X X X T100\|P Callitriche stagnalis Scop. NAT X X LC Calluna vulgaris (L.) Hull NAT X X LC Borges Silva L et al 16 Scientific Name CS Forest type Island Conservation NF EW PF SMG TER PIC DLR15 H B UICN Others Cardamine caldeirarum Guthnick ex Seub. END X X Carex divulsa Stokes NAT X X X X X Carex hochstetteriana J.Gay ex Seub. END X X X X Carex pendula Huds. NAT X X Carex vulcani Hochst. ex Seub. END X X X X Culcita macrocarpa C.Presl NAT X X X X X X X X X NT T100 Daphne laureola L. NAT X X Deschampsia foliosa Hack. END X X Diplazium caudatum (Cav.) Jermy NAT X X X X X X LC Dryopteris aemula (Aiton) Kuntze NAT X X X X X X LC Dryopteris affinis (Lowe) Fraser-Jenk. NAT X X Dryopteris azorica (Christ) Alston END X X X X X X Dryopteris crispifolia Rasbach, Reichst. & Vida END X X X X X X LC Elaphoglossum hirtum (Sw.) C.Chr. NAT X X X X EN Erica azorica Hochst. ex Seub. END X X X X X X X X Euphorbia stygiana H.C.Watson subsp. stygiana END X X X X X T100\|P Festuca francoi Fern.Prieto, C.Aguiar, E.Días & M.I.Gut END X X Fragaria vesca L. NAT X X Frangula azorica Grubov END X X X X X X X LC T100\|P Hedera azorica Carrière END X X X X X X Holcus rigidus Hochst. ex Seub. END X X X Huperzia suberecta (Lowe) Tardieu END X X X X LC R4 Hymenophyllum tunbrigense (L.) Sm. NAT X X X X X LC Hymenophyllum wilsonii Hook. Figure 2. NAT X X X LC Hypericum foliosum Aiton END X X X X LC Ilex azorica Gand. END X X X X X X X LC T100 Juncus effusus L. NAT X X Vascular plant taxa occurrences in exotic woodland and in natural and production ... 17 Scientific Name CS Forest type Island Conservation NF EW PF SMG TER PIC DLR15 H B UICN Others Juniperus brevifolia (Hochst. ex Seub.) Antoine subsp. brevifolia END X X X X X X VU T100\|P Lactuca watsoniana Trel. END X X X X X EN T100\|* Laurus azorica (Seub.) Franco END X X X X X X X LC T100\|P Leontodon filii (Hochst. ex Seub.) Paiva & Ormonde END X X X X T100\|P Leontodon rigens (Aiton) Paiva & Ormonde END X X Luzula purpureosplendens Seub. END X X X X X Lysimachia azorica Hornem. ex Hook. END X X X X X X Morella faya (Aiton) Wilbur NAT X X X X X X LC Myrsine retusa Aiton END X X X X X X Osmunda regalis L. NAT X X X LC Picconia azorica (Tutin) Knobl. END X X X X X X X LC T100\|P Platanthera micrantha (Hochst. ex Seub.) Schltr. END X X X CITES\| P Platanthera pollostantha R.M.Bateman & M.Moura END X X Polypodium macaronesicum subsp. azoricum (Vasc.) Rumsey, Carine & Robba END X X X X X Polystichum setiferum (Forssk.) T.Moore ex Woynar NAT X X X X Potentilla erecta (L.) Raeusch. NAT X X X Prunus lusitanica subsp. azorica (Mouill.) Franco END X X X X X T100\|P Pteridium aquilinum (L.) Kuhn NAT X X X X X X LC Pteris incompleta Cav. NAT X X X X X NT Rubia agostinhoi Dansereau & P.Silva END X X X X X X Rubus hochstetterorum Seub. END X X X LC P Sanicula azorica Guthnick ex Seub. END X X X X X T100\|P Selaginella kraussiana (Kunze) A.Braun NAT X X X X X X LC Sibthorpia europaea L. NAT X X X X X X Borges Silva L et al 18 Scientific Name CS Forest type Island Conservation NF EW PF SMG TER PIC DLR15 H B UICN Others Smilax azorica H.Schaef. Life forms The majority of indigenous and non-indigenous Pteridophyta were hemicryptophytes, 60% and 75%, respectively, while Pinophyta only included one megaphanerophyte and one microphanerophyte. Non-indigenous and indigenous Magnoliopsida included a larger proportion of phanerophytes than Liliopsida (Fig. 4), while geophytes and hemicryptophytes were of some importance in the Liliopsida (Fig. 4). Biogeography of non-indigenous plants Most non-indigenous taxa had a wide geographic distribution. About 75% were Subcosmopolitan and a considerable percentage had a Palaearctic distribution (Fig. 5). Figure 2. & P.Schönfelder END X X X X X X X Struthiopteris spicant (L.) Weis NAT X X X X X X LC Tolpis azorica (Nutt.) P.Silva END X X X X R4 Vaccinium cylindraceum Sm. END X X X X X X X LC T100\|P Vandenboschia speciosa (Willd.) G.Kunkel NAT X X X x X X LC Viburnum treleasei Gand. END X X X X X LC T100\|P Woodwardia radicans (L.) Sm. NAT X X X X X X X X VU Regarding the conservation status of indigenous vascular plant taxa, we found 23 considered as Least Concern (LC) and two as Endangered (EN) in NF and in the three forest types, two as Vulnerable (VU) and two as Near Threatened (NT) (Table 3). We also found particularly rare species such as Lactuca watsoniana Trel. In total, 36 indigenous species (55%) had not been evaluated by IUCN criteria (Table 3). Although, based on DLR no. 15/2012/A (DLR15 2012), 35% of the indigenous species in the current study are covered by measures for conservation and protection (Table 3), of these 60% are proprietary for conversion (P) and all taxa present in NF, 13% EW and 9% PF (Table 3). Usage licence Usage licence: Creative Commons Public Domain Waiver (CC-Zero) Temporal coverage Notes: 2017-4-09 - 2018-7-27 Notes: 2017-4-09 - 2018-7-27 Notes: 2017-4-09 - 2018-7-27 Useful species Almost all non-indigenous Pteridophyta were ornamental plants, while the Pinophyta were forest species (Fig. 6). The Magnoliopsida included a large proportion of ornamental and Vascular plant taxa occurrences in exotic woodland and in natural and production ... 19 ruderal plants. The Liliopsida included a large percentage of ornamental and a small proportion of ruderal taxa (Fig. 6). Figure 4. Frequency (%) of life forms for indigenous and non-indigenous Magnoliopsida and Liliopsida, for the three forest types and the three Islands (90 forests in the Azores). g Frequency (%) of life forms for indigenous and non-indigenous Magnoliopsida and Liliopsida, for the three forest types and the three Islands (90 forests in the Azores). Figure 5. Biogeography of 40 non-indigenous vascular plant taxa sampled at 90 forests in São Miguel, Terceira and Pico Islands, Azores. SCO - Subcosmopolitan, NEO - Neotropical, PAL - Palaearctic, ORI - Oriental, ETH - Ethiopian, HOL – Holarctic. (Example: PALNEO - Palaearctic and Neotropical). Biogeography of 40 non-indigenous vascular plant taxa sampled at 90 forests in São Miguel, Terceira and Pico Islands, Azores. SCO - Subcosmopolitan, NEO - Neotropical, PAL - Palaearctic, ORI - Oriental, ETH - Ethiopian, HOL – Holarctic. (Example: PALNEO - Palaearctic and Neotropical). Biogeography of 40 non-indigenous vascular plant taxa sampled at 90 forests in São Miguel, Terceira and Pico Islands, Azores. SCO - Subcosmopolitan, NEO - Neotropical, PAL - Palaearctic, ORI - Oriental, ETH - Ethiopian, HOL – Holarctic. (Example: PALNEO - Palaearctic and Neotropical). Borges Silva L et al 20 Figure 6. Percentage of non-indigenous vascular plants (naturalised or casual) with human utilisation (ornamental, forest species and food crops) or ruderal, for the three forest types in São Miguel, Terceira and Pico Islands, Azores. The data labels correspond to the number of taxa. Figure 6. Percentage of non-indigenous vascular plants (naturalised or casual) with human utilisation (ornamental, forest species and food crops) or ruderal, for the three forest types in São Miguel, Terceira and Pico Islands, Azores. The data labels correspond to the number of taxa. Percentage of non-indigenous vascular plants (naturalised or casual) with human utilisation (ornamental, forest species and food crops) or ruderal, for the three forest types in São Miguel, Terceira and Pico Islands, Azores. The data labels correspond to the number of taxa. Data resources Data package title: Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the Islands of São Miguel, Terceira and Pico (Azores). Resource link: https://www.gbif.org/dataset/158e0f0d-26e3-4883-bf5e-6040c1bb1ff4 Alternative identifiers: http://ipt.gbif.pt/ipt/resource?r=plants-azo-foresteco2&v=1.2 Number of data sets: 2 Data set name: Event Table Character set: UTF-8 Download URL: http://ipt.gbif.pt/ipt/resource?r=plants-azo-foresteco2&v=1.2 Data format: Darwin Core Archive format Data package title: Vascular plant taxa occurrences in exotic woodland and in natural an production forests on the Islands of São Miguel, Terceira and Pico (Azores). Resource link: https://www.gbif.org/dataset/158e0f0d-26e3-4883-bf5e-6040c1bb1ff4 Alternative identifiers: http://ipt.gbif.pt/ipt/resource?r=plants-azo-foresteco2&v=1.2 Number of data sets: 2 Data set name: Event Table Character set: UTF-8 Download URL: http://ipt.gbif.pt/ipt/resource?r=plants-azo-foresteco2&v=1.2 Data format: Darwin Core Archive format Data package title: Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the Islands of São Miguel, Terceira and Pico (Azores). Resource link: https://www.gbif.org/dataset/158e0f0d-26e3-4883-bf5e-6040c1bb1ff Vascular plant taxa occurrences in exotic woodland and in natural and production ... 21 Data format version: Version 1.2 Description: The dataset was published in Global Biodiversity Information Facility platform, GBIF (Borges Silva et al. 2023).The following data table includes all the records for which a taxonomic identification of the species was possible. The dataset submitted to GBIF is structured as a sample event dataset that has been published as a Darwin Core Archive (DwCA), which is a standardised format for sharing biodiversity data as a set of one or more data tables. The core data file contains 90 records (eventID). This IPT (integrated publishing toolkit) archives the data and thus serves as the data repository. The data and resource metadata are availabe for download from Borges Silva et al. (2023). Column label Column description eventID Identifier of the events, unique for the dataset. https://dwc.tdwg.org/terms/ #dwc:eventID datasetName The name identifying the data set from which the record was derived. https:// dwc.tdwg.org/terms/#dwc:datasetName habitat The habitat for an Event. https://dwc.tdwg.org/terms/#dwc:habitat samplingProtocol The sampling protocol used to capture the species. https://dwc.tdwg.org/terms/ #dwc:samplingProtocol sampleSizeValue The numeric amount of time spent in each sampling. https://dwc.tdwg.org/terms/ #dwc:sampleSizeValue sampleSizeUnit The unit of the sample size value. https://dwc.tdwg.org/terms/#dwc:sampleSizeUnit samplingEffort The amount of time of each sampling. https://dwc.tdwg.org/terms/ #dwc:samplingEffort eventDate Date of the sampling. https://dwc.tdwg.org/terms/#dwc:eventDate locationID Identifier of the location. https://dwc.tdwg.org/terms/#dwc:locationID islandGroup Name of the archipelago. https://dwc.tdwg.org/terms/#dwc:islandGroup island Name of the island. https://dwc.tdwg.org/terms/#dwc:island country Country of the sampling site. https://dwc.tdwg.org/terms/#dwc:county countryCode The standard code for the country of the sampling site. https://dwc.tdwg.org/terms/ #dwc:countryCode stateProvince Name of the region of the sampling site. https://dwc.tdwg.org/terms/ #dwc:stateProvince municipality Municipality of the sampling site. https://dwc.tdwg.org/terms/#dwc:municipality locality Name of the locality. https://dwc.tdwg.org/terms/#dwc:locality locationRemarks Comments or notes about the Location. https://dwc.tdwg.org/terms/ #dwc:locationRemarks 22 Borges Silva L et al 22 minimumElevationInMetres The lower limit of the range of elevation (altitude, usually above sea level), in metres. https://dwc.tdwg.org/terms/#dwc:minimumElevationInMeters maximumElevationInMetres The upper limit of the range of elevation (altitude, usually above sea level), in metres. https://dwc.tdwg.org/terms/#dwc:maximumElevationInMeters verbatimCoordinates Original coordinates recorded. https://dwc.tdwg.org/terms/ #dwc:verbatimCoordinates decimalLatitude Approximate centre point decimal latitude of the field site in GPS coordinates. https://dwc.tdwg.org/terms/#dwc:decimalLatitude decimalLongitude Approximate centre point decimal longitude of the field site in GPS coordinates. https://dwc.tdwg.org/terms/#dwc:decimalLongitude geodeticDatum The ellipsoid, geodetic datum or spatial reference system (SRS) upon which the geographic coordinates given in decimalLatitude and decimalLongitude are based. https://dwc.tdwg.org/terms/#dwc:geodeticDatum coordinateUncertaintyInMetres Uncertainty of the coordinates of the centre of the sampling plot in metres. https:// dwc.tdwg.org/terms/#dwc:coordinateUncertaintyInMeters georeferenceSources Method used to obtain coordinates. Data format version: Version 1.2 https://dwc.tdwg.org/terms/ #dwc:georeferenceSources Data set name: Occurrence Table Character set: UTF-8 Download URL: http://ipt.gbif.pt/ipt/resource?r=plants-azo-foresteco2&v=1.2 Data format: Darwin Core Data format version: Version 1.2 Data set name: Occurrence Table Character set: UTF-8 Data set name: Occurrence Table Data format version: Version 1.2 Description: The dataset was published in Global Biodiversity Information Facility platform, GBIF (Borges Silva et al. 2023). The following data table includes all the records for which a taxonomic identification of the species was possible. The dataset submitted to GBIF is structured as a occurrence table that has been published as a Darwin Core Archive (DwCA), which is a standardised format for sharing biodiversity data as a set of one or more data tables. The core data file contains 1150 records (occurrenceID). This IPT (integrated publishing toolkit) archives the data and thus serves as the data repository. The data and resource metadata are available for download from Borges Silva et al. (2023). Vascular plant taxa occurrences in exotic woodland and in natural and production ... 23 23 Column label Column description licence Reference to the licence under which the record is published. https://dwc.tdwg.org/ terms/#dcterms:license institutionID The identity of the institution publishing the data. https://dwc.tdwg.org/terms/ #dwc:institutionID institutionCode The code of the institution publishing the data. https://dwc.tdwg.org/terms/ #dwc:institutionCode basisOfRecord The nature of the data record. https://dwc.tdwg.org/terms/#dwc:basisOfRecord occurrenceID Identifier of the record, coded as a global unique identifier. https://dwc.tdwg.org/terms/ #dwc:occurrenceID recordedBy A list of names of the people who performed the sampling of the specimens. https:// dwc.tdwg.org/terms/#dwc:recordedBy organismQuantity A number or enumeration value for the quantity of organisms. https://dwc.tdwg.org/ terms/#dwc:organismQuantity organismQuantityType The type of quantification system used for the quantity of organisms. https:// dwc.tdwg.org/terms/#dwc:organismQuantityType establishmentMeans The process of establishment of the species in the location, using a controlled vocabulary: 'native', 'introduced'. https://dwc.tdwg.org/terms/#dwc:establishmentMeans eventID Identifier of the events, unique for the dataset. https://dwc.tdwg.org/terms/#dwc:eventID identifiedBy A list of names of people who assigned the Taxon to the subject. https://dwc.tdwg.org/ terms/#dwc:identifiedBy dateIdentified Date on which the record was identified. https://dwc.tdwg.org/terms/#dwc:dateIdentified scientificName Complete scientific name including author. https://dwc.tdwg.org/terms/ #dwc:scientificName kingdom Kingdom name. https://dwc.tdwg.org/terms/#dwc:kingdom phylum Phylum name. https://dwc.tdwg.org/terms/#dwc:phylum class Class name. https://dwc.tdwg.org/terms/#dwc:class order Order name. https://dwc.tdwg.org/terms/#dwc:order family Family name. https://dwc.tdwg.org/terms/#dwc:family genus Genus name. https://dwc.tdwg.org/terms/#dwc:genus specificEpithet Specific epithet. https://dwc.tdwg.org/terms/#dwc:specificEpithet infraspecificEpithet Infraspecific epithet, when available. https://dwc.tdwg.org/terms/ #dwc:infraspecificEpithet taxonRank Lowest taxonomic rank of the record. https://dwc.tdwg.org/terms/#dwc:taxonRank 24 Borges Silva L et al scientificNameAuthorship Name of the author of the lowest taxon rank included in the record.https://dwc.tdwg.org/ terms/#dwc:scientificNameAuthorship taxonRemarks Comments or notes about the taxon or name. https://dwc.tdwg.org/terms/ #dwc:taxonRemarks dynamicProperties A list of additional measurements, facts, characteristics or assertions about the record. Meant to provide a mechanism for structured content. https://dwc.tdwg.org/terms/ #dwc:dynamicProperties Conclusions and prospects 25 tolerant species. They are characterised by a high percentage of endemic species, trees covered by epiphytes and a complex vertical structure with several layers (Elias et al. 2016). Meanwhile, the role of exotic woodland and, to a lesser extent, of production forest, as reservoirs of invasive species is also confirmed (Borges Silva L et al. 2022b). The low plant diversity noted in EW and PF could be explained by the dominance of a single species (Pittospotum undulatum and Cryptomeria japonica, respectively), contributing with nearly 90% of the total number of trees per plot and dominating the canopy where only ferns and a few invasive species that tolerate low levels of light intensities below 1% full sunlight at ground level (e.g. Hedychium gardnerianum) are found (Gleadow and Ashton 1981, Gleadow et al. 1983, Cordeiro and Silva 2003, Borges Silva L et al. 2022b). Regarding anthropogenic action, NF have less human influence and are hard to access (Ramos 1996). Disturbances are limited to minor harvesting of non-timber forest products (Borges Silva L et al. 2022b). Nevertheless, active and persistent conservation measures are needed to ensure the preservation of the natural forests in the Azores. In the case of PF and the spread of invasive species in EW, an intensive management regime resulted in a decrease in plant diversity levels (Silva et al. 2008, Castro et al. 2010, Kueffer et al. 2010 , Gil et al. 2013, DRRF 2017). While plantations are known for high timber productivity, their potential to harbour plant diversity is low (Borges et al. 2019). In the Azores, the new production forests already include a buffer zone with native elements (DRRF 2017). However, management plans should be developed for exotic woodland, following the guidelines already established for the renovation of Cryptomeria japonica production forests in the Azores. Finally, we consider that our dataset and the derived conclusions will be useful for future conservation and research activities, as well as for forest managers, in the development of more comprehensive action plans, particularly on islands. Conclusions and prospects For the three forest types and for all Islands, the dataset included 105 vascular plant taxa, 62% indigenous and 38% non-indigenous, distributed by 60 families belonging to 91 genera, each family and genus contributing only with a small number of taxa. Regarding Magnoliopsida, NF showed the highest number of endemic and native taxa, while EW and PF showed the highest values of invasive taxa, with EW also showing a high number of naturalised and casual taxa. For Liliopsida, again NF showed the highest values of endemism, despite also including naturalised and invasive taxa. For Pteridophyta, all three forest types showed similar values of endemic, native and invasive taxa. The frequency of indigenous vascular plant taxa was highest for NF (55% endemic and 38% native) and lowest for EW (23% endemic and 25% native) and PF (17% endemic and 18% native). Pico Island displayed the highest number of indigenous species in NF and the lowest number in PF. The results of this study agree with data from previous investigations (Borges Silva L et al. 2022b). As expected, natural forests correspond to indigenous plant diversity hotspots and exotic woodland works as a source of invasive taxa. According to our results, 35% of the indigenous plant taxa in the current study are covered by conservation regulations. The list of the vascular plants found in our study devoted to natural and production forests and to exotic woodland in three Azores Islands, clarifies the type of flora to be expected in the forested areas in the Azores, emphasising the relevant role of the former as hotspots of native biodiversity, which agrees with previous studies for the Azores (Silva et al. 2010, Marcelino et al. 2013, Mendonça 2013, Marcelino et al. 2014, Elias et al. 2016, Borges 2018, Borges Silva et al. 2018, Borges Silva L et al. 2022b). As stated by Borges Silva L et al. (2022b), natural forests mainly corresponded to montane forests which occur in the thermotemperate-hyperhumid and ultrahyperhumid belts, from 600 to 1000 m a.s.l., in areas with high rainfall (3000 to 5000 mm year ) and occult precipitation (cloud forests) (Gabriel and Bates 2005, Elias et al. 2016). These forests have small stature and are frequently subjected to natural disturbances (Elias et al. 2011). This favours plant diversity by allowing the existence of both light-demanding and shade −1 Vascular plant taxa occurrences in exotic woodland and in natural and production ... Acknowledgements This research was funded by Project FOREST-ECO2-Towards an Ecological and economic valorization of the Azorean Forest ACORES-01-0145-FEDER-000014-Azores 2020 PO, 2016–2019; FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and by National Funds through FCT-Foundationfor Science and Technology under the UID/BIA/50027/2019 and POCI-01-0145-FEDER-006821. Additionally, by the Portal da Biodiversidade dos Açores - PO Azores Project - M1.1.A/ INFRAEST CIENT/001/2022. We thank students Angela Vieira, Celia Canizares, Mireia Mari, Ruben Rego, Gonçalo Almeida and Bruno Alves for support in field and lab work. We are grateful to Paulo Borges and Rui Figueira for helping with the Darwin Core Archive. We are grateful to Roberto Resendes, technician of AZB Herbarium in the University of the Azores. We would also like to thank Natural Parks of Terceira and Pico Islands and Direcção Regional dos Recursos Florestais for providing GIS data on the distribution of each forest type. Borges Silva L et al 26 Author contributions LBS with conceptualisation and writing original draft, carried out field and lab work, data collection and analysis, writing-review and editing. LS conceived the project, carried out fieldwork, data collection, writing-review and editing. DP carried out field, lab work, data collection. 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https://openalex.org/W4241637694	https://clok.uclan.ac.uk/25922/1/25922%20Tyrer%20H%202014%20A%20murine%20Parasit%20Vectors.pdf	English	null	A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis	Parasites & vectors	2,014	cc-by	13,479	RESEARCH Open Access © 2014 Halliday et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis Alice Halliday1, Ana F Guimaraes1, Hayley E Tyrer1, Haelly Mejane Metuge2, Chounna Ndongmo Winston Patrick2,3, Kengne-Ouafo Jonas Arnaud2,3, Tayong Dizzle Bita Kwenti2,3, George Forsbrook1, Andrew Steven1, Darren Cook1, Peter Enyong2, Samuel Wanji2,3, Mark J Taylor1 and Joseph D Turner1* Abstract Background: New drugs effective against adult filariae (macrofilaricides) would accelerate the elimination of lymphatic filariasis and onchocerciasis. Anti-Onchocerca drug development is hampered by the lack of a facile model. We postulated that SCID mice could be developed as a fmacrofilaricide screening model. Methods: The filaricides: albendazole (ABZ), diethylcarbamazine (DEC), flubendazole (FBZ), ivermectin (IVM) and the anti-Wolbachia macrofilaricide, minocycline (MIN) were tested in Brugia malayi (Bm)-parasitized BALB/c SCID mice vs vehicle control (VC). Responses were compared to BALB/c wild type (WT). Onchocerca ochengi male worms or onchocercomata were surgically implanted into BALB/c SCID, CB.17 SCID, BALB/c WT mice or Meriones gerbils. Survival was evaluated at 7–15 days. BALB/c SCID were tested to evaluate the responsiveness of pre-clinical macrofilaricides FBZ and rifapentine (RIFAP) against male Onchocerca. Results: WT and SCID responded with >95% efficacy following ABZ or DEC treatments against Bm larvae (P < 0.0001). IVM was partially filaricidal against Bm larvae in WT and SCID (WT; 39.8%, P = 0.0356 and SCID; 56.7%, P = 0.026). SCID responded similarly to WT following IVM treatment of microfilaraemias (WT; 79%, P = 0.0194. SCID; 76%, P = 0.0473). FBZ induced a total macrofilaricidal response against adult Bm in WT and SCID (WT; P = 0.0067, SCID; P = 0.0071). MIN induced a >90% reduction in Bm Wolbachia burdens (P < 0.0001) and a blockade of microfilarial release (P = 0.0215) in SCID. Male Onchocerca survival was significantly higher in SCID vs WT mice, but not gerbils, after +15 days (60% vs 22% vs 39% P = 0.0475). Onchocercoma implants had engrafted into host tissues, with evidence of neovascularisation, after +7 days and yielded viable macro/microfilariae ex vivo. FBZ induced a macrofilaricidal effect in Onchocerca male implanted SCID at +5 weeks (FBZ; 1.67% vs VC; 43.81%, P = 0.0089). Wolbachia loads within male Onchocerca were reduced by 99% in implanted SCID receiving RIFAP for +2 weeks. Conclusions: We have developed a ‘pan-filarial’ small animal research model that is sufficiently robust, with adequate capacity and throughput, to screen existing and future pre-clinical candidate macrofilaricides. Pilot data suggests a murine onchocercoma xenograft model is achievable. Keywords: Anti-filarial, Lymphatic filariasis, Onchocerciasis, Macrofilaricide, Brugia, Onchocerca, Wolbachia Keywords: Anti-filarial, Lymphatic filariasis, Onchocerciasis, Macrofilaricide, Brugia, Onchocerca, Wolbachia Central Lancashire Online Knowledge (CLoK) Title A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis Type Article URL https://clok.uclan.ac.uk/25922/ DOI https://doi.org/10.1186/s13071-014-0472-z Date 2014 Citation Halliday, Alice, Guimaraes, Ana F, Tyrer, Hayley orcid iconORCID: 0000- 0002-4447-828X, Metuge, Haelly Mejane, Patrick, Chounna Ndongmo Winston, Arnaud, Kengne-Ouafo Jonas, Kwenti, Tayong Dizzle Bita, Forsbrook, George, Steven, Andrew et al (2014) A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis. Parasites & Vectors, 7 (472). pp. 1-14. Creators Halliday, Alice, Guimaraes, Ana F, Tyrer, Hayley, Metuge, Haelly Mejane, Patrick, Chounna Ndongmo Winston, Arnaud, Kengne-Ouafo Jonas, Kwenti, Tayong Dizzle Bita, Forsbrook, George, Steven, Andrew, Cook, Darren, Enyong, Peter, Wanji, Samuel, Taylor, Mark J and Turner, Joseph D Central Lancashire Online Knowledge (CLoK) Title A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis Type Article URL https://clok.uclan.ac.uk/25922/ DOI https://doi.org/10.1186/s13071-014-0472-z Date 2014 Citation Halliday, Alice, Guimaraes, Ana F, Tyrer, Hayley orcid iconORCID: 0000- 0002-4447-828X, Metuge, Haelly Mejane, Patrick, Chounna Ndongmo Winston, Arnaud, Kengne-Ouafo Jonas, Kwenti, Tayong Dizzle Bita, Forsbrook, George, Steven, Andrew et al (2014) A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis. Parasites & Vectors, 7 (472). pp. 1-14. Creators Halliday, Alice, Guimaraes, Ana F, Tyrer, Hayley, Metuge, Haelly Mejane, Patrick, Chounna Ndongmo Winston, Arnaud, Kengne-Ouafo Jonas, Kwenti, Tayong Dizzle Bita, Forsbrook, George, Steven, Andrew, Cook, Darren, Enyong, Peter, Wanji, Samuel, Taylor, Mark J and Turner, Joseph D Central Lancashire Online Knowledge (CLoK) Central Lancashire Online Knowledge (CLoK) Title A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis Type Article URL https://clok.uclan.ac.uk/25922/ DOI https://doi.org/10.1186/s13071-014-0472-z Date 2014 Citation Halliday, Alice, Guimaraes, Ana F, Tyrer, Hayley orcid iconORCID: 0000- 0002-4447-828X, Metuge, Haelly Mejane, Patrick, Chounna Ndongmo Winston, Arnaud, Kengne-Ouafo Jonas, Kwenti, Tayong Dizzle Bita, Forsbrook, George, Steven, Andrew et al (2014) A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis. Parasites & Vectors, 7 (472). pp. 1-14. Creators Halliday, Alice, Guimaraes, Ana F, Tyrer, Hayley, Metuge, Haelly Mejane, Patrick, Chounna Ndongmo Winston, Arnaud, Kengne-Ouafo Jonas, Kwenti, Tayong Dizzle Bita, Forsbrook, George, Steven, Andrew, Cook, Darren, Enyong, Peter, Wanji, Samuel, Taylor, Mark J and Turner, Joseph D It is advisable to refer to the publisher’s version if you intend to cite from the work. https://doi.org/10.1186/s13071-014-0472-z It is advisable to refer to the publisher’s version if you intend to cite from the work. https://doi.org/10.1186/s13071-014-0472-z All outputs in CLoK are protected by Intellectual Property Rights law, including Copyright law. Copyright, IPR and Moral Rights for the works on this site are retained by the individual authors and/or other copyright owners. Terms and conditions for use of this material are defined in the http://clok.uclan.ac.uk/policies/ Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 http://www.parasitesandvectors.com/content/7/1/472 * Correspondence: joseph.turner@lstmed.ac.uk 1Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Full list of author information is available at the end of the article Background Presently, only larval Onchocerca life cycle stages are routinely used to screen filaricidal drug candi- dates in mice. Infectious stage (L3) larvae of O. volvulus can be implanted within micro-chambers to achieve only abbreviated development to the L4 larval stage [17]. Microfilarial infections of the skin, utilising purified cattle Onchocerca mf, can be established in inbred laboratory mouse strains and have been utilised in pre-clinical filarici- dal testing [18]. However, no reliable macrofilarial Oncho- cerca small animal model has been described. Previous attempts to utilize inbred mouse strains, including lym- phopenic strains, to establish Onchocerca macrofilarial in- fections from infectious stage (L3) larval inoculations, has thus far proven unsuccessful [19]. However, Rajan and col- leagues demonstrated that O. volvulus female worm ‘loops’, exposed from surrounding nodular encasement in excised nodules, could remain viable following implantation into Severe-Combined ImmunoDeficiency (SCID) mice [20]. The same laboratory has also demonstrated that full devel- opment of Brugia can occur in SCID strains [21]. Further, our laboratory has previously demonstrated that O. lienalis mf infections can persist for at least 100 days in SCID recipients without waning [22]. Existing Onchocerca macrofilaricides have either too low selective toxicity (suramin) [7], are known to induce severe adverse reactions contraindicating usage at doses required to be effective (e.g. diethylcarbamazine or high dose iver- mectin) [8,9] or presently cannot be delivered orally (e.g. flubendazole) [10]. Whilst targeting the O. volvulus endo- symbiont, Wolbachia, with tetracycline antibiotics has proven safe in delivering macrofilaricidal activity, includ- ing in individuals co-infected with L. loa [11,12], present protracted treatment lengths (4–6 weeks) and contraindi- cations in certain population groups (pregnant women and children <8 years old) precludes the wide-scale use of this class of anti-Wolbachia drug in large scale MDA [1]. Stimulated by renewed philanthropic investment by a number of stakeholders, a number of drug discovery and development programmes have been initiated to identify new candidate macrofilaricidal therapeutics that can effectively and safely target the macrofilarial stage of O. volvulus or its endosymbiont in a timeframe compatible with MDA delivery (generally considered an orally adminis- tered course not exceeding seven days). The development of Onchocerca macrofilaricides is hindered by the lack of an appropriate small animal laboratory model to robustly evaluate candidates in vivo at the pre-clinical stage. A long term model of brugian lymphatic filariasis (B. malayi/B. Background with ivermectin (IVM) target the transmissive stages of O. volvulus, microfilariae (mf), in the skin, with limited ef- ficacy against adult (macrofilariae) fertility or survival [4]. Whilst effective at reducing disease, due to repopulation of the skin with mf post-treatment, IVM has to be admin- istered periodically. In addition, IVM has to be delivered for protracted periods (17-20+ years) due to the longevity of macrofilarial infections (10–15 years). This presents a challenge in sustainability with high population coverage in mass drug administration programmes (MDA) to The tissue dwelling filaria, Onchocerca volvulus, infects an estimated 37 million people, predominantly in Sub- Saharan Africa [1]. Ocular onchocerciasis (river blindness) is the second leading cause of worldwide preventable blindness [2] and is a priority neglected tropical disease (NTD) targeted for elimination [3]. Current treatments Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 2 of 14 patients [15,16]. Traditionally, the use of cattle naturally infected with bovine Onchocerca (O. dukei, O. gutterosa, O. ochengi, O. lienalis) has been exploited as a pre-clinical system to scrutinise Onchocerca-specific macrofilaricidal activity. Throughput is severely restricted by the logistics of identifying and experimenting on parasitized cattle in sub- Saharan Africa and the large quantities of drug required to dose up to 0.5 metric tonne animals. Further, without challenging empirical pharmacological evaluation, re- quiring robust sample sizes, atypical pharmacokinetic profiles within ruminants may lead to erroneous con- clusions regarding efficacy. maintain control of disease and eventually break the trans- mission cycle. Further, the emergence of sub-optimal responders to IVM following repetitive treatments [5] and the risk of severe adverse reactions in patients co-infected with the bloodborne filaria, Loa loa [6], exert additional challenges on maintaining control of onchocerciasis. Drugs effective at killing adult Onchocerca (macrofilari- cides), would be desirable to address this pressing global health problem and accelerate the ultimate elimination of onchocerciasis. New macrofilaricides may well also have useful indications against the causative agents of lymphatic filariasis; LF (Wuchereria bancrofti, Brugia malayi, B. timori), especially to reduce the long tail of ‘endgame mop-up’ in countries that have completed extensive elimination programmes or in ‘hard to reach’ areas [1]. A potential solution to the current challenges facing pre-clinical onchocerciasis macrofilaricide evaluation is the development of a small animal model of macrofilarial onchocerciasis. Background pahangi) is available in the susceptible gerbil host Meriones unguiculatus [13] and certain strains of inbred mice are susceptible to the filaria, Litomosoides sigmodontis [14]. Whilst these ro- dent filariasis models are undoubtedly useful in identify- ing efficacious filaricidal compounds, overt differences in the biology of Onchocerca species requires confirmation of effectiveness at the pre-clinical stage before informed decisions can be made about clinical development for onchocerciasis indications. A current example of this has been the lack of translation of macrofilaricidal activity of the macrocyclic lactone, moxidectin, from pre-clinical testing against Brugia in gerbils and dogs, when evaluated by a phase II clinical trial in onchocerciasis Thus, in this paper, we evaluated the suitability of the SCID mouse as a pre-clinical model to test macroflarici- dal activity against filariae, using laboratory maintained Brugia malayi to assess responsiveness to a range of filaricidal compounds before trialling survival and drug responses against Onchocerca ochengi implants. Brugia malayi parasites The life cycle of B. malayi (Bm) was maintained in mosquitoes and susceptible Meriones gerbils at LSTM. To generate infective Bm larvae (BmL3) for infections, female adult Aedes aegypti mosquitoes were fed with Bm microfilariae (mf) collected from infected gerbils by catheterisation, as previously described [23], followed by mixing with human blood and feeding through an artifi- cial membrane feeder (Hemotek®). Blood-fed mosquitoes were reared for 14 days to allow for development to L3. The L3 were collected from infected mosquitoes by crushing and concentration using a Baermann’s appar- atus and RPMI medium. For Onchocerca implants, rodents were placed under surgical anaesthesia using i.p. injections of ketamine and medetomidine. Onchocercomata were thoroughly cleaned of bovine tissue, washed in several changes of RPMI + PSN and small sections of the adult worms were exposed by partial rupture of the capsule. Groups of 8–15 motile O. ochengi male macrofilariae or 4 prepared O. ochengi nodules were implanted into the peritoneal cavity or to the cutaneous tissue of the upper side of the neck (for s.c nodule implants). All wounds were re-sutured after surgery and animals were individually housed and closely monitored for the recovery period (7 days post-op). Drug efficacy assessments Developing larvae, macrofilariae and released mf were recovered by peritoneal washings and enumerated by mi- croscopy. Motile, unencapsulated worms were scored as viable. Bm mf were centrifuged before being resuspended in a known volume and a sample enumerated. For Bm mf microfilaraemias, 30 μl thick films on uncoated glass slides (Corning) were prepared from freshly-collected blood (tail bleeds or cardiac punctures post-mortem) in 5000 U/ml Implantation of adult macrofilariae Bm macrofilariae were collected from infected SCID mice and were grouped into batches of 13 adults. Male BALB/c SCID and BALB/c WT mice were placed under surgical anaesthesia using isofluorane and were given s.c. injection of bruprenorphine before 13 Bm macrofilariae were placed into the peritoneal cavity by making a small incision to the skin and abdominal cavity wall in the upper right quad- rant. The incisions were re-sutured after implant and animals were individually housed after surgery. Methods A i l Male BALB/c SCID were purchased from Harlan La- boratories, UK, while male CB.17 SCID mice and BALB/c WT mice were purchased from Charles River, UK. Male Meriones unguiculatus (Mongolian gerbils; jirds) were purchased from either Charles River, UK or Janvier La- boratories, France. Rodents shipped to REFOTDE, Buea, Cameroon, were maintained in conventional housing with Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 3 of 14 daily cage cleaning and changing of food. Food, water and bedding were sterilised by autoclaving. For B. malayi experiments, animals were kept at the Biomedical Services Unit (BSU), University of Liverpool, UK in specific pathogen-free (SPF) conditions. All experiments carried out in Cameroon were approved by the Animal Care Committee, REFOTDE. All experiments on animals in the UK were approved by the ethical committees of the University of Liverpool and LSTM, and were conducted according to Home Office (UK) requirements. purified by PD10 column size exclusion chromatography (Amersham). Bm mf were enumerated by microscopy, concentrated by centrifugation and 0.125 × 106 mf were inoculated into the circulation via the lateral tail vein as described previously [24]. Onchocerca ochengi parasites O. ochengi nodules were obtained from the skins of nat- urally infected zebu cattle from the Adamawa region of Cameroon through existing commercial practice for meat production. At abbatoirs in the South West Province, Cameroon, female cattle were checked for O. ochengi infec- tion by palpation of the skin of the umbilical region for onchocercomata. Positive skins were collected after slaugh- ter. The tissue was then transported to REFOTDE, Buea within 2 hours of collection. Skins were washed several times and the hair was removed by shaving. Onchocerco- mata were excised from dermal tissue using sterile scalpels and forceps, and were placed in RPMI containing penicillin, streptomycin and neomycin (RPMI + PSN). To obtain free adult males, onchocercomata were cut gently to expose the adult worms using sterile scalpels and were incubated in petri dishes containing RPMI + PSN for 4 hours at 37°C, 5% CO2, to allow males to escape into the medium. Free, intact and motile adult males were confirmed by visualisa- tion of posterior anatomy, were washed several times in fresh RPMI + PSN and kept at 37°C, 5% CO2 overnight. Drug treatments Th d d The drug doses in mg/kg and routes used in this study were: Albendazole (ABZ; 50 mg/kg qd po), diethylcarbam- azine (DEC; 50 mg/kg qd po), flubendazole (FBZ; 10 mg/ kg qd sc), ivermectin (IVM; 5 mg/kg qd ip or 15 mg/kg qd po), minocycline (MIN; 25 mg/kg bid po) and rifapentine (RIFAP; 15 mg/kg qd po). Drugs were dissolved in stand- ard suspension vehicle (0.5% sodium carboxymethyl cellu- lose; 0.5% benzyl alcohol; 0.4% Tween 80; 0.9% NaCl), with the exception of MIN, which was dissolved in water, RIFAP which was dissolved in 55% polyethylene glycol 300; 25% propylene glycol; 20% water and parenteral IVM which was dissolved in 1% DMSO. Oral drugs were ad- ministered in volumes of 100-200 μl by gavage. All drugs were purchased from Sigma Aldrich. PCR quantification of Wolbachia Individual adult filariae were fixed in RNAlater and stored at 4°C. For Wolbachia enumerations, DNA was extracted from worm samples using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer’s instructions. Levels of Bm Wolbachia wsp and Bm gst gene copy numbers were quantified using qPCR with Bm-specific primers, as reported elsewhere [25]. Levels of O. ochengi-specific wsp and gst were estimated using identical PCR conditions, with the wsp primer pairs: wsp420TGTTGGT(AG)TTGGT(GC)TTGGTG, wsp583 AACCAAA(AG)TAGCGAGC(CT)CCA and the gst pri- mer pairs: gst175ATTGAAGCGCTTATTAGTCTGC, gst3 05TGTCGTTTCCATTTCATTTTC. To further assess responses of the SAFD, IVM, against mf in the circulation, Bm mf were inoculated via the tail vein into BALB/c SCID mice or BALB/c immune competent controls. Initial parasitaemias were measured at a baseline of +48 h after inoculation, be- fore groups of mice were administered with a single parenteral dose of 5MK IVM or VC. IVM induced rapid reductions in microfilaraemia +24 h post-treatment and levels further declined +7d post-treatment in both WT and SCID mice (79%; P = 0.0194 and 76%; P = 0.0473 median reductions +7d, respectively, Figure 2A). In SCID mice, the levels of mf recovered from the cardiac puncture at termination were also significantly reduced in IVM-treated mice (89% median reduction, P = 0.0286, Figure 2B). B. malayi experimental infections For B. malayi (Bm) L3 infection, 100 freshly collected, motile Bm L3 were injected via the intra-peritoneal route. Efficiencies of inoculations were confirmed by needle washout. For mf perfusion, Bm mf were harvested by peri- toneal washings of patently infected Meriones gerbils under isoflurane anaesthesia, washed in RMPI + PSN and Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 4 of 14 infectious third stage (L3) of Brugia malayi (Bm) via the intra-peritoneal route. Infected mice were orally admin- istered with SAFD or vehicle control (VC) at indicated doses for seven days, before Bm larvae were recovered by peritoneal lavage (+24 h following the final drug exposure) and motile parasites enumerated. Results are shown in Figure 1. ABZ and DEC treatment delivered an almost complete filaricidal activity in WT mice (100% and >99%, respectively). These potent filaricidal effica- cies were emulated within SCID hosts (ABZ; 98%, DEC; 100% reductions, P < 0.0001). IVM was partially filarici- dal at the dose administered (15 mk/kg qd) in WT mice (39.8% mean reduction, P = 0.0356). In SCID mice, a partial filaricidal response was also evident (56.7% mean reduction, P = 0.026). lithium heparin anti-coagulant (Sigma Aldrich). Slides were air dried, de-haemaglobinised in tap water, fixed in 80% MeOH and stained with Giemsa (BDH). Bm mf were enumerated by microscopy. For recovery of O. ochengi nodules, the nodule tissue was excised from the area of tissue engraftment post-mortem. Nodules were dissected and incubated in RPMI + PSN, 37°C, 5% CO2 for +4 h before the presence of motile worms or mf was evaluated by microscopy. Statistical analysis Where intra-group data was skewed, non-parametric analyses were used to compare statistical differences between two independent groups (Mann Whitney test) or three or more independent groups (Kruskal Wallis with Dunn’s post-hoc tests). Where intra-group data dis- played a normal distribution, parametric 1way ANOVA with Bonferroni post-hoc tests were used to examine statistical differences between 3 or more groups. Paired T tests evaluated changes in time within individual ani- mals. Correlations were assessed for significance using Pearson Correlation test. Significance is indicated by P < 0.05, P < 0.01* and P < 0.001*. Whilst infectious stage Bm L3 are reported to develop into mf-producing macrofilariae in SCID mice, WT mice are refractory to full Bm development [21,26]. For the purposes of validating direct FBZ macrofi- laricidal efficacy in immunodeficient mice compared with immunosufficient controls, we implanted a defined num- ber of Bm macrofilariae sourced from +35 day ip infec- tions of SCID donors into either WT or SCID recipients. Following +2 day recovery from surgery, mice were placed on parenteral FBZ suspension (10 mk/kg sub- cutaneous, qd) or matching VC for a total of five days and macrofilaricidal efficacy was evaluated +5 weeks following the last drug exposure. Comparing VC groups, there was a marginally non-significant de- creased recovery of Bm macrofilariae in BALB/c WT versus BALB/c SCID mice (P = 0.069). FBZ treatments were 100% efficacious in both WT and SCID BALB/c mice implanted with Bm macrofilariae (Figure 3A; aver- age recovery of 0% FBZ treatment vs. 38%, P = 0.0067 for VC WT and 46% P = 0.0071 for VC SCID). En- trapped immotile macrofilariae encapsulated within leukocytic granulomas were frequently evident in WT but not SCID mice (median recovery 4, WT vs 0, SCID, P = 0.0104; Figure 3B). Onchocerca macrofilariae survival following implantation into WT/SCID mice and Mongolian gerbils Using the intraperitoneal surgical implant technique as detailed above for Bm, we implanted isolated motile male Onchocerca macrofilariae into various laboratory rodent models and evaluated survival following +15 days (Figure 6). As well as comparison of WT vs SCID mice, we tested survival in Mongolian gerbils (Meriones ungui- culatus), an outbred laboratory rodent susceptible to a number of other filariae. After 15 days post-surgical im- plant, we successfully recovered parasites from all rodent recipients with the exception of one WT BALB/c mouse. Male Onchocerca macrofilariae recovered from rodent im- plants were monitored for viability ex vivo in culture after recovery from rodent hosts and motility comparable to freshly isolated adult males was observable up to 7 days. At +15 days, recoveries of male macrofilariae were, on average, significantly lower in WT BALB/c versus SCID BALB/c mice (22% vs 60% median recovery, P = 0.0475), whilst the average recovery of Onchocerca male macrofi- lariae was not significantly different in Meriones gerbils vs WT or SCID BALB/c mice (38%). We took advantage of the full development of Bm in SCID mice to examine the response to oral treatment with the anti-Wolbachia tetracycline, MIN, against the macrofilarial stage of the parasite. SCID BALB/c mice were infected with 100 infectious stage BmL3 larvae ip and dosing with MIN or vehicle control (VC) com- menced at +6wk post-infection, when parasites had reached the juvenile adult stage. Mice were left for two weeks following the last drug exposure and adult Bm and released mf were collected at the early onset of patency (+12wks post-infection). The average recovery of adult Bm in control mice was 9.4 +/−2.6 SEM (n = 5) and recoveries did not vary in MIN treated animals (Figure 4A). Numbers of Wolbachia were quantified following DNA extraction of individual female worms (n = 10/group, pooled from individual mice; Figure 4B). In the control group, average Wolbachia numbers per female were 5.865 × 107 +/−0.72 × 107. The average number of Wol- bachia in female worms exposed to MIN in vivo in SCID mice had been significantly reduced to 1.837 × 106 +/−0.702 × 106. This equated to a 98.4% reduction in Wolbachia (P < 0.0001). The majority of control mice also contained peritoneal mf (4/5 mice; Figure 4C). Mf production was enumerated per female worm and, on average, each 12 week old female worm had produced We also trialled the implantation of isolated O. Evaluating the performance of reference filaricidal compounds against B. malayi in SCID mice We adapted mouse models of Brugian filariasis [26-28] to scrutinise whether the standard anti-filarial drugs (SAFD) albendazole (ABZ), diethylcarbamazine (DEC) and ivermec- tin (IVM) or drugs with demonstrated macrofilaricidal properties, flubendazole (FBZ) and the tetracycline anti- wolbachia antibiotic, minocycline (MIN), displayed predict- able efficacy in a Severe-Combined Immuno-Deficiency (SCID) mouse model system. For SAFD, which predominantly target the larval stages of filariae in clinical and veterinary indications, we inoculated either BALB/c SCID or BALB/c wild type (WT) mice, as an immune-sufficient control, with 100 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 5 of 14 WT VC WT+ABZ WT+DEC WT+IVM SCID VC SCID+ABZ SCID+DEC SCID+IVM 0 20 40 60 80 % recovery BmL4 Figure 1 Comparative larvicidal activity of SAFDs against Brugia in WT versus SCID mice. Percentage recoveries of BmL4 larvae from the peritoneal cavities of mice eight days after inoculation with 100 BmL3 and commencement of a seven day oral regimen of 50 mg/kg qd albendazole (ABZ), diethylcarbamazine (DEC) or 15 mg/kg qd ivermectin (IVM). Circle plots are recoveries from BALB/c WT mice whilst square plots are derived from BALB/c SCID mice. Error bars are mean +/−SEM% recoveries per group. Data is pooled from two individual experiments for WT and one experiment for SCID mice (n = 4/group). Significant differences (1 way ANOVA with Bonferroni multiple comparison) are indicated P < 0.001, P < 0.01, P < 0.05. 373.3 (+/−204.4) mf. Contrastingly, there was a complete absence of mf in the peritoneal cavity of MIN treated SCID mice (P = 0.0215), consistent with loss of embryonic development following the sterilisation of Wolbachia from adult Bm filarial tissues. Yields of Onchocerca macrofilariae from naturally parasitized cattle Zebu cattle, naturally infected with the bovine Oncho- cerca species, O. ochengi, were identified within herds moved from pasture land in the Adamawa Region and slaughtered for meat production in abattoirs located in the South West region of Cameroon. Over a 48-day col- lection period, a total of 2612 nodules were collected from 28 infected hides (mean 105.5 +/−28.4 SEM, Figure 5), which were processed to purify motile male Onchocerca macrofilariae. A positive correlation between numbers of nodules recovered and numbers of adult males was ob- served (Pearson R = 0.869; P < 0.0001) and, on average, 69.7 +/−16.4 SEM motile male macrofilariae were recov- ered per infected cattle hide processed. This was equiva- lent to an average ratio of 0.66:1 male macrofilariae recovered per harvested nodule. Figure 1 Comparative larvicidal activity of SAFDs against Brugia in WT versus SCID mice. Percentage recoveries of BmL4 Figure 1 Comparative larvicidal activity of SAFDs against Brugia in WT versus SCID mice. Percentage recoveries of BmL4 larvae from the peritoneal cavities of mice eight days after inoculation with 100 BmL3 and commencement of a seven day oral regimen of 50 mg/kg qd albendazole (ABZ), diethylcarbamazine (DEC) or 15 mg/kg qd ivermectin (IVM). Circle plots are recoveries from BALB/c WT mice whilst square plots are derived from BALB/c SCID mice. Error bars are mean +/−SEM% recoveries per group. Data is pooled from two individual experiments for WT and one experiment for SCID mice (n = 4/group). Significant differences (1 way ANOVA with Bonferroni multiple comparison) are indicated P < 0.001*, P < 0.01, P < 0.05. Figure 1 Comparative larvicidal activity of SAFDs against Brugia in WT versus SCID mice. Percentage recoveries of BmL4 larvae from the peritoneal cavities of mice eight days after inoculation with 100 BmL3 and commencement of a seven day oral regimen of 50 mg/kg qd albendazole (ABZ), diethylcarbamazine (DEC) or 15 mg/kg qd ivermectin (IVM). Circle plots are recoveries from BALB/c WT mice whilst square plots are derived from BALB/c SCID mice. Error bars are mean +/−SEM% recoveries per group. Data is pooled from two individual experiments for WT and one experiment for SCID mice (n = 4/group). Significant differences (1 way ANOVA with Bonferroni multiple comparison) are indicated P < 0.001, P < 0.01, P < 0.05. Onchocerca macrofilariae survival following implantation into WT/SCID mice and Mongolian gerbils ochengi onchocercomata, containing a mixture of cow tissue plus both female and male Onchocerca macrofi- lariae, into SCID BALB/c or SCID CB.17 strains. In this pilot study, four onchocercomata were implanted into the peritoneal cavity or sub-cutaneously at the nape of the neck of each SCID recipient. Table 1 and Figure 7 detail the parasitological observations +7 days Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 6 of 14 y http://www.parasitesandvectors.com/content/7/1/472 A B baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c SCID baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c SCID + 5MK IVM baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c + 5MK IVM baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c BALB/c SCID VC BALB/c SCID IVM 0 20000 40000 60000 Bm mf/ml cardiac circulation +7d BALB/c WT VC BALB/c WT IVM 0 20000 40000 60000 Bm mf/ml cardiac circulation +7d Figure 2 Comparative microfilaricidal activity of IVM against Brugia in WT versus SCID mice. Bm mf densities in peripheral circulating blood (A) or cardiac circulation (B) at ‘baseline’ (48 h following intravenous perfusion with 1.25 × 105 purified mf) or at indicated time points following treatment with 5 mg/kg ivermectin (IVM) or vehicle control (VC) via the intraperitoneal route. Top panels are data derived from WT BALB/c, bottom panels are data from BALB/c SCID mice. Error bars indicate median mf densities and interquartile range. Data is from an individual experiment (n = 4/group). Significant differences with time (paired T Test) or between groups (Mann Whitney Test) are indicated P < 0.05. A baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c + 5MK IVM baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c B A Bm mf/ml cardiac circulation +7d baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c SCID baseline +24h +7d 0 500 1000 1500 2000 Bm mf/ml peripheral blood BALB/c SCID + 5MK IVM BALB/c SCID VC BALB/c SCID IVM 0 20000 40000 60000 Bm mf/ml cardiac circulation +7d Bm mf/ml peripheral blood Bm mf/ml peripheral blood Bm mf/ml cardiac circulation + Figure 2 Comparative microfilaricidal activity of IVM against Brugia in WT versus SCID mice. Onchocerca macrofilariae survival following implantation into WT/SCID mice and Mongolian gerbils Bm mf densities in peripheral circulating blood (A) or cardiac circulation (B) at ‘baseline’ (48 h following intravenous perfusion with 1.25 × 105 purified mf) or at indicated time points following treatment with 5 mg/kg ivermectin (IVM) or vehicle control (VC) via the intraperitoneal route. Top panels are data derived from WT BALB/c, bottom panels are data from BALB/c SCID mice. Error bars indicate median mf densities and interquartile range. Data is from an individual experiment (n = 4/group). Significant differences with time (paired T Test) or between groups (Mann Whitney Test) are indicated P < 0.05. following nodule implantation. At necropsy, the major- ity of SCID BALB/c recipients (4/6) had evidence of engraftment of some or all implanted onchocercomata into abdominal visceral tissues, mainly connective tissues continuous with the mesenteries and visceral fat pads. In engrafted onchocercomata, neovacularisation could be observed (9/24 implanted nodules), manifest as capillary beds distributed across the capsule surface, stemming from proximal host vascular networks. Onchocercomata were extracted, partially dissected to expose loops of female macrofilariae and cultured ex vivo for 4 hours. Ob- servations of motility during this period revealed that in 5/ 6 SCID recipients, 100% of implanted nodules contained motile female Onchocerca macrofilariae. Transplanted onchocercomata yielded motile male Onchocerca macrofilariae after recovery from 4/6 SCID recipients at a ratio similar to that observed from freshly isolated nodules (i.e. ~1 male/2 nodules). Following sub cutaneous implantations in SCID CB.17 mice, 100% of onchocerco- mata (n = 8) in 2/2 recipients displayed engraftment into the sub cutaneous layer with evidence of neovascularisa- tion. Vessel-like structures containing blood cells, prox- imal to female worms, were identifiable within implanted onchocercomata by histology. All onchocercomata recov- ered from sub cutaneous implants at +7 days contained motile female macrofilariae when dissected and cultured ex vivo. A single motile male macrofilaria was released from nodules derived from 1/2 recipient mice. Evidence of Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 7 of 14 to evaluate macrofilaricide efficacy. We applied the opti- mised FBZ screen verified in BALB/c SCID Bm implants (Figure 3) to compare suitability of this system for macrofilaricide testing. An increased number of 15 mo- tile male Onchocerca macrofilariae were implanted into each SCID mouse recipient to mitigate against decline in survival over 5 weeks. Onchocerca macrofilariae survival following implantation into WT/SCID mice and Mongolian gerbils Error bars indicate median and interquartile range. Data is from an individual experiment (n = 5/group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.05, *P < 0.01. Figure 3 Comparative macrofilaricidal activity of FBZ against Brugia in WT versus SCID mice. (A) % recoveries of Bm embryogenesis within implanted female Onchocerca uteri was apparent by histological examination, including the presence of inter-uterine stretched mf. Further, released motile microfilariae were evident in the culture medium following ex vivo culture of implanted sub cutaneous onchocercomata derived from 2/2 SCID CB.17 recipients. We also tested a lead anti-Wolbachia rifamycin anti- biotic, rifapentine (RIFAP), for activity against Onchocerca male worms in CB.17 SCID mice. Groups of two mice were given oral doses of RIFAP (15 mg/kg) or standard suspension vehicle (VC) daily for 14 days, starting from 1–2 days recovery after surgery. Implanted Onchocerca were retrieved +2 weeks after last drug exposure. Total re- coveries of Onchocerca macrofilariae were not affected by Onchocerca macrofilariae survival following implantation into WT/SCID mice and Mongolian gerbils Also, daily isolates of macrofilar- iae (from individual cattle hides) were divided equally between recipients consigned to FBZ or VC treatments to avoid inter-group bias in quality and age of male worms that might affect survival. Further, to mitigate against inter-group variation in survival rates over a pro- tracted period post-implantation masking macrofilaricidal effects, group sizes were increased to 7–8 mice. Treatment groups were maintained for up to 31 days (4–5 wks post implant) before recovery of parasites (Figure 8). FBZ in- duced a significant, almost total macrofilaricidal response, with mean survival of 1.67% (+/−1.09, n = 8), compared with a mean survival of 43.81% (+/−11.44, n = 7) recovery in the VC group (P = 0.0089). In the treated group, single motile worms were recovered in 2/8 recipients, whilst the other six mice had either an absence of infection or recov- ery of completely immotile worms. The two motile worms derived from FBZ treated mice were moribund with irregular and retarded ‘twitching’ motility, compared to the motility rate observed of macrofilariae recovered from VC mice or of freshly isolated onchocercomata. A B WT FBZ SCID FBZ 0 1 2 3 4 5 6 granuloma recovery WT VC WT FBZ SCID VC SCID FBZ 0 20 40 60 80 % recovery adult Bm Figure 3 Comparative macrofilaricidal activity of FBZ against Brugia in WT versus SCID mice. (A) % recoveries of Bm macrofilariae and (B) numbers of granulomas recovered +6 weeks after intraperitoneal surgical implantation into BALB/c WT or BALB/c SCID mice and commencement of a 5 day 10 mg/kg qd sub-cutaneous dosing with flubendazole (FBZ) or matching vehicle control (VC). Error bars indicate median and interquartile range. Data is from an individual experiment (n = 5/group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.05, P < 0.01. A WT VC WT FBZ SCID VC SCID FBZ 0 20 40 60 80 % recovery adult Bm * ** B WT FBZ SCID FBZ 0 1 2 3 4 5 6 granuloma recovery * B Figure 3 Comparative macrofilaricidal activity of FBZ against Brugia in WT versus SCID mice. (A) % recoveries of Bm macrofilariae and (B) numbers of granulomas recovered +6 weeks after intraperitoneal surgical implantation into BALB/c WT or BALB/c SCID mice and commencement of a 5 day 10 mg/kg qd sub-cutaneous dosing with flubendazole (FBZ) or matching vehicle control (VC). Evaluation of the SCID model of onchocerciasis as a pre-clinical macrofilaricidal drug screen Because of initial, reproducible, high recoveries of viable male Onchocerca macrofilariae in BALB/c SCID mice, we evaluated this model as an anti-Onchocerca in vivo macrofilaricidal drug screen where survival over a more protracted time frame (4–5 weeks) would be necessary A B C VC MIN 0 5 10 15 20 % recovery B. malayi @ +12 wks VC MIN 105 106 107 108 wsp copy number / female B. malayi @ +12 wks *** (98.4%) VC MIN 1 10 100 1000 10000 Mf (+1) / female B. malayi @ +12 wks * Figure 4 Anti-Wolbachia activity against Brugia macrofilariae in SCID mice. (A) % recoveries of Bm macrofilariae (B) Wolbachia loads per female Bm and (C) numbers of released mf per female Bm in BALB/c SCID mice +12 weeks after intraperitoneal infection with 100 BmL3 and +6 weeks after commencement of 4 week oral 25 mg/kg bid minocycline (MIN) or vehicle control (VC). Error bars indicate median and interquartile range. Dashed horizontal lines in (B) indicate 1 and 2 log reductions compared with median VC levels. Median % reduction in (B) is indicated in parentheses. Data is from an individual experiment (n = 5/group; individual worms pooled per group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.05, P < 0.001. A VC MIN 0 5 10 15 20 % recovery B. malayi @ +12 wks B VC MIN 105 106 107 108 wsp copy number / female B. malayi @ +12 wks * (98.4%) C VC MIN 1 10 100 1000 10000 Mf (+1) / female B. malayi @ +12 wks * B Figure 4 Anti-Wolbachia activity against Brugia macrofilariae in SCID mice. (A) % recoveries of Bm macrofilaria Figure 4 Anti-Wolbachia activity against Brugia macrofilariae in SCID mice. (A) % recoveries of Bm macrofilariae (B) Wolbachia loads per female Bm and (C) numbers of released mf per female Bm in BALB/c SCID mice +12 weeks after intraperitoneal infection with 100 BmL3 and +6 weeks after commencement of 4 week oral 25 mg/kg bid minocycline (MIN) or vehicle control (VC). Error bars indicate median and interquartile range. Dashed horizontal lines in (B) indicate 1 and 2 log reductions compared with median VC levels. Median % reduction in (B) is indicated in parentheses. Data is from an individual experiment (n = 5/group; individual worms pooled per group). Evaluation of the SCID model of onchocerciasis as a pre-clinical macrofilaricidal drug screen Significant differences between groups (Mann Whitney Test) are indicated P < 0.05, *P < 0.001. Figure 4 Anti-Wolbachia activity against Brugia macrofilariae in SCID mice. (A) % recoveries of Bm macrofilariae (B) Wolbachia loads per female Bm and (C) numbers of released mf per female Bm in BALB/c SCID mice +12 weeks after intraperitoneal infection with 100 BmL3 and +6 weeks after commencement of 4 week oral 25 mg/kg bid minocycline (MIN) or vehicle control (VC). Error bars indicate median and interquartile range. Dashed horizontal lines in (B) indicate 1 and 2 log reductions compared with median VC levels. Median % reduction in (B) is indicated in parentheses. Data is from an individual experiment (n = 5/group; individual worms pooled per group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.05, **P < 0.001. Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 8 of 14 A B C D Nodules Free adult males 0 100 200 300 400 Count 0 100 200 300 400 0 50 100 150 200 motile male macrofilariae nodules Figure 5 Yields of female and male Onchocerca macrofilariae isolated from naturally infected cattle. A) Typical yield of excised O. ochengi onchocercomata from a parasitized cattle hide B) A pair of liberated, motile male O. ochengi worms +4 h after disruption of onchocercoma (nodule) capsule and culture at 37°C/5%CO2. Scale bar =1 cm. C) Relationship between numbers of onchocercomata recovered per cattle hide and number of motile male O. ochengi harvested. Dashed line is linear regression best fit. D) Recoveries of onchocercomata (closed plots) and male O. ochengi (open plots) per cattle hide (n = 29). Error bars are mean +/−SEM. A A B B C 0 100 200 300 400 0 50 100 150 200 motile male macrofilariae nodules D Nodules Free adult males 0 100 200 300 400 Count 0 C D D Figure 5 Yields of female and male Onchocerca macrofilariae isolated from naturally infected cattle. A) Typical yield of excised O. ochengi onchocercomata from a parasitized cattle hide B) A pair of liberated, motile male O. ochengi worms +4 h after disruption of onchocercoma (nodule) capsule and culture at 37°C/5%CO2. Scale bar =1 cm. C) Relationship between numbers of onchocercomata recovered per cattle hide and number of motile male O. ochengi harvested. Dashed line is linear regression best fit. Evaluation of the SCID model of onchocerciasis as a pre-clinical macrofilaricidal drug screen D) Recoveries of onchocercomata (closed plots) and male O. ochengi (open plots) per cattle hide (n = 29). Error bars are mean +/−SEM. treatment regimen (56.65%, VC and 58.35%, median recovery, RIFAP; Figure 9A). Numbers of the single copy Wolbachia surface protein (wsp) gene per isolated male Onchocerca marofilariae were quantified as a surrogate measurement of endosymbiont density within nematode tissues (Figure 9B). A highly significant 99% reduction in Wolbachia number was recorded in Onchocerca macrofi- lariae obtained from CB.17 SCID mice treated with rifa- pentine for 14 days (4.6 × 106 median wsp copies, VC vs. 0.0452 × 106, RIFAP, P < 0.001). Wolbachia reductions were preserved when adjusted for potential variation in size and age of adult male Onchocerca derived from cattle onchocercomata, by normalisation to a single copy filarial gene, gst (Figure 9C). throughput to address current demand. For these rea- sons we decided to develop and validate a small animal model of onchocerciasis. We chose to trial SCID mice as a compatible host for the cattle Onchocerca, O. ochengi. We selected this particular parasite after identifying an abundant and relatively convenient sampling source of O. ochengi macrofilariae in cattle herds derived from the Adamawa region of Northern Cameroon used for com- mercial meat production. Our experience over a three- month evaluation period indicated that the prevalence of infected female cattle being moved for slaughter in the South West Province was typically between 5-10% and that, with around 10–20 cattle being processed daily at a local abattoir, there was frequent availability of infected cow tissues. Because we typically collected >100 O. ochengi macrofilariae from a single hide, this provided an adequate daily supply line of Onchocerca for in vivo drug testing. The implantation of macrofilariae into groups of rodents from a single cow effectively increased capacity 4–5 fold in terms of biological units available for drug testing. Further, considering the much reduced space and costs demanded for long-term husbandry of rodents vs cattle, the rodent model should further facilitate increased Discussion The development of macrofilaricides against onchocer- ciasis is currently hampered by a lack of a facile pre- clinical infection model. Extrapolating efficacy of drug candidates against lymphatic filariae in susceptible ro- dents may not necessarily translate into effective oncho- cerciasis indications and traditional pre-clinical testing in cattle onchocerciasis does not possess the required Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 9 of 14 BALB/c WT BALB/c SCID GERBIL 0 20 40 60 80 % recovery male Onchocerca Figure 6 Survival of male Onchocerca macrofilariae in laboratory rodents. Percentage recoveries +15 days post-intraperitoneal implantation with 8–12 isolated, motile male Onchocerca macrofilariae in BALB/c WT, BALB/c SCID and Meriones unguiculatus gerbils. Error bars are median recoveries with interquartile range. For BALB/c SCID, data is pooled from two individual experiments (n = 3/4). For BALB/c WT and Meriones, data is from a single experiment (n = 3/4). Significant differences between groups (Kruskal-Wallis Test with Dunn’s multiple comparison) is indicated P < 0.05. The selection of SCID mice was based on extensive use of this lymphopenic mouse system for xenograft transfers in cancer and malaria chemotherapy pre-clinical testing [30,31] and our own and other laboratory’s observations that SCID mice are permissive hosts for non-murine fil- ariae, including full development of the human lymphatic filariae, B. malayi [21] and stage-specific Onchocerca spp. infections [20,22]. [ ] A potential caveat to the application of SCID mice for anti-filarial drug screening is the absence of adaptive im- mune responses, which might be important in interacting with candidate filaricidal compounds to induce macrofilar- icidal effects. Whilst it is inconclusive whether inflamma- tory reactions in patients post-treatment with anti-filarial drugs (e.g. “Mazzoti reactions”) are a necessary compo- nent in the death of filariae or merely a response to dead and damaged worms [16] and the concomitant liberation of somatic antigens and Wolbachia endobacteria [32,33], the former has been proposed due to a general lack of effi- cacy of SAFD at physiological levels in vitro against filariae [34,35]. Further, various ‘immuno-pharmacological’ modes of action have been proposed for SAFD, including the prevention of immuno-modulatory secretions from mf by IVM [36] and the induction of host inducible nitric oxide and cyclooxygenase pathways by DEC [24]. Thus, it was important to validate anti-filarial responses in the SCID model against an immunologically intact WT control, where possible. otility assessed by dissection microscope +4 h after excision of nodule grafts, disruption of nodule capsule and culture in complete me – not done. Discussion For this we took advantage of WT BALB/ c mice as an immune competent background strain that, whilst resistant to chronic patent infections, accommo- dates life cycle stages of B. malayi for periods sufficient to evaluate filaricidal drug effects [24,27]. The results of our studies, comparing larvicidal responses of the SAFD: ABZ, DEC and IVM, illustrated no difference in the level of ob- served efficacy of these reference anti-filarials against in- fectious Bm L3 larvae, or in the case of IVM, bloodborne mf, in the absence of adaptive immune responses. In fact, for mf, we observed a 2.5 fold higher peripheral circulating mf and 6.4 fold increased cardiac blood levels in SCID vs Figure 6 Survival of male Onchocerca macrofilariae in Figure 6 Survival of male Onchocerca macrofilariae in laboratory rodents. Percentage recoveries +15 days post-intraperitoneal implantation with 8–12 isolated, motile male Onchocerca macrofilariae in BALB/c WT, BALB/c SCID and Meriones unguiculatus gerbils. Error bars are median recoveries with interquartile range. For BALB/c SCID, data is pooled from two individual experiments (n = 3/4). For BALB/c WT and Meriones, data is from a single experiment (n = 3/4). Significant differences between groups (Kruskal-Wallis Test with Dunn’s multiple comparison) is indicated P < 0.05. capacity for simultaneous or overlapping Onchocerca drug screening within the same laboratory, in a cost-effective manner. The ability to culture the male O. ochengi macro- filariae ex vivo for durations of at least five days, also means that this method of collection could be applied to in vitro testing of novel macrofilaricides, similar to the published male O. gutterosa drug screen [29]. Table 1 Parasitological observations of Onchocerca ochengi onchocercomata post-implantation into SCID mice Mouse strain/ID Number/location Vascularisation Motile females* Motile males* Motile mf* BALB/c SCID 1 4 ip 4/4 4/4 2/4 nd BALB/c SCID 2 4 ip 2/4 4/4 0/4 nd BALB/c SCID 3 4 ip 0/4 4/4 0/4 nd BALB/c SCID 4 4 ip 0/4 4/4 2/4 nd BALB/c SCID 5 4 ip 1/4 0/4 2/4 nd BALB/c SCID 6 4 ip 2/4 4/4 2/4 nd CB.17 SCID 1 4 sc 4/4 2/2 0/2 yes CB.17 SCID 2 4 sc 4/4 3/3 1/3 yes Intraperitoneal implantation (ip), sub cutaneous implantation (sc). motility assessed by dissection microscope +4 h after excision of nodule grafts, disruption of nodule capsule and culture in complete medium at 37°C/5%CO2. nd – not done. ( p), p ( ) ection microscope +4 h after excision of nodule grafts, disruption of nodule capsule and culture in complete medium at 37°C/5%CO2. p p ( p), p ( ) motility assessed by dissection microscope +4 h after excision of nodule grafts, disruption of nodule capsule and culture in complete medium at 37°C/5%CO2. nd – not done. Discussion ical observations of Onchocerca ochengi onchocercomata post-implantation into SCID mice Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 Page 10 of 14 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 y http://www.parasitesandvectors.com/content/7/1/472 Figure 7 Engraftment of Onchocerca onchocercomata in SCID mice. Engrafted peritoneal (A&B) or sub cutaneous (C&D) onchocercomata in situ or excised nodules from the peritoneum (D) or sub cutaneous tissue (E) +7 days after implantation into BALB/c or CB.17 SCID mice. Haematoxylin and eosin staining of sub cutaneous engrafted onchocercomata illustrating embryogenesis (F) and putative murine host vessel-like structures (H). High magnification images of inter-uterine stretched microfilarae (G) and vessel-like structure (I). Key: dermis (d), embryos (e), visceral fat (f), mesentery (m), microfilariae (mf), nodule (n), female Onchocerca ochengi (o), uteris (u), vessel (v). Arrows indicate zones of neovascularisation. Scale bars are 50 μm (F&H) and 10 μm (G&I). Figure 7 Engraftment of Onchocerca onchocercomata in SCID mice. Engrafted peritoneal (A&B) or sub cutaneous (C&D) onchocercomata in situ or excised nodules from the peritoneum (D) or sub cutaneous tissue (E) +7 days after implantation into BALB/c or CB.17 SCID mice. Haematoxylin and eosin staining of sub cutaneous engrafted onchocercomata illustrating embryogenesis (F) and putative murine host vessel-like structures (H). High magnification images of inter-uterine stretched microfilarae (G) and vessel-like structure (I). Key: dermis (d), embryos (e), visceral fat (f), mesentery (m), microfilariae (mf), nodule (n), female Onchocerca ochengi (o), uteris (u), vessel (v). Arrows indicate zones of neovascularisation. Scale bars are 50 μm (F&H) and 10 μm (G&I). We extended our evaluation of the SCID mouse, tak- ing advantage of the full development of Brugia in this immunocompromised system, to assess the appropriate- ness of its use as a facile anti-Wolbachia drug screen. For this we chose a point of oral administration to drug with the anti-Wolbachia reference tetracycline, MIN, once parasites had undergone the final moult to become immature macrofilariae. At this point in the Bm life cycle, Wolbachia numbers have completed a log phase expansion and are representative of levels in mature Brugia (1-5 × 107 Wolbachia per female worm) [25]. Fur- ther, initiating anti-Wolbachia drugging at the immature macrofilarial stage allowed us to simultaneously and rapidly discern downstream effects on embryogenesis via initial mf release, at the immediate onset of patency (+10-11 weeks in mice). Discussion We could consistently recover Bm macrofilariae in infected and dosed SCID animals, supporting prior observations of full permissiveness of this model [21] and allowing for reliable production of the required numbers of adult worms and mf release for assessment of Wolbachia efficacy. Oral MIN administra- tion in infected SCID mice for 28 days reduced Wolba- chia loads within female Bm below the 90% threshold deemed to irreversibly sterilise filarial tissues in clinical studies, leading to macrofilaricidal effects [33,42,43]. The >90% levels of depletion observed in female macro- filariae derived from drugged SCID Bm infections were consistent with in vivo effects reported following pro- tracted tetracycline treatments in patent Bm infections of gerbils [44] and were reflected in a complete block- ade in mf release in drugged animals. WT control groups at 9 days post-tail vein perfusion, which allowed us to discern a more obvious treatment effect of single dose IVM. We also observed similar mf levels in CB.17 SCID mice (data not shown). The elevated persistence of circulating mf in SCID mice may be due to a lack of initiation of an effective stage-specific adaptive immune response. Certainly, microfilariaemic brugian filariasis patients have hyporesponsive filarial antigen- specific peripheral blood mononuclear cell responses vs amicrofilaraemic infected individuals [37]. Splenic clear- ance has been demonstrable in controlling bloodborne microfilaraemias in mice [38] and baboons [39] which suggests that SCID mice may have a defective mechanism at this secondary lymphoid organ. This data extends experiments undertaken in athymic nude mice that demonstrates a lack of a role for T lym- phocytes/lymphocyte ‘help’ in a DEC mode of action [40] and also suggests T and B lymphocyte-mediated adaptive immunity is dispensable for the effects of the benzimid- azole (BZ) and macrocyclic lactone families of anti-filarial drugs. Further, a complete FBZ macrofilaricidal response was apparent in both WT and SCID animals using a dose regimen previously reported as effective in the Meriones gerbil Brugia macrofilariae implant model [41]. FBZ macrofilaricidal efficacy generally proceeded without overt leukocytic granuloma formation in SCID mice suggesting that these reactions are a response to dead worms but are not integral to the mode of action of BZ anthelmintics. Whether this extends to other classes of SAFD that have macrofilaricidal activity against Bm, e.g. DEC and MOX, remain to be evaluated. Halliday et al. Discussion However, should drugs emerge with putative T or B cell adaptive immune-pharmacological mechanisms, the avail- ability of comparative Bm and Onchocerca macrofilaricide screens in WT mice, SCID mice and gerbils would serve Figure 8 Macrofilaricidal activity of parenteral FBZ against Onchocerca macrofilariae in SCID mice. Percentage recoveries +5 weeks after intraperitoneal surgical implantation of 15 isolated, motile male Onchocerca macrofilariae in BALB/c SCID mice and commencement of a 5 day 10 mg/kg qd sub-cutaneous dosing with flubendazole (FBZ) or matching vehicle control (VC). Error bars indicate median and interquartile range. Data is from an individual experiment (n = 7-8/group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.01. Figure 8 Macrofilaricidal activity of parenteral FBZ against Onchocerca macrofilariae in SCID mice. Percentage recoveries g +5 weeks after intraperitoneal surgical implantation of 15 isolated, motile male Onchocerca macrofilariae in BALB/c SCID mice and commencement of a 5 day 10 mg/kg qd sub-cutaneous dosing with flubendazole (FBZ) or matching vehicle control (VC). Error bars indicate median and interquartile range. Data is from an individual experiment (n = 7-8/group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.01. A B C VC RIFAP 0 20 40 60 80 100 % recovery male Onchocerca VC RIFAP 102 103 104 105 106 107 108 wsp copies / male Onchocerca (99.0%) * VC RIFAP 0.1 1 10 100 1000 wsp/gst / male Onchocerca (99.3%) * Figure 9 Anti-Wolbachia activity against Onchocerca macrofilariae in SCID mice. (A)% recoveries, (B) Wolbachia loads per motile male Onchocerca macrofilaria measured by wsp gene copy number or (C) Wolbachia loads per motile male Onchocerca macrofilaria as a ratio wsp/gst filarial gene copy number +29 days after intraperitoneal implantation in CB.17 SCID mice and +28 days after commencement of 2 week oral 15 mg/kg rifapentine (RIFAP) or vehicle control (VC). Error bars indicate median and interquartile range. Dashed horizontal lines indicate 1 and 2 log reductions compared with median VC levels. Median % reduction is indicated in parentheses. For B&C, data from individual worms are pooled from groups of two mice. Significant differences between groups (Mann Whitney Test) are indicated *P < 0.001. Discussion Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 11 of 14 VC FBZ 0 20 40 60 80 100 % recovery male Onchocerca Figure 8 Macrofilaricidal activity of parenteral FBZ against Onchocerca macrofilariae in SCID mice. Percentage recoveries +5 weeks after intraperitoneal surgical implantation of 15 isolated, motile male Onchocerca macrofilariae in BALB/c SCID mice and commencement of a 5 day 10 mg/kg qd sub-cutaneous dosing with flubendazole (FBZ) or matching vehicle control (VC). Error bars indicate median and interquartile range. Data is from an individual experiment (n = 7-8/group). Significant differences between groups (Mann Whitney Test) are indicated P < 0.01. Having validated the SCID model system as suitably responsive to a range of filaricidal reference compounds, we tested survival rates of male Onchocerca macrofilariae in either SCID BALB/c or WT BALB/c mice. The results of a two-week implant pilot study indicated that male Onchocerca survived significantly better in the immuno- compromised system, suggesting that adaptive immune responses begin to exert attritional effect on survival by this stage. This is consistent with the kinetics of immune- attrition against larval Bm infections where adaptive immune responses begin to exert significant filaricidal activity at +2 weeks post-infection [26]. We were also able to preliminarily evaluate survival in the Brugia susceptible outbred rodent, Meriones, which indicated that male Onchocerca macrofilariae could persist at a similar level as within SCID mice. As our focus was on development of a flexible ‘pan-filarial’ SCID pre-clinical model, we did not investigate survival in Meriones gerbils further. However our initial findings suggest Meriones may also be a suitable laboratory host for male Onchocerca implants and further investigation is warranted to explore the length of persist- ence in this immune-intact outbred rodent. It is debatable whether macrofilaricides with a total reliance on adaptive immunity would be suitable for indications in chronic fil- ariasis patients who typically display hyporesponsive T cell profiles to filarial antigens [37,45,46]. Furthermore, identi- fication of ‘hits’ within in vitro culture screening assays [29,47] would fail to identify drugs with a reliance on host adaptive immune responses and as such, these candidates would be suitable for in vivo evaluation in SCID mice. Discussion This would offer a desirable refinement to the male Onchocerca SCID implant model and control for testing drug exposure effects at a more naturalistic site of sub-cutaneous parasit- ism and/or emulate drug targeting of worms within a complex tissue surrounding of the onchocercoma. For this reason we undertook a pilot study whereby onchocerco- mata were implanted both into the peritoneal cavity and sub-cutaneously. It was observed that, at both sites, many of the nodules had become attached to host tissues and the majority of nodules had evidence of rapid host neovas- cularization. Vascularisation with both blood and lymph- atic vessels is a consistent feature of O. volvulus nodule microarchitecture [50-52]. Whether neovascularisation is actively induced by pro-angiogenic parasite secretions [53] or is part of an innate inflammatory response to foreign material, remains to be resolved. Analogous to solid tu- mours, that can be successfully targeted by antiangiogenic therapies, neovascularisation is possibly a pre-requisite for more protracted survival, as a source of nutrients and oxy- gen supply deep within the nodule. Whilst only evaluated for seven days, our pilot data is encouraging; 83% of the im- planted nodules contained motile female macrofilariae and 25% also yielded motile male macrofilariae ex vivo. Whilst no mf were observed from the culture of skin from the ear or tail in SCID recipients of sub-cutaneous nodules, motile mf were liberated after culturing recovered implants. This suggests that female macrofilariae are fertile and produ- cing mf post-implant in vivo. Because O. lienalis mf show a cumulative increase in recruitment to the skin during the first three weeks following inoculations in SCID mice [22] extended experiments are required to determine the dynamics of mf recruitment to the skin from implanted nodules. As lymphatic growth into nodular tissue has been proffered as an exit route for mf migration into the skin [52], this process may be dependent on lymphangiogenic responses developing within implanted nodules. These experiments are ongoing and require further optimization and refinement to test whether survival can as useful pre-clinical assessment tools to dissect the mode of action and spectrum of anti-filarial effects. By applying the efficacious FBZ regimen demonstrable to exert macrofilaricidal effects against Bm adults, we could reproduce the profound effect of this drug against im- planted adult male Onchocerca macrofilariae within SCID mice. This tallies with the reported macrofilaricidal effect of the modified FBZ, UMF078, against O. Discussion ochengi in the nat- ural cattle host [48]. Thus, our experiment demonstrates ‘proof of principle’ that the SCID mouse Onchocerca macrofilariae implant model is sufficiently robust to test macrofilaricide activity over a 5 week time frame. Because single, moribund yet motile worms could be isolated in a minority of FBZ treated mice, a slightly extended period may be warranted to determine maximum macrofilaricidal activity. Male macrofilariae were reproducibly recovered from 100% of 22 SCID mouse recipients in our experi- ments (discounting FBZ treatments), assessed between 2–5 weeks post implant (mean survival = 49.22% +/−5.08). It is probable that more protracted durations of survival are achievable and this requires further assessment. Confirming our validation experiments with Bm, the male Onchocerca macrofilariae SCID implant model was also assessed as a suitable anti-Wolbachia screen, with strong Wolbachia signal being reliably detected from indi- vidual male Onchocerca worms and >98% depletions of Wolbachia from filarial tissues recorded following ‘gold- standard’ oral RIFAP treatment. This is consistent with the rapid effects of the rifamycin class of antibiotics observable in the B. malayi infection model in SCID mice (manuscript in preparation). The matching anatomical site of Bm adult parasitism and Onchocerca macrofilariae implantation within the peritoneal cavity of the pan-filarial SCID pre-clinical screen, along with availability of both Brugia and Oncho- cerca macrofilariae ex vivo screening systems, offers a comprehensive suite of pre-clinical tools for robust in- terrogation of novel macrofilaricide and anti-Wolbachia drugs in development. This is further aided by a high throughput cell screening assay for anti-Wolbachia drug discovery [49]. The pan-filarial murine SCID host de- scribed here will therefore provide a useful tool to facili- tate the construction of rational PK/PD models and help drive iterative medicinal chemistry to further improve promising classes of drugs. Perhaps the most important advance this model provides is the ability to more accur- ately discern comparative drug class effects against differ- ent genera of medically important filariae in a controlled experimental system. Should the Bm life cycle be estab- lished in sub-Saharan African laboratories, this raises the possibility of undertaking dual Bm and Onchocerca macrofilarial implants within the same host, which would powerfully address the issue of drug effect on different filarial genera. Discussion C VC RIFAP 0.1 1 10 100 1000 wsp/gst / male Onchocerca (99.3%) * C VC RIFAP 0.1 1 10 100 1000 wsp/gst / male Onchocerca (99.3%) * B VC RIFAP VC RIFAP 102 103 104 105 106 107 108 wsp copies / male Onchocerca (99.0%) * B VC RIFAP 102 103 104 105 106 107 108 wsp copies / male Onchocerca (99.0%) * C Figure 9 Anti-Wolbachia activity against Onchocerca macrofilariae in SCID mice. (A)% recoveries, (B) Wolbachia loads per motile male Onchocerca macrofilaria measured by wsp gene copy number or (C) Wolbachia loads per motile male Onchocerca macrofilaria as a ratio wsp/gst filarial gene copy number +29 days after intraperitoneal implantation in CB.17 SCID mice and +28 days after commencement of 2 week oral 15 mg/kg rifapentine (RIFAP) or vehicle control (VC). Error bars indicate median and interquartile range. Dashed horizontal lines indicate 1 and 2 log reductions compared with median VC levels. Median % reduction is indicated in parentheses. For B&C, data from individual worms are pooled from groups of two mice. Significant differences between groups (Mann Whitney Test) are indicated P < 0.001. Figure 9 Anti-Wolbachia activity against Onchocerca macrofilariae in SCID mice. (A)% recoveries, (B) Wolbachia loads per motile male Onchocerca macrofilaria measured by wsp gene copy number or (C) Wolbachia loads per motile male Onchocerca macrofilaria as a ratio wsp/gst filarial gene copy number +29 days after intraperitoneal implantation in CB.17 SCID mice and +28 days after commencement of 2 week oral 15 mg/kg rifapentine (RIFAP) or vehicle control (VC). Error bars indicate median and interquartile range. Dashed horizontal lines indicate 1 and 2 log reductions compared with median VC levels. Median % reduction is indicated in parentheses. For B&C, data from individual worms are pooled from groups of two mice. Significant differences between groups (Mann Whitney Test) are indicated **P < 0.001. Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 12 of 14 Given that SCID mice have an impaired mechanism of foreign tissue rejection and are routinely used for grafting human tumours [30] it was hypothesized that O. ochengi onchocercomata from the dermis of infected cows could survive upon engraftment into these mice, giving rise to the possibility of developing a murine model containing adult males, females and mf and keeping the natural ana- tomical structure of the parasitic niche intact. Competing interests The authors declare that they have no financial or other competing interests. 13. Ash LR: Chronic Brugia pahangi and Brugia malayi infections in Meriones unguiculatus. J Parasitol 1973, 59(3):442–447. Received: 19 August 2014 Accepted: 2 October 2014 Received: 19 August 2014 Accepted: 2 October 2014 reduced biomass and less dense extra-cellular matrix of ochengi vs. volvulus onchocercomata may facilitate an in- crease in perfusion of host solutes, as well as our observa- tions of neovascularization, to support protracted survival of implanted female macrofilariae. Acknowledgements We thank Professor Charles MacKenzie for helpful advice regarding FBZ dosing and critical appraisal of the manuscript. This work was supported by a Bill and Melinda Gates Foundation (BMGF) funded Grand Challenges Explorations Phase I award (OPP1086755) to JDT, MJT and SW and the BMGF funded A.WOL drug development programme (OPP1045261) to LSTM. 18. Townson S, Dobinson A, Connelly C, Muller R: Chemotherapy of Onchocerca lienalis microfilariae in mice: a model for the evaluation of novel compounds for the treatment of onchocerciasis. J Helminthol 1988, 62(3):181–194. 19. Townson S, Bianco AE, Owen D: Attempts to infect small laboratory animals with the infective larvae of Onchocerca lienalis. J Helminthol 1981, 55(4):247–249. Authors’ contributions 14. Petit G, Diagne M, Marechal P, Owen D, Taylor D, Bain O: Maturation of the filaria Litomosoides sigmodontis in BALB/c mice; comparative susceptibility of nine other inbred strains. Ann Parasitol Hum Comp 1992, 67(5):144–150. AH designed the study, carried out experimental infections/implantation studies, undertook statistical analyses and assisted in drafting the manuscript. ACG assisted in experimental infections and undertook molecular assays. HT undertook experimental infections, molecular assays and histology analysis. HMM/CNW/KOJA/TDBK assisted in implantation studies. GF/AS/DC assisted in experimental infections. PE assisted in implantation studies. SW/MJT conceived and designed the study. JDT conceived and designed the study, assisted in implant studies, undertook statistical analyses and wrote the manuscript. All authors read and approved the final manuscript. 15. Awadzi K, Opoku NO, Attah SK, Lazdins-Helds J, Kuesel AC: A randomized, single-ascending-dose, ivermectin-controlled, double-blind study of moxidectin in Onchocerca volvulus infection. PLoS Negl Trop Dis 2014, 8(6):e2953. 16. Geary TG, Mackenzie CD: Progress and challenges in the discovery of macrofilaricidal drugs. Expert Rev Anti Infect Ther 2011, 9(8):681–695. 17. Taylor MJ, Van Es RP, Shay K, Folkard SG, Townson S, Bianco AE: Protective immunity against Onchocerca volvulus and O. lienalis infective larvae in mice. Trop Med Parasitol 1994, 45(1):17–23. Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Page 13 of 14 Page 13 of 14 References Awadzi K: Clinical picture and outcome of serious adverse events in the treatment of Onchocerciasis. Filaria J 2003, 2(Suppl 1):S6. 8. Bird AC, El-Sheikh H, Anderson J, Fuglsang H: Visual loss during oral diethylcarbamazine treatment for onchocerciasis. Lancet 1979, 2(8132):46. 9. Fobi G, Gardon J, Kamgno J, Aimard-Favennec L, Lafleur C, Gardon-Wendel N, Duke BO, Boussinesq M: A randomized, double-blind, controlled trial of the effects of ivermectin at normal and high doses, given annually or three-monthly, against Onchocerca volvulus: ophthalmological results. Trans Roy Soc Trop Med Hyg 2005, 99(4):279–289. 6. Gardon J, Gardon-Wendel N, Demanga N, Kamgno J, Chippaux JP, Boussinesq M: Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet 1997, 350(9070):18–22. 6. Gardon J, Gardon-Wendel N, Demanga N, Kamgno J, Chippaux JP, Boussinesq M: Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet 1997, 350(9070):18–22. 6. Gardon J, Gardon-Wendel N, Demanga N, Kamgno J, Chippaux JP, Boussinesq M: Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet 1997, 350(9070):18–22. 7. Awadzi K: Clinical picture and outcome of serious adverse events in the treatment of Onchocerciasis. Filaria J 2003, 2(Suppl 1):S6. 8. Bird AC, El-Sheikh H, Anderson J, Fuglsang H: Visual loss during oral diethylcarbamazine treatment for onchocerciasis. Lancet 1979, 2(8132):46. 9. Fobi G, Gardon J, Kamgno J, Aimard-Favennec L, Lafleur C, Gardon-Wendel N, Duke BO, Boussinesq M: A randomized, double-blind, controlled trial of the effects of ivermectin at normal and high doses, given annually or three-monthly, against Onchocerca volvulus: ophthalmological results. Trans Roy Soc Trop Med Hyg 2005, 99(4):279–289. 7. Awadzi K: Clinical picture and outcome of serious adverse events in the treatment of Onchocerciasis. Filaria J 2003, 2(Suppl 1):S6. Conclusions SCID mice can be successfully utilised to maintain all life cycle stages of the lymphatic filariae, Brugia malayi and adult stages of the cattle filaria, Onchocerca ochengi with protracted survival. A range of reference anti- filarial drugs including macrofilaricides targeting nema- tode or Wolbachia endosymbionts have been tested against Bm and O. ochengi in SCID mice. These refer- ence drugs perform with matching efficacy compared with either immune-competent controls or between the two filarial genera. Thus, we have established a ‘pan- filarial’ in vivo pre-clinical tool suitable to screen novel macrofilaricides against both lymphatic and Onchocerca filarial genera of medical importance. 10. Mackenzie CD, Geary TG: Flubendazole: a candidate macrofilaricide for lymphatic filariasis and onchocerciasis field programs. Expert Rev Anti Infect Ther 2011, 9(5):497–501. 11. Turner JD, Tendongfor N, Esum M, Johnston KL, Langley RS, Ford L, Faragher B, Specht S, Mand S, Hoerauf A, Enyong P, Wanji S, Taylor MJ: Macrofilaricidal activity after doxycycline only treatment of Onchocerca volvulus in an area of Loa loa co-endemicity: a randomized controlled trial. PLoS Negl Trop Dis 2010, 4(4):e660. 12. Wanji S, Tendongfor N, Nji T, Esum M, Che JN, Nkwescheu A, Alassa F, Kamnang G, Enyong PA, Taylor MJ, Hoerauf A, Taylor DW: Community- directed delivery of doxycycline for the treatment of onchocerciasis in areas of co-endemicity with loiasis in Cameroon. Parasit Vectors 2009, 2(1):39. References 1. Taylor MJ, Hoerauf A, Bockarie M: Lymphatic filariasis and onchocerciasis. Lancet 2010, 376(9747):1175–1185. 1. Taylor MJ, Hoerauf A, Bockarie M: Lymphatic filariasis and onchocerciasis. Lancet 2010, 376(9747):1175–1185. 1. Taylor MJ, Hoerauf A, Bockarie M: Lymphatic filariasis and onchocerciasis. Lancet 2010, 376(9747):1175–1185. The full development of B. malayi and the protracted survival of implanted O. ochengi in SCID mice raises the possibility that this research model might be exploited for drug screening against other medically important fil- ariae. Recently, it has been demonstrated that L. loa can develop to adulthood in immunodeficient mice (BALB/c IL-4Rα−/−/IL-5−/−), although patent infections producing circulating mf were not achieved [54]. Because the avail- ability of a L. loa microfilaraemic mouse model would supplement the identification of safe macrofilaricides against onchocerciasis, an ongoing area of investigation is the testing of L. loa development and survival within inbred SCID mouse lines. 2. Babalola OE: Ocular onchocerciasis: current management and future prospects. Clin Ophthalmol 2011, 5:1479–1491. 2. Babalola OE: Ocular onchocerciasis: current management and future prospects. Clin Ophthalmol 2011, 5:1479–1491. 3. Turner HC, Walker M, Churcher TS, Osei-Atweneboana MY, Biritwum NK, Hopkins A, Prichard RK, Basanez MG: Reaching the London declaration on neglected tropical diseases goals for Onchocerciasis: an economic evaluation of increasing the frequency of ivermectin treatment in Africa. Clin Infect Dis 2014, 59:923–932. 3. Turner HC, Walker M, Churcher TS, Osei-Atweneboana MY, Biritwum NK, Hopkins A, Prichard RK, Basanez MG: Reaching the London declaration on neglected tropical diseases goals for Onchocerciasis: an economic evaluation of increasing the frequency of ivermectin treatment in Africa. Clin Infect Dis 2014, 59:923–932. 4. Basanez MG, Pion SD, Boakes E, Filipe JA, Churcher TS, Boussinesq M: Effect of single-dose ivermectin on Onchocerca volvulus: a systematic review and meta-analysis. Lancet Infect Dis 2008, 8(5):310–322. 4. Basanez MG, Pion SD, Boakes E, Filipe JA, Churcher TS, Boussinesq M: Effect of single-dose ivermectin on Onchocerca volvulus: a systematic review and meta-analysis. Lancet Infect Dis 2008, 8(5):310–322. 5. Churcher TS, Pion SD, Osei-Atweneboana MY, Prichard RK, Awadzi K, Boussinesq M, Collins RC, Whitworth JA, Basanez MG: Identifying sub-optimal responses to ivermectin in the treatment of River Blindness. Proc Natl Acad Sci U S A 2009, 106(39):16716–16721. 6. Gardon J, Gardon-Wendel N, Demanga N, Kamgno J, Chippaux JP, Boussinesq M: Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet 1997, 350(9070):18–22. 7. Discussion These experiments are ongoing and require further optimization and refinement to test whether survival can be achieved for longer periods in vivo, and thus whether an onchocercoma xenograft (OX) model could also serve as a platform for the screening of novel therapeutics. Rajan et al. previously described that exposed ‘loops’ of O. volvu- lus female worms embedded in onchocercomata could remain viable and contain developing mf for up to 20 wks post-implant in SCID mice [20], demonstrating the long- term feasibility of this approach. We speculate that the Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 antifilarial therapy has macrofilaricidal activity in onchocerciasis: a randomized placebo-controlled study. Med Microbiol Immunol 2008, 197(3):295–311. 21. Nelson FK, Greiner DL, Shultz LD, Rajan TV: The immunodeficient scid mouse as a model for human lymphatic filariasis. J Exp Med 1991, 173(3):659–663. 44. Turner JD, Langley RS, Johnston KL, Egerton G, Wanji S, Taylor MJ: Wolbachia endosymbiotic bacteria of Brugia malayi mediate macrophage tolerance to TLR- and CD40-specific stimuli in a MyD88/ TLR2-dependent manner. J Immunol 2006, 177(2):1240–1249. 22. Folkard SG, Taylor MJ, Butcher GA, Bianco AE: Protective responses agains skin-dwelling microfilariae of Onchocerca lienalis in severe combined immunodeficient mice. Infect Immun 1997, 65(7):2846–2851. 23. Griffiths KG, Alworth LC, Harvey SB, Michalski ML: Using an intravenous catheter to carry out abdominal lavage in the gerbil. Lab Animal 2010, 39(5):143–148. 45. Satoguina J, Mempel M, Larbi J, Badusche M, Loliger C, Adjei O, Gachelin G, Fleischer B, Hoerauf A: Antigen-specific T regulatory-1 cells are associated with immunosuppression in a chronic helminth infection (onchocerciasis). Microbes Infect 2002, 4(13):1291–1300. 24. McGarry HF, Plant LD, Taylor MJ: Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway. Filaria J 2005, 4:4. 46. Akue JP, Devaney E: Transmission intensity affects both antigen-specific and nonspecific T-cell proliferative responses in Loa loa infection. Infect Immun 2002, 70(3):1475–1480. 25. McGarry HF, Egerton GL, Taylor MJ: Population dynamics of Wolbachia bacterial endosymbionts in Brugia malayi. Mol Biochem Parasitol 2004, 135(1):57–67. 47. 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Parasitol Today 1988, 4(8):226–228. doi:10.1186/s13071-014-0472-z Cite this article as: Halliday et al.: A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis. Author details 1 1Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK. 2Research Foundation for Tropical Diseases and the Environment, Buea, Cameroon. 3Department of Microbiology and Parasitology, Parasite and Vector Research Unit, University of Buea, Buea, Cameroon. 20. Rajan TV, Nelson FK, Cupp E, Schultz LD, Greiner DL: Survival of Onchocerca volvulus in nodules implanted in immunodeficient rodents. J Parasitol 1992, 78(1):160–163. Page 14 of 14 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Halliday et al. Parasites & Vectors 2014, 7:472 http://www.parasitesandvectors.com/content/7/1/472 Parasites & Vectors 2014 7:472. 35. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 39. Orihel TC, Eberhard ML: Loa loa: development and course of patency in experimentally-infected primates. Trop Med Parasitol 1985, 36(4):215–224. 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Debrah AY, Mand S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Buttner M, Adjei O, Buttner D, Hoerauf A: Macrofilaricidal effect of 4 weeks of treatment with doxycycline on Wuchereria bancrofti. Trop Med Int Health 2007, 12(12):1433–1441. 43. Hoerauf A, Specht S, Buttner M, Pfarr K, Mand S, Fimmers R, Marfo-Debrekyei Y, Konadu P, Debrah AY, Bandi C, Brattig N, Albers A, Larbi J, Batsa L, Taylor MJ, Adjei O, Büttner DW: Wolbachia endobacteria depletion by doxycycline as
https://openalex.org/W3031138232	https://pure.bond.edu.au/ws/files/108337381/Partnering_ethics_and_chemistry.pdf	English	null	Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements	Acta bioethica	2,020	cc-by	3,264	Bond University Research Repository Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements Bramstedt, Karina Published in: Acta Bioethica DOI: 10.4067/S1726-569X2020000100101 Licence: CC BY Link to output in Bond University research repository. Recommended citation(APA): Bramstedt, K. (2020). Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements. Acta Bioethica, 26(1), 101-106. https://doi.org/10.4067/S1726-569X2020000100101 Bond University Research Repository Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements Bramstedt, Karina Published in: Acta Bioethica DOI: 10.4067/S1726-569X2020000100101 Licence: CC BY Link to output in Bond University research repository. Recommended citation(APA): Bramstedt, K. (2020). Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements. Acta Bioethica, 26(1), 101-106. https://doi.org/10.4067/S1726-569X2020000100101 Bond University Research Repository Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements Bond University Research Repository Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements Bramstedt, Karina Bramstedt, Karina Published in: Acta Bioethica DOI: 10.4067/S1726-569X2020000100101 Licence: CC BY Link to output in Bond University research repository. Recommended citation(APA): Bramstedt, K. (2020). Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements. Acta Bioethica, 26(1), 101-106. https://doi.org/10.4067/S1726-569X2020000100101 Recommended citation(APA): Bramstedt, K. (2020). Partnering Ethics and Chemistry in Secondary and University STEM Education via an Innovatively Designed Periodic Table of Chemical Elements. Acta Bioethica, 26(1), 101-106. https://doi.org/10.4067/S1726-569X2020000100101 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. mation, or if you believe that this document breaches copyright, please contact the Bond University research repository For more information, or if you believe that this document breaches copyright, please contact the Bond University research repository coordinator. Download date: 24 Oct 2024 Download date: 24 Oct 2024 Acta Bioethica 2020; 26 (1): 101-106 Karina Bramstedt1 Abstract: As declared by the United Nations, 2019 is the International Year of the Periodic Table of Chemical Elements. Accordingly, an innovative new Periodic Table of Chemical Elements and Ethics [PETE] has been developed as an educational tool for secondary and university STEM (Science, Technology, Engineering and Mathematics) education. The tool is designed to be used in a case-based approache that is feasible to both individual and small group learners. Specifically, with the new periodic table, STEM becomes aligned with STSE (Science and Technology on Society and Environment), laying a curricular pathway for integrating discussions of ethics into the teaching of chemistry. Presented is the table, as well as sources for pairable ethics cases, and guidance for teachers to create curriculum which facilitates connections between ethics, chemistry, and society. Keywords chemistry; education; ethics; science/education; educational technology Ética y química asociadas en la educación secundaria y universitaria STEM, a través de una tabla periódica de elementos químicos de diseño innovador Resumen: Según lo declarado por las Naciones Unidas, 2019 es el Año Internacional de la Tabla Periódica de Elementos Químicos. En consecuencia, se ha desarrollado una nueva tabla periódica innovadora de elementos químicos y ética [PETE] como una herramienta educativa para la educación secundaria y universitaria STEM (Ciencia, Tecnología, Ingeniería y Matemáticas). La herramienta está diseñada para usarse en un enfoque basado en casos que sea factible tanto para estudiantes individuales como para grupos pequeños. Específicamente, con la nueva tabla periódica, STEM se alinea con STSE (Ciencia y Tecnología en Sociedad y Medio Ambiente), estableciendo una vía curricular para integrar las discusiones de ética en la enseñanza de la química. Se presenta la tabla, así como las fuentes de casos éticos deseables y orientación para que los maestros creen un plan de estudios que facilite las conexiones entre la ética, la química y la sociedad. Palabras clave: química; educación; ética; enseñanza de las ciencias; tecnologia Educacional 1 Luxembourg Agency for Research Integrity (LARI) 6, Luxembourg. Bond University Faculty of Medicine and Health Sciences, Queens land, Australia Correspondence: txbioethics@yahoo.com PARTNERING ETHICS AND CHEMISTRY IN SECONDARY AND UNIVERSITY STEM EDUCATION VIA AN INNOVATIVELY DESIGNED PERIODIC TABLE OF CHEMICAL ELEMENTS Karina Bramstedt1 Introduction of Chemical Elements termed the Periodic Table of Chemical Elements and Ethics [PETE.] PETE retains the standard features of the Periodic Table of Chemical Elements(4) (i.e., atomic number, el ement symbol, element name, atomic mass) while pairing one word relating to ethics or research eth ics along with the chemical element. The word pairing aligns to the letters of the element symbol (118 unique pairs). In this way, the geometric lay out and scientific content of the Periodic Table of Chemical Elements are retained and visually in tact, and the ethics content is added. In 2016, Dr Astrid Steele argued in The Journal of Science Teacher Education that there was the “need for a moral component for science educa tion”(1:357). She further stated that an ethical framework would “inform decisions and direc tions of [STEM] teachers, and teacher educa tors”(1:359). Concurring, an innovative new de sign of the Periodic Table of Chemical Elements has been created which facilitates both ethics and chemistry education in secondary schools as well as universities (Figure 1). Specifically, with the new periodic table, STEM content aligns with STSE (Science and Technology on Society and Environ ment) content, facilitating a curricular pathway to integrate discussions of ethics into the teaching of chemistry. 2019 was declared the International Year of the Periodic Table of Chemical Elements by the United Nations(2), thus the timely creation of this new table for STEM is fitting [See next page]. Some of the word-element pairs have an imme diate ethical connection. For example, potassium (element 19, symbol K) is paired with the ethics word “kind”. In this context, students could ex plore the case of three authors who unkindly pla giarized the work of others who had also researched potassium(5). With regard to element 98 (Califor nium, symbol Cf), this is paired with the ethics word “confidentiality” and students could explore the National Aeronautics and Space Administra tion’s Aviation Safety Reporting System(6) in the context of Californium’s use in the detection of aircraft metal fatigue. Copper (element 29, sym bol Cu) is paired with the ethics word “culture” and this can be explored in the context of mining harms to indigenous peoples(7). For the word-el ement pairings that do not [currently] have direct ethical connections, analysis of the pairings in the context of element groups and historical cases is proposed. The Evolution of the Periodic Table of Chemi cal Elements What is known today as the Periodic Table of Chemical Elements had its origination in the mid-1800s by several scientists, including chemist Dmitri Ivanovich Mendeleev and physician Julius Lothar Meyer. Even the contributions and ulti mate authorship credit of the table is an ethical is sue worthy of debate(3). Due to the table’s unique ability to be a visual teaching tool, many variations have been created. These variations are notably different from the original in that they do not aim to also teach chemistry concurrently, but rather the Periodic Table of Chemical Elements becomes only a geometric template with the chemistry con tent removed. Such examples include the Periodic Table of the Presidents (https://periodicpresidents. com/product/ptotp/), Periodic Table of Figures of Speech (https://visual.ly/community/infographic/ education/periodic-table-figures-speech), and the Pe riodic Table of Musical Instruments (https://www. kickstarter.com/projects/1913274728/periodic-table- of-musical-instruments). Teaching chemistry involves exploring the chemi cal elements according to their group (vertical column of elements that generally have similar physical and chemical properties), period (hori zontal row of elements that have the same num ber of atomic orbitals), and blocks (table segments relating to electrons). Table 1 collates the ethics words according to element group to facilitate a potential curricular package that can further pair with a chemistry-related ethics case(8) that ei ther the teacher presents for analysis, or that the students search for using the Internet as part of an individual or small group project. Reflective writing could also be added in addition to case discussions(9). Online sources for case studies are presented in Table 2. Associando ética e química na educação stem secundária e universitária via uma tabela periódica de element químicos desenvolvida de forma innovadora Resumo: Como declarado pelas Nações Unidas, 2019 é o Ano Internacional da Tabela Periódica de Elementos Químicos. Portanto, uma inovadora e nova Tabela Periódica de Elementos Químicos e Ética (PETE, sigla em inglês) foi desenvolvida como uma ferramenta educacional para educação STEM (sigla em inglês para Ciência, Tecnologia, Engenharia e Matemática) secundária e universitária. A ferramenta é elaborada para ser usada em uma abordagem baseada em casos, viável tanto para aprendizagem individual como de pequenos grupos. Especificamente, com a nova tabela periódica, STEM se alinha com STSE (sigla em inglês para Ciência e Tecnologia na Sociedade e Ambiente), estabelecendo uma via curricular para integrar discussões de ética no ensino de química. Apresenta-se a tabela, bem como fontes de casos éticos pareados, e orientação para professores criarem um currículo que facilite conexões entre ética, química e sociedade. Palavras chave: química, educação, ética, ciência/educação, tecnologia educacional 101 Innovatively Designed Periodic Table of Chemical Elements - Karina Bramstedt Meet PETE Presented here is a newly designed Periodic Table 102 Acta Bioethica 2020; 26 (1): 101-106 Acta Bioethica 2020; 103 Innovatively Designed Periodic Table of Chemical Elements - Karina Bramstedt Table 1. Element Groups and the Ethics Terms/ Atomic Number Group # Ethics Terms 1 Hypothesis/1; Listen/3; Nature/11; Kind/19; Robust/37; Codes/55; Fair/87 2 Benefit/4; Mitigate/12; Careful/20; Scru ples/38; Balance/56; Rapport/88 3 Secure/21; Yielding/39; Lawful/57; Cere bral/58; Proper/59; Need/60; Permis sion/61; Smile/62; Equipoise/63; Good/64; Timber/65; Duty/66; Honest/67; Earnest/68; Teamwork/69; Verifiability/70; Luminant/71; Accurate/89; Thank/90; Pauseful/91; Util ity/92; Non-Partisan/93; Purpose/94; Ami able/95; Circumspect/96; Benchmark/97; Confidentiality/98; Ethos/99; Fathom/100; Methodical/101; Novel/102; Liberty/103 4 Timely/22; Zoiatrics/40; Helpful/72; Refer ence/104 5 Veracity/23; Noble/41; Tactful/73; De brief/105 6 Credible/24; Moral/42; Worthy/74; Sport ing/106 7 Manners/25; Toxicity/43; Reliable/75; Bioeth ics/107 8 Feedback/26; Rule/44; Onus/76; Humans/108 9 Collegial/27; Rights/45; Irenic/77; Men tored/109 10 Nice/28; Prudent/26; Patience/78; Dis close/110 11 Culture/29; Agile/47; Authorized/79; Regu lated/111 12 (Grey) Zone/30; Credit/48; High Road/80; Consent/112 13 Bona Fide/5; Altruism/13; Gatekeeper/31; In sight/49; Truthful/81; Neighborly/113 14 Courage/6; Sincere/14; Genuine/32; Sound/50; Publish/82; Fly Right/114 15 Norm/7; Professional/15; Ask/33; Stable/51; Biosafety/83; Meticulous/115 16 Objective/8; Sharing/16; Search/34; Temper ate/52; Polite/84; Level-headed/116 17 Futility/9; Clear/17; Belmont Report/35; In clusion/53; Attentive/85; Trust/117 18 Humane/2; Neutral/10; Archive/18; Kin dred/36; Xenial/54; Randomization/86; Ob ligation/118 Table 2. Online Sources for Chemistry-Ethics Case Studies Source Location News stories Google search (e.g., search terms chemist/chemistry, ethics/miscon duct, lab/laboratory, poison) Full journal articles dis cussing chemistry-ethics cases http://www.hyle.org/journal/issues/ special/ethical-cases.html Fictional chemistry- ethics cases created by the American Chemical Society https://www.acs.org/content/acs/ en/about/governance/committees/ ethics/ethics-case-studies.html Fictional cases in e- book, chapter 7, The Ethical Chemist https://global.oup.com/academic/ product/the-ethical-chemist- 9780190668648?cc=lu&lang=en& Catalogue of chemistry- related retracted journal articles https://retractionwatch.com/cat egory/by-subject/physical-sciences- retractions/chemistry-retractions/ Misconduct cases from the US Office of Re search Integrity https://ori.hhs.gov/content/case- summary-endo-matthew https://ori.hhs.gov/content/case- summary-baughman-brandi-m https://ori.hhs.gov/content/case- summary-ramadugu-venkata-sud heer-kumar Online chemistry maga zine with news content which includes ethics cases https://www.chemistryworld.com/ Table 1. forming their attitudes, values, and behaviors. forming their attitudes, values, and behaviors. forming their attitudes, values, and behaviors. Examples for connecting elements to cases include the following: Strontium (element 39, group 2) could be discussed along with concepts in eth ics and nuclear chemistry, specifically, balancing benefits and risks in various settings (medicine, war) and the careful use of mitigation to minimize harm(10). Carbon (element 6, group 14) could be discussed in the context of the ethics of the Pheramor dating app (https://www.pheramor.com/), specifically exploring how the concepts of sincer ity, genuineness, soundness and evidenced-based science thread among marketing of “health” and social apps. Silver (element 47, group 11) could be discussed in the context of the ethical complex ity of silver nanoparticles, raising concerns about regulations and authorized use (medical use in hospitals vs personal use in socks)(11). Manga nese (element 25, group 7) could be discussed in the context of the improperly formulated metal storage vats of the 1919 catastrophic molasses flood, along with the concepts of reliability and professionalism (a synonym for manners)(12). The level of complexity of cases and concepts should be steered by the teacher during curriculum devel opment so as to match the learning environment (secondary vs higher education). Indeed, the many examples of ethics cases illus trate there are frequent possibilities for ethical di lemmas in the field of chemistry. Memorization of element symbols, atomic numbers and atomic mass, are devoid of the innate contextual connec tion of chemistry to society, and the ethical mat ters therein. The use of the PETE in chemistry education is a potential tool for luminating the connection and facilitating discussion and de construction. When students make links to real world cases, this can help chemistry seem real and alive, and there is support for this ethics teaching method among chemistry educators(19). For the students who become scientists, “[they] have an obligation to further the conversation about the implications of their work, because they possess more information about the advances that create these ethical questions”(20:59). A potential limitation of this work is that the eth ics words chosen for the PETE table are gener ally positive or neutral, rather than negative. For example, element 87 (francium, Fr) was assigned the ethics word “FAIR” rather than “FRAUD”. Similarly, element 27 (cobalt, Co) was assigned “COLLEGIAL” rather than “CONFLICT OF INTEREST”. Why Bring Ethics to Chemistry? The European Chemical Society established the Working Party on Ethics in Chemistry(13) with objectives that include increasing the awareness of the moral complexity of chemistry activities(14). More specifically, they view the ethical issues as crossing several domains, including good scientific practice, publishing, chemical safety (inside and outside the laboratory), chemical synthesis, educa tion, mentoring, and sustainability(14). Other is sues in chemistry include professionalism(15,16) and dual use technologies(11). The ALL European Academies Permanent Work ing Group on Science and Ethics argued in their statement on Ethics Education in Science(17), that ethics education should include the ethical aspects of how society interconnects with science. Edu cation about the ethical connections in chemistry has also been promoted by others(18). Yet while the Working Group’s focus is the university set ting, it seems appropriate to begin these discus sions prior to university, where early learners are forming their attitudes, values, and behaviors. The PETE table is intentionally created with a positive tone to show the benefi cent aspects of ethics; however, through case dis cussions, the negative aspects of ethical dilemmas will also emerge. For example, when discussing element 82 (lead, Pb, group 14, ethics word “pub lish”) chemistry teachers can include the concept of plagiarism. This notably links to other ethics words in group 14, such as “COURAGE” and “GENUINE”. Another potential limitation of this work is that it has not been formally studied as a curriculum tool. It is hoped that the publica tion of PETE in open-access form will encourage teachers to use it and assess its value with empiri cal research methods. Why Bring Ethics to Chemistry? Meet PETE Element Groups and the Ethics Terms/ Atomic Number Group # Ethics Terms 1 Hypothesis/1; Listen/3; Nature/11; Kind/19; Robust/37; Codes/55; Fair/87 2 Benefit/4; Mitigate/12; Careful/20; Scru ples/38; Balance/56; Rapport/88 3 Secure/21; Yielding/39; Lawful/57; Cere bral/58; Proper/59; Need/60; Permis sion/61; Smile/62; Equipoise/63; Good/64; Timber/65; Duty/66; Honest/67; Earnest/68; Teamwork/69; Verifiability/70; Luminant/71; Accurate/89; Thank/90; Pauseful/91; Util ity/92; Non-Partisan/93; Purpose/94; Ami able/95; Circumspect/96; Benchmark/97; Confidentiality/98; Ethos/99; Fathom/100; Methodical/101; Novel/102; Liberty/103 4 Timely/22; Zoiatrics/40; Helpful/72; Refer ence/104 5 Veracity/23; Noble/41; Tactful/73; De brief/105 6 Credible/24; Moral/42; Worthy/74; Sport ing/106 7 Manners/25; Toxicity/43; Reliable/75; Bioeth ics/107 8 Feedback/26; Rule/44; Onus/76; Humans/108 9 Collegial/27; Rights/45; Irenic/77; Men tored/109 10 Nice/28; Prudent/26; Patience/78; Dis close/110 11 Culture/29; Agile/47; Authorized/79; Regu lated/111 12 (Grey) Zone/30; Credit/48; High Road/80; Consent/112 13 Bona Fide/5; Altruism/13; Gatekeeper/31; In sight/49; Truthful/81; Neighborly/113 14 Courage/6; Sincere/14; Genuine/32; Sound/50; Publish/82; Fly Right/114 15 Norm/7; Professional/15; Ask/33; Stable/51; Biosafety/83; Meticulous/115 16 Objective/8; Sharing/16; Search/34; Temper ate/52; Polite/84; Level-headed/116 17 Futility/9; Clear/17; Belmont Report/35; In clusion/53; Attentive/85; Trust/117 18 Humane/2; Neutral/10; Archive/18; Kin dred/36; Xenial/54; Randomization/86; Ob ligation/118 4 Timely/22; Zoiatrics/40; Helpful/72; Refer ence/104 104 Acta Bioethica 2020; 26 (1): 101-106 References 1. Steele A. Troubling STEM: Making a case for an ethics/STEM partnership. Journal of Science Teacher Education 2016; 27(4): 357-371. 2. United Nations Educational, Scientific and Cultural Organization. 2019 IYPT International Year of the Periodic Table of Chemical Elements Opening Ceremony. Paris, France. January 2019. Available from: https://en.unesco.org/sites/ default/files/iypt2019-programme-opening_en.pdf Accessed 10 April 2019. i g g 3. Kaji M. Social background of the discovery and the reception of the periodic law of the elements: recognizing the con tributions of Dmitri Ivanovich Mendeleev and Julius Lothar Meyer. Annals of the New York Academy of Sciences 2003; 988: 302-306. 4. IUPAC \| International Union of Pure and Applied Chemistry. IUPAC Periodic Table of the Elements; 2018. Available from: https://iupac.org/what-we-do/periodic-table-of-elements/#a9 Accessed 10 April 2019. 5. Yan X, Lv S, Guo R. Retraction: A New Indicator for Potassium Ions at Physiological pH by Using a Macrocyc nescent Metal Complex. Chemistry: A European Journal 2013; 19(19): 5783. p y p 6. National Aeronautics and Space Administration. Aviation Safety Reporting System: Confidentiality and Incentives to Report. Available from: https://asrs.arc.nasa.gov/overview/confidentiality.html Access 1 June 2019. 7. Woollard J. Overcoming the challenges of legacy mines in the Northern Territory. AUSIMM Bulletin 2014; 3: 55-56. 8. Fisher ER, Levinger NE. A directed framework for integrating ethics into chemistry curricula and programs using real and fictional case studies. Journal of Chemical Education 2008; 85(6): 796-801. 9. Coppola BP. Targeting entry points for ethics in chemistry teaching and learning. Journal of Chemical Education 2000; 77(11): 1506-1511. 10. Mast L. Meet Katsuko Saruhashi, a resilient geochemist who detected nuclear fallout in the Pacific; 2019. Availa https://massivesci.com/articles/katsuko-saruhashi-geochemistry-seawater-japan/ Accessed 10 April 20 11. Calderón-Jiménez B, Johnson ME, Montoro Bustos AR, et al. Silver nanoparticles: Technological advances, so cietal impacts, and metrological challenges. Frontiers in Chemistry 2017; 5: 6 Published 2017 Feb 21. doi:10.3389/ fchem.2017.00006. 12. Rossow MP. The Molasses Flood of 1919 and Other Ethical Failures in Engineering; 2015. Available from: https://pdhon line.com/courses/r151/Molasses%20flood%20Study%20Guide.pdf Accessed 10 April 2019. 13. European Chemical Society. Statutes; 2016. Available from: https://www.euchems.eu/wp-content/uploads/2017/10/ Statutes-WP-on-Ethics.pdf Accessed 10 April 2019. 14. Mehlich J, Moser F, Van Tiggelen B, et al. The ethical and social dimensions of chemistry: reflections, considera clarifications. Chemistry: A European Journal 2017; 23: 1210-1218. 15. Kovac J. Ethics in science: The unique consequences of chemistry. Accountability in Research 2015; 22(6): 312-329. 16. Tragger R. Chemistry graduate student gets seven years for poisoning co-worker. Chemistry World, 13 December 2018. Acknowledgements The author thanks retired science teacher Kathleen Schenk (Evansville, IN USA) for the open-access Excel template (http://www.sciencegeek.net/tables/ tables.shtml) that was used to build the Periodic Table of Chemical Elements and Ethics. 105 Innovatively Designed Periodic Table of Chemical Elements - Karina Bramstedt References Available from: https://www.chemistryworld.com/news/chemistry-graduate-student-gets-seven-years-for-poi soning-co-worker/3009898.article Accessed 10 April 2019. 17. ALL European Academies Permanent Working Group on Science and Ethics. Ethics Education in Science; 2013. Available from: https://www.allea.org/wp-content/uploads/2015/07/Statement_Ethics_Edu_web_final_2013_10_10. pdf Accessed 10 April 2019. 18. Köster V, Mehlich J. Bringing ethics in chemistry to universities. ChemViews 2018, 4 September. DOI: chemv.201800076 19. Kovac J. Scientific ethics in chemical education. Journal of Chemical Education 1996; 73(10): 926-928. 20. Wright MB. The role of ethics in stem education: A course proposal (Honor’s thesis, Baylor University, Houston, TX, USA); 2017. Available from: https://baylor-ir.tdl.org/handle/2104/9974 Accessed 10 April 2019. Received: July 5, 2019 Accepted: October 18, 2019 Received: July 5, 2019 Accepted: October 18, 2019 Received: July 5, 2019 Accepted: October 18, 2019 Accepted: October 18, 2019 106
https://openalex.org/W2167734792	https://www.cifor.org/publications/pdf_files/Articles/ACapistrano0601.pdf	English	null	Forest governance and institutional structure: an ignored dimension of community based forest management in the Philippines	International forestry review	2,006	cc-by	11,879	Gestion des forêts et structure institutionelle: une dimension peu connue de la gestion forestière communautaire aux Philippines Gestion des forêts et structure institutionelle: une dimension peu connue de la gestion forestière communautaire aux Philippines G. R. DAHAL et D. CAPISTRANO Cet article identifie les faiblesses stratégiques du processus de politique de dévolution dans la gestion forestière, et il analyse leurs raisons sous-jacentes. Il établit ensuite la relation des résultats de la politique de dévolution avec la gestion et les structures institutionelles. La recherche sur le terrain a été entreprise dans les Philippines, en considérant six cas de sites de gestion forestière communautaire ( CBFM) dans la province de Nueva Vzcaya et Quirino, et en employant une technique qualitative pour recueillir les données et leur interprétation. L’étude démontre que le processus de politique de dévolution a deux faiblesses stratégiques interdépendantes majeures: l’une est une articulation inadéquate de la politique, et l’autre, la réalité complexe de la mise en pratique.En se basant sur l’analyse des faiblesses stratégiques dans le processus de la politique de dévolution dans les Philippines, cet article démontre que le degré de succès des résultats des politiques dépend de l’interdépendance entre les niveaux de dévolution et une articulation claire des lignes de conduite d’une part; et la qualité de la gestion et des structures institutionelles d’autre part. Gestión forestal y estructuras institucionales: una dimensión olvidada del manejo forestal comunitario en Filipinas Forest governance and institutional structure: an ignored dimension of community based forest management in the Philippines G. R. DAHAL and D. CAPISTRANO Centre for International Forest Research, Forests and Governance Programme, Bogor Barat 16680, Indonesia Email: g.dahal@cgiar.org Email: g.dahal@cgiar.org SUMMARY This paper identifies strategic weaknesses in the devolution policy process in forest management and analyses the reasons behind them. Further, it establishes the relationship of devolution policy outcomes with governance and institutional structures. The field research was undertaken in the Philippines, taking six cases of community based forest management (CBFM) sites in the province of Nueva Vizcaya and Quirino and employing a qualitative technique for data collection and interpretation. The study demonstrates that the devolution policy process has two major interrelated strategic weaknesses: one is inadequate policy articulation and the other is a set of differences between policy and the complex reality of implementation. Drawing upon this analysis of strategic weaknesses in the devolution policy process in the Philippines the paper argues that the level of success of policy outcomes is dependent on the interrelation between the levels of devolution with clear policy articulation on the one hand and quality of governance and institutional structures on the other. Keywords: Philippines, forest policy, governance, institutions, community 377 International Forestry Review Vol.8(4), 2006 377 International Forestry Review Vol.8(4), 2006 PAPERS INTRODUCTION Most developing countries have initiated decentralisation reforms over the past two decades. In decentralisation, central governments transfer some of their fiscal, political and administrative responsibilities to lower-level government units, local institutions, corporate agencies, community groups and the private sectors (World Bank 1997, Ribot 2004). The primary rationale for decentralisation is to overcome the failure of central government in addressing people’s needs and priorities. Other driving forces behind decentralisation are: to achieve allocative efficiency in resource distribution in the context of different local preferences and priorities, to maintain equity and social justice, to increase the competitiveness of government in providing services to their citizens, to neutralise the possible pressure of regional autonomy and provide better and more stable governance (Rondinelli 1981, Cheema and Rondinelli 1983, Smith 1985, Burns et al. 1994, Mayers and Bass 1999). a series of factors such as: the need to overcome increasing forest degradation due to a history of government failure in protection and promotion of the forest, the need to reduce the cost of central bureaucracies, the desire to comply with the concept of economic liberalisation and market orientation, the desire to increase access and control of local community and to ensure equity (Shepherd 1992, Hobley 1996, Fisher 2000, Edmunds and Wollenberg 2001, Colfer and Capistrano 2005). For the last two decades, conservation and natural resource management approaches have been shifting from costly state control systems to ones in which local people are actively involved in the process. The new system includes the participation of resource users in management decisions and the sharing of benefits through restructuring power relations between the state and communities by transferring management authority to the local level (Chambers 1995, 1997; Shackleton et al. 2002). The approach of decentralisation is also linked with neoliberal ideas, which consider that the state is not the only provider of public goods and services, but the private and civil society domain of development could be equally efficient or even better in providing such services (Turner and Hulme 1997, Manor 1999). These objectives are further linked with establishment of good governance, UNDP (1997) views decentralisation as the key to good governance and a logical application of the major characteristics of good governance. Despite the aforementioned rationales of decentralisation and devolution in forestry, there is a quantity of literature that views the success of forest devolution as limited in practice (Anderson 2000, Enters et al. INTRODUCTION 2000, Fisher 2000, Edmunds and Wollenberg 2002). The effort towards devolution so far is insufficient, as a result, a number of setbacks have been encountered in the implementation process (Shepherd 1992, and Anderson 2000). This indicates that there is still a lack of meaningful devolution in practice. Edmunds and Wollenberg (2001:190) stated that ‘regarding devolution in forestry from the perspective of the poorest farmers, who solely depend on nearby forest for their livelihood through collection of fodder, fuel wood, woodcarving, charcoal making, livestock raising the results are almost disappointing’. In most cases, the livelihood of the poor forest dependents is challenged due to the influence of elites in decision-making about forest management (Shepherd 1992). These all indicate that there are some weaknesses in the devolution process in forestry and as a result the achievement so far is limited or even negative in many cases (Gilmour and Fisher 1997, Roy and Tisdell 1998, Agrawal and Ostrom 2001, Djeumo and Fomete 2001). In the forestry sector, the decentralisation and devolution are dominant themes of policy discussion in most developing countries. Many countries in Asia and Africa have recently developed legislation and policies to address the core value of decentralisation and devolution. Some of them are considered progressive in terms of devolving authority and power from central government to local government or community institutions. However, in most cases the genuine transfer of power and authority is not happening as stipulated in the policy and legislation (Fisher 1999, Ostrom 1999, Fisher 2000, Enters and Anderson 2000, Ribot 2002 and 2004). Against this backdrop, this research analyses the strategic weaknesses in the devolution policy process in forest management and assesses the reasons for such weaknesses. It also explores the possible links between those reasons and the issues of governance and institutional structure in collective action. For this research, the case of community based forest management (CBFM) in the Philippines is taken as a manifestation of devolution policy in forestry. alta calidad de gobierno y de estructuras institucionales. alta calidad de gobierno y de estructuras institucionales. Gestión forestal y estructuras institucionales: una dimensión olvidada del manejo forestal comunitario en Filipinas G. R. DAHAL y D. CAPISTRANO Este artículo identifica los problemas estratégicos del proceso político de delegación de poderes en el manejo forestal, y analiza su origen, además de establecer la relación entre los resultados de la política de transferencia de poderes, y la administración y las estructuras institucionales. La investigación sobre el terreno se llevó a cabo en Filipinas, basada en seis casos de manejo forestal comunitario en las provincias de Nueva Vizcaya y Quirino, y empleó una técnica cualitativa para la recolección e interpretación de datos. El estudio demuestra que el proceso de delegación de poderes tiene dos debilidades estratégicas importantes, interrelacionadas entre sí: en primer lugar, la coordinación política insuficiente, y en segundo lugar, las diferencias entre la política misma y la realidad compleja de su implementación. Basándose en este análisis de las debilidades estratégicas del proceso de delegación de poderes en Filipinas, el estudio sugiere que el grado de éxito de las políticas depende de la interrelación entre los niveles de delegación, junto con una coordinación política transparente y una G.R. Dahal and D. Capistrano 378 Post colonial period (1947 to date) or local government) and civil society (community based institutions) at the local level. Both hinder the process of devolving power, functions, and resources on the one hand and contribute to deviation of policy implementation from its original thrust on the other hand. The framework also indicates that devolution policy is a form of broader governance process (as a manifestation of the relationship between state and civil society) operating within a broader governance framework of state, civil society and market. However, this study concentrates only on the governance and institutional part within the devolution policy process, basically focusing on quality or process of governance. The framework also shows that governance and institutional structure are interdependent and mutually reinforcing. Even after the end of colonial period, the government continued to attack upland dwellers against their practice of ‘slash and burn’ agriculture inside the forest. A new thinking emerged when a FAO research project gave a report in 1957 stating that Kaingin could be continued through the application of a fallow system, which would not pose a threat to the forest ecology, but contribute to the livelihoods of the communities. Further to this report, an additional recommendation was made through a national conference on Kaingin in 1965, where the social dimension of Kaingin was highlighted (Magno 2001). In 1972, President Ferdinand Marcos declared Martial Law in the Philippines. During this period the president allowed his cronies and relatives to operate concessions to harvest the trees under Timber License Agreements (TLAs) in areas of over 100,000 hectares (Porter and Ganapin 1988, Vitug 2000). During the Marcos era the Philippine forest had one of the worst deforestation rates (as in Table 1) in the Asia Pacific region, losing on average 316,000 hectares of forest a year from 1980-1990 (ADB 1992 1994, Bagadian 1993, MPFS 2001). GENERAL CONTEXT OF DECENTRALISATION/ DEVOLUTION POLICY IN FORESTRY In the case of forestry, devolution is commonly understood as the transfer of the role of the central state in managing forest under specified terms and conditions to local government units or communities. Devolution policy in forest management has been gaining popularity in most developing countries since 1990 as a strategy to achieve the goal of sustainable forest management and biodiversity conservation (Toha and Barros 1997). The motivation for devolution in forestry is driven by Based on the above discussion a hypothesis was developed (Figure 1) against which the results of the study are assessed. It states that there are weaknesses at two levels in devolution policy process. First lies between central government and local government. Second lies between the state (local administration Forest governance and institutional structure 379 FIGURE 1 Framework of the research FIGURE 1 Framework of the research FIGURE 1 Framework of the research D E V O L U T I O N P O L I C Y D E V O L U T I O N O U T C O M E Governance Institutional Structure Devolution policy process Central government Power Functions Resources Local administration Local government Power Functions Resources Community institutions Partnership Negotiation Implementation W E A K N E S S E S W E A K N E S S E S State Civil society Market Colonial period (1521-1947) FIGURE 1 Framework of the research D E V O L U T I O N P O L I C Y D E V O L U T I O N O U T C O M E Governance Institutional Structure Devolution policy process Central government Power Functions Resources Local administration Local government Power Functions Resources Community institutions Partnership Negotiation Implementation W E A K N E S S E S W E A K N E S S E S State Civil society Market The colonisers viewed the Philippines forest as state property, so the government always attempted to maintain control over the forest against the indigenous/tribal upland dwellers living inside the forest. For this reason, the government formulated a number of Laws and Acts to evict the indigenous people from the forest. In 1863, the “Inspeccion General de Montes” (IGM) was established as a first forest agency to control and manage the forest in the Philippines (Magno 2001, Poffenberger and McGean 1993). In order to prohibit the slash and burn practice of agriculture (Kaingin) inside the forest, particularly in upland areas, the colonial government formulated a Definite Forest Laws and Regulation (Royal Decree of the King of Spain) in 1889. Later in 1901, a much stronger law was drawn up to control Kaingin, which was termed as the Kaingin Law (Act No. 274). Market DECENTRALISATION/DEVOLUTION OF FOREST MANAGEMENT IN THE PHILIPPINES Devolution policy for forestry in the Philippines has a long history, however, more specific attempts to implement forest devolution have been made over the last decade. Until the early 1980s the Philippines’ forest policies were characterised as highly regulatory, centrally controlled, and industry oriented (Brillantes 2000, Contreras 2000, Pulhin 2003). Forest policy change in the Philippines falls into three distinctive periods as shown below. Year Forest Cover (in millions of hectares) Percent of total area 1575 27.5 92 1863 20.9 70 1920 18.9 64 1934 17.8 57.3 1970 10.9 36.3 1980 7.4 24.7 1990 6.7 20.7 2001 5.4 18 TABLE 1 Forest cover decline in the Philippines Source: RMPFD 2003 TABLE 1 Forest cover decline in the Philippines Pre-colonial period (before 1521) This is the period before the arrival of Spanish colonisers in 1521. According to Sanvictores (1997), forest management then was based on communal cooperation among tribal members, who were residing inside or around the forestland. Forest was taken as a sacred creation of God. So, people worshipped the forest as a part of their livelihood gift. Over 92 percent of the total land was under forest cover with naturally grown dipterocarps, narra, mahogany, mangrove swamps, pine and mossy forest. Source: RMPFD 2003 380 G.R. Dahal and D. Capistrano To respond to the destructive outcome of the uncontrolled timber harvesting by TLA Concessions and huge conversion of forestland to agriculture, which caused rapid deforestation, a new approach was introduced to rehabilitate areas through reforestation. Some of the related initiatives include the formulation of policies on Forest Ecosystem Management in 1976, Communal Tree Farming in 1979 and the Family Approach to Reforestation in 1981. In 1982, the Integrated Social Forestry (ISF) policy was introduced to include the upland dwellers as partners in resource development and conservation. The emphasis was put on the issuance of secured long-term tenure stewardship contracts for 25 years (Poffenberger and McGean 1993, Magno 2001). from the Department of Environment and Natural Resources (DENR) to the Local Government Units (DENR 1992). Second, the enactment of Executive Order 263 in 1995 by the then President Fidel Ramos declared CBFM as a national strategy to achieve sustainable forest management and social justice. Under this the DENR transferred their role and authority over forest protection to the organised local communities, under community based forest management agreements (CBFMA) for 25 years and conditionally renewable for another 25 years. Since then there have been more than 1500 CBFM agreements made in the Philippines to manage 1.5 million hectares of forestland (Table 2). The third is the Indigenous People’s Rights Act (IPRA) of 1997. This Act mandated a National Commission for Indigenous People (NCIP) to hand over specified ancestral The Marcos regime was overthrown after a historical people’s power movement commonly known as ‘EDSA’1 The Marcos regime was overthrown after a historical people’s power movement commonly known as ‘EDSA’1 TABLE 2 Status of CBFM agreements in the Philippines TABLE 2 Status of CBFM agreements in the Philippines Source: DENR 2004 Regions No. of CBFM agreement sites Tenured area (Hectares) No of households No. 1 Epifanio De Los Santos Avenue 2 POs- People’s Organisation, who undertake a CBFM agreement with the DENR 3 ARMM- Autonomous Region of Muslim Mindanao 4 CAR- Cordillera Administrative Region Pre-colonial period (before 1521) of PO2 ARMM3 10 22,861 2,365 10 CAR4 67 48,045 11,909 67 01 126 40,080 14,205 126 02 93 272,509 92,099 93 03 120 78,066 11,544 120 04-A 31 16,914 2,944 31 04-B 79 96,602 10,122 79 05 52 41,703 10,542 52 06 104 42,656 16,978 104 07 133 45,476 11,901 133 08 111 107,557 12,693 111 09 121 66,298 12,030 121 10 295 214,209 30,376 295 11 92 195,396 25,895 92 12 48 88,645 10,197 48 13 95 197,788 28,150 95 Total 1,577 1,574,812 303,950 1,577 Source: DENR 2004 domain to the indigenous cultural communities to manage, protect and use the resources in their domain forever. domain to the indigenous cultural communities to manage, protect and use the resources in their domain forever. movement in 1986. The changed political circumstances slightly helped to strengthen the devolution process in forestry as government started to formulate new policies and guidelines. In this endeavour, the following three main policy reforms were initiated in the Philippines. In theory, the preamble to all these milestone policies stated that local institutions would be given full authority and power to make independent decisions at the local level in terms of management of forests, the policies would ensure that the tenure and use rights of local communities were secured. However, in practice, a considerable literature First is the Local Government Code (also known as Republican Act, RA 7160), which transferred certain roles of previously operating integrated social forestry (ISF) sites Forest governance and institutional structure 381 RESEARCH SITES, THEIR CONTEXT AND METHODS indicates that the authority, power and resources are not devolved, benefits to the real users of the forest have been limited (Guiang 1996, Bagadion 2000, Uting 2000, Fisher 2000, Borlagdan et al. 2001, Contreras 2001, Edmunds and Wollenberg 2001, Pulhin 2003). In this research, the devolution policy process related to community based forest management is taken as the major field of analysis. In order to select the research sites a set of criteria was established which includes: selection of sites covering all types of intervention (state, foreign donor, or community themselves), sites operating under Local Government Code (LGC) 1991, Executive Order (EO) 263 and IPRA Law 1997, and sites having experience with the commercial harvesting of timber. Under these criteria, the following sites were deliberately selected in the province of Nueva Vizcaya and Quirino (Figure 2). Against this backdrop this study aims to analyse empirically the important issues pertaining to the devolution policy process in reference to forest management in the Philippines. This study focuses on the six cases of community based forest management in northern Luzon Island, Region II and has the following objectives. • Identify the strategic weaknesses5 in the devolution policy process in forestry in the Philippines. A total of 87 semi structured interviews, 6 focus group discussions and 18 key informant interviews were taken during the study. The research employed a qualitative technique for data collection, analysis and interpretation. The major tools used for data collection were: document analysis, observation, and interviews as described above. • Analyse the reasons behind the strategic weaknesses in the devolution policy process. • Assess and establish the relationship of the devolution policy outcomes to the quality of governance and institutional structures. FIGURE 2 Research sites in the province of Nueva Vizcaya (NV) and Quirino FIGURE 2 Research sites in the province of Nueva Vizcaya (NV) and Quirino FIGURE 2 Research sites in the province of Nueva Vizcaya (NV) and Quirino Alicia Buena vista Kinacao Bitnong Kalahan DMP 5 Strategic weakness: lack of strength, power or determination to achieve particular purpose as per plan or to gain an advantage. Buena vista Kinacao Bitnong Kalahan Alicia 5 Strategic weakness: lack of strength, power or determination to achieve particular purpose as per plan or to gain an advantage. G.R. Dahal and D. RESEARCH SITES, THEIR CONTEXT AND METHODS Capistrano 382 TABLE 3 Key features of the research sites in Nueva Vizcaya and Quirino province Research sites Key features Federation of Vista Hill, Kalongkong and Kakilingan Upland Farmers Association Inc. Buenavista, NV ITTO funded, mixed tribal community, pastureland before CBFM, plenty of physical resources of PO, numbers of collective enterprises, tenure instruments are CBFMA and CSC6. 3000 hectares of forest managed by 212 households under CBFM since 1999 Bitnong Guijo Greeners Association Inc., Bitnong, Dupax del Norte, NV DENR regular intervention site, people from three barangay are using the forest, pastureland before, less plantation effort. Tenure instruments are CBFMA and CSC. Total of 300 hectares of forest has been managed by 86 households under CBFM since 1999. Kalahan Education Foundation, Imugan, Sta Fe, Nueva Vizcaya Community initiated, ancestral domain of Kalanguya indigenous community, PO operating livelihood enterprises and community services such as: health and education, local rules in operation, type of tenure: CADC-CBFMA7. Total of 15000 hectares of forestland has been managed by 2000 households since 1974 as community forestry. Kinacao Upland Planters Association Inc., Kinacao, Baratbet Nueva Vizcaya Devolved ISF site, managed by the LGU8, no communal tenure, type of tenure: CSC. Total of 87 hectares of forestland has been managed by 66 households since 1991 as a devolved ISF site. Don Mariano Perez (DMP) Farmers Multi Purpose Cooperative Diffun, Quirino German funded site, timber potential forest, having RUP9 to harvest timber by the PO, type of tenure: CBFMA and CSC. Total of 135 households have managed 3100 hectares of forest since 1991. Alicia Sustainable Resource Development Cooperative, Quirino It is partially funded by the German project, timber potential forest, having RUP to harvest timber by the PO, type of tenure: CBFMA and CSC. Total of 5520 hectare of forest has been managed by 125 households since 2002. TABLE 3 Key features of the research sites in Nueva Vizcaya and Quirino province Devolved ISF site, managed by the LGU8, no communal tenure, type of tenure: CSC. Total of 87 hectares of forestland has been managed by 66 households since 1991 as a devolved ISF site. German funded site, timber potential forest, having RUP9 to harvest timber by the PO, type of tenure: CBFMA and CSC. Total of 135 households have managed 3100 hectares of forest since 1991. MAJOR FINDINGS AND DISCUSSION outcomes the CBFM implementation at the community level carries many weaknesses and limitations. Table 4 summarises the issues that emerged from the study and their status in CBFM implementation at the PO/community level. The status of CBFM implementation at PO/community level gives an overall picture of what is happening with devolution in forestry in the Philippines. The variables that emerged from the field study based on individual responses are mixed in nature. Most of them are related to the institutional process of POs, and enabling factors for implementation while some are related to the outcome of CBFM intervention at the community level. Based on issues that emerged and their status, it is clear that besides having limited positive Similarly, the issues that emerged at the provincial and central level indicate that the outcome of CBFM implementation is closely associated with the prevailing status of enabling factors at the provincial and national offices of DENR and LGU. Despite numerous coordination forums designed to avoid confusion and maintain the relationship between the DENR and LGU, there is still confusion on Implementation issues Status Institutional process of POs Sharing of benefits and resources Inequitable, discriminatory Participation, incentives and motivation Fewer incentives, poor motivation paid participation, lack volunteerism Volunteer culture replaced by project incentive Membership and community representation in CBFM PO Eligible members not included, CBFM more PO based than community based Accountability, decision making and power exercise Poor compliance with their obligations Structural and policy problems, Elite controlled the decision making and the process of decision making is non participatory Information, communication and transparency Poor transparency mainly with funds, no system of auditing in practice Prevalence of conflicts Boundary, role and power conflict Unclear policy creating conflict among actors Enabling factors related to CBFM implementation Security of tenure and property rights Time bond security, CBFM members feel insecure and are reluctant to contribute in CBFM activities, inequitable land distribution Resources and competencies Limited financial and human resources Project dependent Coordination, network, social relations Poorly functioning coordination and network Too many forums, but mostly non functional Leadership and personal qualities Majority with poor qualities and attitude Process of timber harvesting under resource utilisation permit (RUP) Complex paper processing, insufficient market information Red tape and loopholes for unethical behaviour Ethnicity, culture, and norms Indigenous cultural community with strong social capital. RESEARCH SITES, THEIR CONTEXT AND METHODS It is partially funded by the German project, timber potential forest, having RUP to harvest timber by the PO, type of tenure: CBFMA and CSC. Total of 5520 hectare of forest has been managed by 125 households since 2002. FIGURE 3 Theme building process in N6- coding, search and exploring the texts FIGURE 3 Theme building process in N6- coding, search and exploring the texts FIGURE 3 Theme building process in N6- coding, search and exploring the texts FIGURE 3 Theme building process in N6- coding, search and exploring the texts g p g, p g The themes that emerged from the qualitative data were identified using computer software QSR N6. For this, the text of individual interviews was coded and kept in an appropriate node. The data were analysed using content analysis technique discussing the themes that emerged from coding of the text. Themes carrying similar meaning were clustered under a single overarching theme. The QSR N6 is a computer package designed and developed by the Computer Assisted Qualitative Data Analysis Software (CAQDAS) group to aid researchers in handling textual data for qualitative analysis. Certain tools are provided for managing and exploring data and developing and testing ideas and theories. The QSR N6 version of Nvivo computer software was used to analyse the primary data (Figure 3). The text unit for coding was taken as line and paragraph. 6 CSC- Certificate of Stewardship Contract 7 CADC-CBFMA- Certificate of Ancestral Domain Claim- Community Based Forest Management Agreement 8 LGU- Local Government Unit 9 RUP- Resource Utilisation Permit Forest governance and institutional structure 383 MAJOR FINDINGS AND DISCUSSION Heterogeneous communities with weak social capital Ethics and standard of conduct Stakeholders involved in corruption, and other unethical illegal activities Outcome of CBFM implementation Condition of forest Better protected in project intervention sites, but poor in regular sites Support for people’s livelihood and enterprise development Project based, insufficient options available Poor support in technology and marketing Traditional livelihood options are displaced Collective enterprises are mostly failed TABLE 4 State of CBFM implementation at the PO/community level TABLE 4 State of CBFM implementation at the PO/community level G.R. Dahal and D. Capistrano 384 policy per se about CBFM implementation. The Table 5 summarises the state of CBFM implementation at provincial and central levels. has the greatest knowledge and information about the needs and priorities of community people. Hence, the exclusion of the barangay10 from the whole process of devolution in forest management has made the devolution attempts incomplete. In this regard, one of the barangay captains stated: The evidence collected from the six research sites, the provinces and the centre indicates that despite there being some positive outcomes from CBFM implementation, there are a number of limitations and weaknesses remaining at all levels. To ensure the effective implementation of CBFM these limitations and weaknesses need to be understood well and addressed in a proper way. “I have seen that there are some technical problems with implementation. We are not legally recognised as the partner for CBFM implementation. There is problem in policy also. When CBFM was formed at that time they asked us to sign in TABLE 5 State of CBFM implementation: provincial and central levels Resources and competencies Limited resources to address increasing numbers of CBFM sites, insufficient skills Monitoring and supervision Project oriented, traditional and irregular Resources lacking for monitoring Planning and policy formulation Top down planning, inconsistent policy Coordination and cooperation More forums but less action Tension rather than cooperation Incentives, motivation and service quality Little incentive, poor motivation and quality, no special incentives for CBFM, apathy Accountability structure Policy conflict, unclear structure, poor accountability in practice Ethics and standards of conduct Involvement of key actors in corruption, illegal logging and unethical practices TABLE 5 State of CBFM implementation: provincial and central levels MAPPING THE STRATEGIC WEAKNESSES IN DEVOLUTION POLICY PROCESS an agreement paper as a kind of witness rather than having a role. We are not legally required to attend any meeting. We are taking care of all development in the barangay, so, then why are we not given a role in forest management?” The emerged issues that have been presented in earlier sections indicate that there are a number of weaknesses in the devolution policy process in forestry in the Philippines. Looking on the type of these issues, it revealed that the weaknesses are of strategic in nature. Broadly, these strategic weaknesses are mapped into two parts as in Figure 4. The first is attributed to an inadequate policy articulation in devolving the management role from the state to the LGU or POs and is referred to as strategic weakness domain one. The second is a set of differences between idealised policy and the complex realities of implementation, which is associated with the issue of poor governance practice and weak institutional structures and is referred to as strategic weakness domain two. Second, the power of supervision and control of any forest management activities devolved either to LGU or PO is still retained by the DENR. Under the provision of the LGC 1991, for all the devolved ISF activities, supervision and control is remaining with the DENR, which is against the principle of devolution. In such cases implementation of devolution policy under the LGC seems incomplete, which is due to existing incomplete devolution policy per se. One of the Governors from the project site stated: “The power and authority devolved to the LGU is to enforce the forestry law within community based forest management as it is subject to control, supervision and monitoring by DENR. In this case it looks like the Governor is the police to protect forest and will enforce the forest law in the province but the head of the police is DENR/ PENRO. How can it be like that?” 10 Barangay- the lowest political unit of local government Strategic weakness domain one The study identified the strategic weaknesses domain one as a result of an incomplete and inadequate policy articulation about devolving power and function from the centre to the local level, which is reflected in the following evidence from the field data. Third, the property rights of the PO members under ‘devolution’ in CBFM are limited to 25 years, and tenure rights of the communal area under CBFMA and individual actual land tilling as CSC by the members of CBFM are not guaranteed for further extension after 25 years. First, the devolution under LGC 1991 is only to the municipality level as it does not include the barangay, the lowest level of the local government unit, which is the closest structure of the state dealing with civil society and 10 Barangay- the lowest political unit of local government Forest governance and institutional structure 385 DENR plays a vital role, whereas the POs comply and act as legitimate agents of the state to implement its agendas. Besides the preparation of documents, it is the DENR that takes action against the defaulters in the POs. The harvesting of any timber and non-timber forest products from the CBFM is also subject to prior approval by the DENR. Contrary to the thrust of devolution the DENR retains all authority. It is due to incomplete and inadequate articulation of devolution policy. Table 6 further shows that many CBFM members are still not confident enough about present rights and tenure arrangements as they are time bound and subject to change any time in the future along with the change of political scenarios at the national level. Fourth, the DENR’s retention of authority to make final decisions on any actions of the POs has increased the dependency of the POs on the DENR. For example, in the preparation and approval of CRMF11, RUP and AWP12 the FIGURE 4 Di i i f l i f i k i d l i li i f FIGURE 4 Diagrammatic presentation of analysis of strategic weaknesses in devolution policy process in forestry FIGURE 4 Di i i f l i f i k i d l i li i f FIGURE 4 Diagrammatic presentation of analysis of strategic weaknesses in devolution policy process in forestry FIGURE 4 Diagrammatic presentation of analysis of strategic weaknesses in devolution policy process in forestry CCaassee ooff KKaallaahhaann IIPPRRAA SSiittee DDeepptt.. aaddm miinniisstt rraattiivvee oorrddeerr 55 ccaasseess ooff CCBBFFM M SSttrraatteeggiicc wweeaakknneessss ddoom maaiinn oonnee:: SSttrraatteeggiicc W Weeaakknneesssseess == IInnaaddeeqquuaattee ppoolliiccyy aarrttiiccuullaattiioonn SSttrraatteeggiicc wweeaakknneessss ddoom maaiinn ttwwoo:: W Weeaakknneesssseess == PPoooorr ggoovveerrnnaannccee aanndd W Weeaakk iinnssttiittuuttiioonnaall ssttrruuccttuurree DDeevvoolluuttiioonn PPoolliiccyy TThhrruusstt SSttaattee ooff EExxeeccuuttiivvee PPoolliicciieess Devolution Policy O Ouuttccoom mee LLiim miitteedd ssuuppppoorrtt oonn ppeeoopplleess lliivveelliihhoooodd,, eeqquuiittyy pprrooppeerrttyy rriigghhttss aanndd iim mpprroovveem meennttss iinn ffoorreesstt ccoonnddiittiioonn.. 16 19 FIGURE 4 Diagrammatic presentation of analysis of strategic weaknesses in devolution policy process in forestry TABLE 6 Perception of community members about the security of tenure Devolution Policy O Ouuttccoom mee DDeevvoolluuttiioonn PPoolliiccyy TThhrruusstt SSttaattee ooff EExxeeccuuttiivvee PPoolliicciieess SSttrraatteeggiicc wweeaakknneessss ddoom maaiinn ttwwoo:: W Weeaakknneesssseess == PPoooorr ggoovveerrnnaannccee aanndd W Weeaakk iinnssttiittuuttiioonnaall ssttrruuccttuurree TABLE 6 Perception of community members about the security of tenure TABLE 6 Perception of community members about the security of tenure 19 Source: Field study 11 CRMF- Community Resource Management Framework 12 AWP- Annual Work Plan Research sites Research sites Level of confidence in the security of tenure Fully confident Confident Not confident Total Buenavista 0 8 7 15 Bitnong 0 1 6 7 Kalahan 0 1 1 2 Kinacao 1 2 0 3 DMP 1 2 2 5 Alicia 1 2 2 5 Total 3 16 18 37 G.R. Dahal and D. Capistrano 386 TABLE 7 Respondent’s perceptions about prepared CRMF, RUP and AWP TABLE 7 Respondent’s perceptions about prepared CRMF, RUP and AWP Source: Field study Research sites Who prepared CRMF, RUP and AWP DENR PO DENR PO & Project Project Project DENR General Assembly No Idea Total Buenavista 1 1 2 3 4 11 Bitnong 2 1 2 5 Kalahan 5 1 6 Kinacao 1 1 2 DMP 1 1 1 1 1 1 1 7 Alicia 2 1 1 1 5 Total 7 7 3 3 6 1 9 36 Who prepared CRMF, RUP and AWP during extension of RUP it is clear that - no SOP [Standard Operating Procedure- euphemism for illegal activity] no extension. Similarly, sometime DENR delayed the paper processing which is an indirect way to motivate people to go for bribery and support corruption. There are many more other cases, even police, they want lumber without payment, etc. FIGURE 4 Di i i f l i f i k i d l i li i f FIGURE 4 Diagrammatic presentation of analysis of strategic weaknesses in devolution policy process in forestry For example, in Cordon there is a big check point of DENR, the lowest amount a motorist gives them is 500 pesos but it will depend upon the volume of your load. They will say hello, who is your lawyer? Is Aquino [500 pesos] or Marcos [1000 pesos]?” Table 7 shows that most of the decisions are made by the DENR and the project and very few decisions are made by the general assembly of CBFM POs. Furthermore, a member of Bitnong CBFM stated his view as: “We are still depending on DENR for any decisions. Association itself is not authorised to make local decisions about our forest and funds.” Fifth, the present policies of devolution in forestry do not allow community members to harvest forest products, particularly timber. Any decision in this regard is subject to prior approval by the DENR. In all the research sites CBFM members expressed their dissatisfaction about this kind of control mechanism on forest product harvesting. As one of the CBFM PO members from Alicia stated: Not only the DENR, but there are some cases that CBFM POs themselves are also involved in illegal timber poaching activities. For instance, the treasurer of Kalahan Education Foundation in Nueva Vizcaya stated that: “Any decisions are subject to supervision, control, and review by DENR”, which means CBFM implemented by our PO and LGU, should also be the subject of supervision and control by the DENR”. “There are still cases of illegal logging during the night time and selling of timber to Sta Fe and Cabanatuan. But it is much reduced these days. The community themselves control such kinds of activities. Forester Tamano is very strict in controlling the illegal logging. Even the BOT chairman was penalised just now as his son allowed a truckload of timber transported from Malico to the Sta Fe in the name of the municipal office construction, which does not have any permit to carry such amount of timber”. “Yeah there are the cases of corruption, specifically Strategic weakness domain two These are a set of differences between idealised policy and the complex realities of implementation. This hinders the process of transforming policy into practice and the factors associated with such hindrances are related to poor governance and weak institutional structure in the devolution policy process. Some of the factors that have emerged from the study are explained below as evidence of this situation. Second, the exclusion of some community members and discrimination in benefit sharing is a common issue at all the research sites. It is due to the CBFM agreement, which does not require compulsory inclusion of all the potential members in the CBFM. First, the prevalence of corruption and illegal logging at various levels of CBFM implementation signify that the policy practice problem is associated with quality of governance. Unethical practices within POs and the DENR are revealed through different cases noted in the research sites. Illegal timber poaching associated with the exchange of bribes during transportation of timber by the POs is commonly observed in most of the research sites. One of the ex-chairpersons of Alicia CBFM in Quirino stated: Table 8 shows that many members of CBFMs in the research sites still feel that there is discrimination in sharing of benefits and resources within their PO. A huge number of potential households are still not included in CBFM, which further increases the practice of discrimination between members and non-members. A large amount of share capital is necessary to pay for CBFM membership, generally poor farmers are unable to afford the amount and are not registered as a member although they are potential and interested. As one of the non member from Alicia explained: Forest governance and institutional structure Table 12 shows the decreasing trend of budget allocation in the forestry sector and particularly the CBFM programme. Fourth, the existing resources and competencies of POs and the DENR are not compatible with what is required to fulfil the plans of POs. A few attempts are made to generate internal resources and capacities, however, the general trend is to expect support from foreign projects, rather than thinking creatively to generate internal resources. The problem of resource limitation exists widely at community (Table 11), province and central level in both state and civil society. This situation is ultimately hindering the process of implementation of devolution policy. The provincial CBFM coordinator from the DENR Quirino explained the limitation of CBFM implementation in the province as follows: “…that is our challenge. We have very limited staff in Quirino. I am only one taking care of all sites at the provincial level. There are 5 PMO13s assigned in the sites but not only confined in CBFM, they are also assigned to look after the protection of forest, alienation and disposition of land, which are an extra load for them. Forest governance and institutional structure Forest governance and institutional structure 387 “There is no equal sharing. I think it all depends upon the amount of share capital and other benefits are mainly given to its members but the non-members can’t get any benefits. As you have seen, banana suckers were distributed only to the members.” been fully given to making decisions in a participatory way and informing all members of them. The problem of accountability is prevalent in most of the research sites as there is no clear structure to show who is responsible to whom and for what reasons. In such situations the elites and a few powerful position holders are active in making decisions with a poor level of accountability to the member of the POs. Table 9 shows that plenty of potential households in the research sites are still without having CBFM membership. Table 10 shows that decision making in the CBFM PO is mainly done by the DENR and PO officials, however no responses were recorded saying the decisions are made in Third, the institutional practices in terms of maintaining transparency, accountability and participation are not well established within CBFM POs. Attention has not TABLE 8 CBFM member’s perceptions about distribution of forest resources TABLE 9 State of CBFM membership in the research sites TABLE 10 Perceived level of decision making in CBFM PO Source: Field study Research sites Response categories Equal distribution Discrimination Total Buenavista 5 6 11 Bitnong 1 4 5 Kinacao 0 3 3 Kalahan 3 1 4 Alicia 6 2 8 DMP 3 1 4 Total 18 17 35 Research sites Existing CBFM PO member households Non member households Buenavista 212 25 Bitnong 86 30 Kinacao 66 32 Kalahan 514 15 DMP 135 55 Alicia 125 105 Decision making authority Number of responses PO officials 13 PO president or chairman 10 DENR 2 LGU NIL ENRO 1 Board members 4 Management team 2 Project 3 Joint decision by DENR and PO officials 26 Total 61 TABLE 8 CBFM member’s perceptions about distribution of forest resources TABLE 9 State of CBFM membership in the research sites G.R. Dahal and D. Capistrano 388 the general assembly of CBFM PO, which in theory should be the accountable forum to make decisions. the CBFM process. Similarly, within DENR the financial problem is considered as one of the major bottlenecks in promoting CBFM programme. Forest governance and institutional structure We don’t have Except Kalahan the physical and financial resources are only available in the project supported CBFM sites, which indicates that there is lack of required resources to support TABLE 11 Summary of resources of CBFM POs Source: Field study Research sites Infrastructure and physical resource Fund and its sources Trained human resources Buenavista Furnished office building Computers Motorbikes Mobile phones Officials records Revolving Carabao ITTO remaining fund from second phase of the project, Award, Income from enterprises Monthly dues, Membership fee (1.5 million Pesos) Trained members in forest management and enterprises development Paid forest technician Internal auditor Bitnong Few registers and files, no office building Membership fee (29, 500 Pesos) No training given so far Kalahan Furnished office building School Fruit processing unit Donor support Membership Cutting permit fee Selling of product (3.5 million Pesos) Paid forest technician Paid social mobiliser Trained account keeper Kinacao Few register and files, No office building Membership Donation (32,000 Pesos) No training given so far DMP Well furnished office building Consumer goods shop Rice mills Solar system for electrification RP German project Membership and share capital Benefits from rice mills and cooperative store Timber selling under RUP (Fund balance not available) Paid manager, consumer store and rice mill operators Trained members in forest management Alicia Office building Rice mill (not in operation) going to install German project support Membership and share capital Timber selling under RUP operation Donations (2.12 millions Pesos) Trained members in forest management Paid rice mill operator 13 PMO- Project Monitoring Officer Forest governance and institutional structure Forest governance and institutional structure enough funds to travel around and help every site. So POs are complaining us for not visiting their sites. But we have our problem also.” Sixth, the existence of more forums for coordination at all levels does not guarantee the establishment of good coordination of actions. This study has revealed that a number of forums are formed at community level such as BUDAC14 in Buenavista, MOA15 in Quirino, and PENRC16 in both provinces. However, in practice these forums are not much functional in maintaining meaningful coordination. Fifth, the prevalence of conflicts of different types at PO level has acted as a barrier for effective implementation of the devolution policy. Manifestations of conflicts are due to unclear boundary demarcation, discrimination while issuing CSCs, discrimination in resource and benefits sharing, poor leadership qualities, unclear policies etc. Trained human resources 389 Forest governance and institutional structure Seventh, the practice of unequal treatment by the PO in resource distribution and sharing of benefits makes the situation complex in terms of implementing the devolution TABLE 12 Budget allocation for CBFM programme in the Philippines (000 Pesos) Source: JICA-DENR 2004 Years National budget Regional budget Province of Nueva Vizcaya Province of Quirino FMS FMS CBFM DENR/PENRO17 DENR/PENRO 2001 1504805 17089 1556 (9.11%) 1981 1680 2000 1504805 15746 3667 (23.28%) 1981 1680 1999 1467692 13915 3253 (23.37%) 4370 1918 1998 1610430 15651 3257 (20.81%) 1047 808 1997 2025587 21001 5391 (25.68%) 1499 1449 TABLE 12 Budget allocation for CBFM programme in the Philippines (000 Pesos) TABLE 12 Budget allocation for CBFM programme in the Philippines (000 Pesos) TABLE 13 Collective enterprises initiated by CBFM POs and their present status Source: Field study 14 BUDAC- Buenavista Upland Development and Cooperation 15 MOA- Memorandum of Agreement 16 PENRC- Provincial Environment and Natural Resource Committee 17 PENRO- Provincial Environment and Natural Resource Office Research sites Enterprises within CBFM PO Status Buenavista Fossil flower production Cattle dispersal Trading post operation Carabao (buffalo) for hire Not even in breakeven position Running but facing problems Almost closed Problem in maintaining rotation Bitnong None Kalahan Food processing plant Swine production Water purification unit School and health centre Carbon sequestration project Water taxation project Only in breakeven position Already privatised Not yet in operation Not enough fund to run Under process to implement Not operational and legalised yet Kinacao Community fish pond Cooperative marketing unit Stopped due to boundary conflict Conflict about misuse of funds DMP Consumer store operation Swine production Rice mill operation (two) Hardly in breakeven Scale down the size of production 1 in breakeven 1 already stopped Alicia Rice mill operation Purchased but not yet installed TABLE 13 Collective enterprises initiated by CBFM POs and their present status 390 G.R. Dahal and D. Capistrano foresters. Many bureaucrats in the DENR feel devolution of forest management as an act of losing power (Fisher et al. 2000). This attitude creates apathy in the promotion of the CBFM programme. policies. The discrimination between members and non- members, rich and poor, officials and non-officials of POs, etc., creates a situation of conflict and de-motivates community people in participating in the implementation of CBFM. Besides the attitudinal problem, there remains the problem of the structure of the DENR. Corruption as an established covert institution in devolved forest management The evidence presented above shows that corruption in the forestry sector is highly prevalent even in the devolved context. The emergence of new collaborative forms of corruption is revealed in the study, where the DENR and civil society organisations, particularly POs, are jointly involved in such unethical practices in the case of Buenavista. The president of CBFM PO from Buenavista described the case as follows. Unilateral policymaking process The process of policy making at the moment is the unilateral task of the DENR. However, without involving all the actors (civil society, private sector, NGOs and POs) and listening their voices in forest devolution, it is hard to believe that the interests of all the actors are properly accommodated/ reflected in the policies. THE REASONS BEHIND STRATEGIC WEAKNESSES IN DEVOLUTION POLICY PROCESS The research study has revealed that ‘strategic weaknesses domain one’ of the devolution policy process is due to an inadequate policy articulation, but it has also revealed that there are certain factors, which hinder the process of meaningful devolution. Those factors are cross cutting and widespread which are associated with the policymaking process, attitude, structure, priorities of bureaucrats and system of accountability. The reasons for ‘strategic weakness domain two’ on the other hand, are as a result of a set of differences between idealised policy and the complex realities of implementation, linked with poor governance quality and non-supportive institutional structures such as: elites capturing the devolved power, lack of transparency and accountability, lack of trust and cooperation, not giving any recognition to local tradition, culture and systems, bureaucratic apathy, and poor participation of community people in CBFM activities. Trust between state and civil society in devolution of forest management The state has always been a sceptical and unreliable force in the devolution policy process. Many respondents from the study sites were of the opinion that the policy consistency of the government remained always doubtful. The CBFM members are now not sure what will happen after the completion of the 25 year tenurial period of the agreement, as there is a tendency to frequent changes in the forest policies. For example, the policy concerning harvesting of timber by the CBFM PO was withheld unilaterally by the DENR in 1998 and 2005/2006. Forest governance and institutional structure The traditionally designed hierarchical structure of the bureaucracy still exists and is not compatible with the role and responsibilities of the changed context. Similarly, there exists an institutional incompatibility associated with a hierarchical organization in the DENR. For example, the DENR attempting to sponsor and manage a participatory process, however, very little reflected in practice. Making minor changes is unlikely to address the fundamental problem. Eighth, the collective enterprises initiated by the external project are not sustaining in most of the research sites. The practice of volunteer contribution for collective action is replaced by the practice of having remuneration for any participation and contribution to CBFM related activities. The state of collective enterprises in research sites is described below (Table 13). The weakness in CBFM implementation are not confined only to the above-presented factors, but are also present in the overall socio-political and economic context, traditional norms, values, cultural practices, and quality of leadership is also have a direct influence in the implementation process. It is evident that relatively successful CBFM implementation in Kalahan sites is attributed to their practice of a traditional system of accountability and decision-making (Tongtongan: a group of elderly people, who make most of the decisions in Kalahan), a similar cultural identity, strong leadership of Pastor Delbert Rice (A Christian missionary settled in Kalahan since 1965) and a strong network and trust among the members. Referring the evidence from the field some of the broader reasons behind strategic weaknesses are discussed below. GOVERNANCE AND INSTITUTIONAL STRUCTURE: IGNORED DIMENSION GOVERNANCE AND INSTITUTIONAL STRUCTURE: IGNORED DIMENSION GOVERNANCE AND INSTITUTIONAL STRUCTURE: IGNORED DIMENSION As evident from the study, corruption permeates the process of CBFM implementation at all levels. The actors in CBFM implementation have unethical involvements within and around the forest management. Such practices have two implications. First they have reduced the accountability of the actors in performing their roles and functions and second, they have created weak governance practices resulting in poor performance of the policy interventions. The present policies have not concretely addressed these concerns though every actor involved in CBFM implementation is aware of them. The reasons for not taking corruption into account are complex and mixed. First, the practice of corruption in forestry is well established and it is very hard to break the MUTUAL REINFORCEMENT BETWEEN TWO DOMAINS OF STRATEGIC WEAKNESSES People from the research sites perceived CBFM is the target and the need of the DENR rather than as a community need. There is an argument that full devolution will never happen unless it is initiated through demand and pressure from communities themselves (Anderson 2000). No one wants to give up the power, he or she holds. Apart from Kalahan site, none of the research sites demanded any authority and power from the state. In Kalahan, community people jointly exerted pressure on the DENR secretary to cede the authority for managing the forest as community forestry in their ancestral domain. But in other sites the state has introduced the concept of CBFM and induced people to undertake an agreement to fulfil government annual targets, where the relation is of patron and client. Hence, the CBFM programme is unable to make people accountable for their actions and develop a feeling of ownership. The issues that have emerged from this research study ultimately contribute to create two domains of strategic weaknesses, but are interrelated as well. Some examples of such relationship are explained below. For example, the Local Government Code 1991 is a policy document for devolution, which excludes the barangay from the process of devolution of forest management. The implication of this inadequate policy articulation (strategic weakness domain one) has manifested in the form of conflict between barangay councils and PO committees at local level (strategic weakness domain two). Likewise, another example is the unilateral suspension of timber harvesting permit of the POs by the central state, in consequence, local community started feeling sceptical about other provision of CBFM policy as well. Attitude, accountability and structure in government bureaucracy “…We can not deny the illegal activities happening in the area and the assistance of CENRO Bayombong is always regarded in the conduct of the foot patrol and IEC. However, one of the foresters in CENRO18 seems to be providing information to the timber poachers of the planned anti- timber poaching campaign of the CBFM project, giving a chance to the timber poacher to elude possible arrest and confiscation of their equipment and lumbers in exchange The traditional command and control approach of forest management still has some effects on bureaucrats. The orientation of this approach is to exclude community people in the management. This is a techno-centric view of bureaucrats who believe forest is only the domain of technical Forest governance and institutional structure 391 for monetary benefits or the lumber being delivered to the forester’s furniture shop in Bayombong.” the forest department. The inclusion of community people as an actual partner of forest management has hardly happened in the Philippines, instead state perceived local communities as enemies in managing forests. During both colonial and postcolonial periods the politician always used the forest as a means to secure their political position in the government. Issuance of TLAs to political supporters, involvement of political persons in illegal logging of timber and unethical links between forest bureaucrats and politicians are some evidence of the non-supportive political context in devolving the forest management role from the state to the civil society organisations. In addition, some other unethical practices were revealed within CBFM POs, particularly about the mis-use of funds, vehicles and forest resources mostly by the presidents and other executive members of the CBFM POs in the research sites. The existence of such unethical and corrupt practices not only challenges the quality of forest governance in the devolved context, but also reflects the attitude and ethics of the actors involved in the whole process of CBFM implementation in the Philippines. Elites in power centres Similarly, under the present policy it is not obligatory for POs to include all the potential households CBFM members, as only seven members are enough to undertake CBFM agreement with the DENR. This inadequate policy articulation has resulted in the exclusion of plenty of potential members from the CBFM, which has created conflicts and discrimination in benefit sharing at the local level. The leaders handling the decision-making in CBFM POs are mostly from economically wealthy categories, have highly educated backgrounds and are representatives of local political parties. At the beginning of CBFM formation the first approach of the DENR is to contact those socio-economically well off members of the community. This strengthens the link between elite members of the community and the state. This relation further strengthens when the DENR undertakes CBFM agreements and provides the training and exposure to those elites in the process of CBFM implementation. Any benefits and resources devolved to CBFM are enjoyed by well off people in the community whereas poor members have been pessimistic about getting benefits out of the CBFM programme in their areas. 18 CENRO- Community Environment and Natural Resource Office Broader political and historical context of forest policy development Historically, forest management in the Philippines remains under the control of the state authority as the state still believes forest protection and development is only the role of 18 CENRO- Community Environment and Natural Resource Office 392 G.R. Dahal and D. Capistrano • The role transformation process in the state (from a command and control role to the transfer of significant roles to the local people) needs to correspond with a change in attitudes and behaviour from a traditional way of thinking to a more democratic and participatory way of thinking. • The role transformation process in the state (from a command and control role to the transfer of significant roles to the local people) needs to correspond with a change in attitudes and behaviour from a traditional way of thinking to a more democratic and participatory way of thinking. tradition unless there is a strong mechanism to dismantle it. Second, the corruption in forestry is directly related to local and national politics. The network between bureaucrats and politicians in corrupt practices is strong and again is difficult to dismantle as both of them are operating in close partnership as a covert institution to meet their hidden objectives. Hence, along with corruption other issues pertaining to governance such as: accountability, transparency, and participation are also ignored in the whole devolution policy process in the Philippines. Government effort for minor changes to ensure good governance and efficient institution is unlikely to address fundamental problem unless considering the holistic reform. In order to make these recommendations functional, the following framework, Figure 5 is presented, which emphasises the need to support the devolution policy process with clear roles, accountability, transparency, sharing of power and participation of all actors in the process. FIGURE 5 Framework for effective devolution policy process Devolution policy process Policy formulation Policy implementation State Market Civil society Good governance Supportive institutional structure Policy outcome Participation, voice, accountability and power sharing Quality of governance Defined roles and functions FIGURE 5 Framework for effective devolution policy process FIGURE 5 F k f ff ti d l ti li FIGURE 5 Framework for effective devolution policy process WAYS FORWARD TO OVERCOME THE STRATEGIC WEAKNESSES Some recommendations are proposed to overcome the strategic weaknesses identified in the devolution policy process in forestry in the Philippines. • The existing inadequate articulation in devolution policies needs necessary amendment to ensure the transfer of power to make decisions on forest management at the local level. For this, the devolution of roles and functions should correspond with the transfer of power and authority. In order to fulfil this, tenure security should be guaranteed so as to overcome the poor motivation of the CBFM members to manage the forest. Similarly, the authority to harvest forest products, particularly timber, should be given to CBFM POs once their CRMF and RUP are approved. REFERENCES CHAMBERS, R. 1997. Whose Reality Counts?: Putting the First Last. London: Intermediate Technology Publications. AGRAWAL, A. and E. OSTROM 2001. Collective Action, Property Rights, and Decentralisation in Resource Use in Nepal. Politics and Society. 294: 485-514. COLFER J. and D. CAPISTRANO 2005. The Politics of Decentralisation: Forests, Power and People Pp 296- 311. London, Earthscan Publications. ANDERSON, J. 2000. Four Considerations for Decentralised Forest Management: Subsidiarity, Empowerment, Pluralism and Social Capital. RECOFTC Report. Bangkok: FAO and RECOFTC. 18: 11-22. CONTRERAS, A. P. 2000. Rethinking Participation and Empowerment in the Uplands. In: P Utting, ed. Forest Policy and Politics in the Philippines. The Dynamics of Participatory Conservation. Pp. 144-170. Quezon City and Manila: Ateneo de Manila University Press and United Nations Research Institute for Social Development. g ASIAN DEVELOPMENT BANK 1992. Appraisal of the Forestry Sector Project in the Philippines. Manila: Asian Development Bank. ASIAN DEVELOPMENT BANK 1994. ADB Approves New Forest Policy for Asia Pacific Region 6 March. Manila: Asian Development Bank. CONTRERAS, A. P. 2003. �� Creating Space for Local Forest Management in the Philippines: A Synthesis. In: Contreras, A.P. ed.. Creating Space for Local Forest Management. Pp. 211-218. Manila: La Salle Institute of Governance. BABIN, D. and A. BERTRAND 1998. Managing Pluralism: Subsidiarity and Patrimonial Mediation. Unasylva. 194 49: 19-25. BAGADION, Jr. B. C. 1993. Notes and observations on community based resource management. In: community based resource management: perspectives, experiences, and policy issues. F. Fellizar, Jr, ed.. Environment and Resource Management Project. Report No 6. Laguna Philippines: Environment and Resource Management Project. CHEEMA, G. S., and D. A. RONDINELLI 1983. Decentralisation and Development: Policy Implementation in Developing Countries. London: Sage Publications. DENR 1992. Department Administrative Order DAO 92- 30. Guidelines for the Transfer and Implementation of DENR functions Devolved to Local Government Units. Quezon City: Forest Management Bureau. BAGADION, Jr. B. C. 2000. Social and Political Determinants of Successful Community Based Forestry. In: P Utting ed. Forest Policy and Politics in the Philippines: The Dynamics of Participatory Conservation. Pp.117-143. Manila: Ateneo de Manila University Press and United Nations Research Institute for Social Development. DJEUMO, A., and T. FOMETE 2001. The Development of Community Forests in Cameroon: Origins, Current Situation and Constraints. Overseas Development Institute, Rural Development Forestry Network. 25b: 1-28. Rural Development Forestry Network. 25b: 1-28. BORLAGDAN, S.B. 1992. The Cebu Social Forestry Pilot Project: A Case Study in Upland Development. Philippine Journal of Public Administration. CONCLUSION The strategic weakness domain one, as described in the previous section, shows that the policy documents and instruments relating to devolution in forestry are much too narrow in devolving power, functions and responsibilities to the local government and community organisations. Such incomplete policy explanation has given rise to the second domain of weaknesses linked with the policy practice gap. Inadequate policy pronunciation results in an upward accountability structure and is thus a source of poor governance during implementation. Therefore, in the strategic weakness domain two, the reasons for poor implementation of policy are seen as the result of poor quality of governance and non-supportive institutional structures, which are the ignored dimensions in the whole process of transforming policy into practice. • There is a need to pay adequate attention to the issues related to governance such as: clear role and accountability, participation, transparency, fairness, property rights on the one hand, and institutional structures (both formal and informal institutions) such as: norms, network, traditions, ethics, resources and competencies on the other hand. Without more consideration of the above issues it is naïve to expect the desired policy outcomes from devolved forest management in the Philippines. 18 Much research on community based forest management has focused only on the issues associated with poor outcomes in relation to the process of implementation with the presumption that devolution policy in forestry per se is good. They have not given adequate attention to policy matters, and especially, to balancing the relationship between the policies on the one hand and the issues associated with poor outcomes on the other. However, this research has found that the limited successes of devolution policy are not only due to weak implementation but also due to inadequate policy articulation. Thus, as suggested by the present study, the knowledge concerning the reasons for the limited success of devolution policy in forestry is attributed • Devolution should address the issue of people’s livelihoods along with the agenda of environmental protection. Thus, there is a need to balance the priorities of community people’s livelihoods and improving forest conditions. • There is a need to reinforce the restructuring process already started within the state agency, making sure that the state apparatus will have enough capacity to deal with it’s new roles and responsibilities in the context of changed policies. CONCLUSION 393 Forest governance and institutional structure to the inadequate policy articulation on the one hand and a set of differences between policy and the complex realities of implementation on the other hand. 311. London, Earthscan Publications. CHAMBERS, R. 1995. Paradigm Shifts and Practice of Participatory Research and Development. In: Nelson, N. and S. Wright eds. Power and Participatory Development: Theory and Practice. London: Intermediate Technology Publications. REFERENCES 36 4: 367-387. EDMUNDS, D., and E. WOLLENBERG 2001. “Historical Perspectives on Forest Policy Change in Asia: An Introduction.” Environmental History 62: 190-212. BORLAGDAN S.B., ERNESTO S. GUIANG and JUAN M. PULHIN 2001. Community Based Forest Management in the Philippines: A Preliminary Assessment. Quezon City: Institute of Philippine Culture, Ateneo de Manila University. ENTERS, T. and J. ANDERSON 1999. Rethinking the Decentralisation and Devolution of Biodiversity Conservations. In: Unaslyva. 199 50: 6-11. FALCONER, J. 1987. Forestry Extension: A Review of the Key Issues. Social Forestry Network: 1-34. y BRILLANTES, Jr. A. 2000. Decentralisation and Devolution in the Philippines. In: Philippines Rural Development and Natural Resource Management: Trend, Strategy, Implementation, and Framework of Performance Indicator System. Pp. 17-32. A Joint Report of the Government of the Philippines and the World Bank. Manila: The World Bank. FISHER, B. 1999. Creating Space: Development Agencies and Local Institutions in Natural Resource Management. Forests, Trees and People. 22: 4-11. FISHER, R. J. 2000. Decentralisation and Devolution in Forest Management: A Conceptual Overview. In: T. Enters, P.B. Durst, and M.Victor, eds. Decentralisation and Devolution of Forest management in Asia and the Pacific. Pp. 3-10. Bangkok: RECOFTC and FAO. BURNS, D., R. HAMBLETON and P. HOGGETT 1994. The Politics of Decentralisation: Revitalising Local Democracy. Basingstoke: Macmillan Publications. FISHER, R. J., P. B.DURST, T. ENTERS and M. VICTOR 2000. Overview of Themes and Issues in Devolution and Decentralisation of Forest Management in Asia and the Pacific. Bangkok: RECOFTC Report. 18: vi-xi. CAPISTRANO, D. and C. J. PIERCE COLFER 2005. Decentralisation: Issues, Lessons and Reflections. In: Colfer J. and Doris Capistranoeds. The Politics of Decentralisation: Forests, Power and People Pp 296- GILMOUR, D. A., and R. J. FISHER 1997. Evolution in Community Forestry: Contesting Forest Resources. 394 G.R. Dahal and D. Capistrano Community Forestry at Crossroads: Reflections and Future Directions in the Development of Community Forestry. In: M.Victor, C. Lang, and J Bornemeir, eds.. Proceedings of an International Seminar. 17-19 July. Bangkok: RECOFTC Report No. 16: 27- 44. RIBOT, J. C. 2004. Waiting for Democracy: The Politics of Choice in Natural Resource Decentralisation. Washington: World Resource Institute. ROBINSON, N.A. 1993. (Ed.) Agenda 21: Earth Action Plan. New York: Oceana Publications. RONDINELLI, D. A. 1981. Government Decentralisation in Comparative Perspective: The Theory and Practice in Developing Countries. An Article Published in International Review of Administrative Sciences. Public Administration XIVII1: 133-145. GUIANG, E.S. 1996. REFERENCES Guiding National Transition in Forest Management: Some Experience in the Philippines. Paper Presented at the 5th Asia Forest Network Workshop 2- 6 December. What’s Working and What’s Not? New Delhi: AFT. ROY, K. C., and C. A. TISDELL 1998. Good Governance in Sustainable Development: The Impact of Institutions. International Journal of Social Economics. 25 6/7/8: 1310-1325. HOBLEY, M. 1996. Participatory Forestry: The Process of Change in India and Nepal. London, Overseas Development Institute. MAGNO, F. 2001. Forest Devolution and Social Capital: State-Civil Society Relations in the Philippines. In: Environmental History. 6: 264-286. SANVICTORES, E. 1997. Philippines Forestry in 21st Century: Challenges and Prospects. Society of Filipino Foresters: 49th Anniversary and National Conventions. Pp. 16-19. Manila: Society of Filipino Foresters, Inc. MAGNO, F. 2003. Forest Devolution and Social Capital: State-Civil Society Relations in the Philippines. In: Contreras, A.P. ed. Creating Space for Local Forest Management in the Philippines. Pp.17-35. Manila: La Salle Institute of Governance. SHACKLETON, S., B. CAMPBELL, E. WOLLENBERG and D. EDMUNDS 2002. Devolution and Community Based Natural Resource Management: Creating Space for Local People to Participate and Benefits. Overseas Development Institute Briefing Paper. 76: 1-6. MANOR, J. 1999. The Political Economy of Democratic Decentralisation. Washington D.C.: The World Bank. SHEPHERD, G. 1990. Forestry, Social Forestry, Fuelwood and Environment. A tour of the Horizon. Social Forestry Network 11a: 1-12. Overseas Development Institute. MAYERS, J., AND S. BASS 1999. (Eds.). Policy that Works for Forests and People: Real Prospects for Forest Governance and Livelihoods. Policy Overview. London: International Institute for Environment and Development. p SHEPHERD, G. 1992. Forest Policies, Forest Politics. In: G. Shepherd ed Forest Policies, Forest Politics. London: Overseas Development Institutes Publications. Pp. 5-27 SMITH, B. C. 1985. Decentralisation- The Territorial Dimension of the State. London: George Allen and Unwin Publications. SHEPHERD, G. 1992. Forest Policies, Forest Politics. In: G. Shepherd ed Forest Policies, Forest Politics. London: Overseas Development Institutes Publications. Pp. 5-27 MPFS 2001. Master Plan for Forestry Development. Manila: Forest Management Bureau and Food and Agriculture of the United Nations. SMITH, B. C. 1985. Decentralisation- The Territorial Dimension of the State. London: George Allen and Unwin Publications. OSTROM, E. 1999. Self Governance and Forest Resources. CIFOR Occasional Paper No 29. Bogor: CIFOR. TOHA, J., and S. BARROS 1997. The Role of Forest Policies and Institutions in Achieveing Sustainable Forest Development. Unasylva 48 190/191: 69-78. POFFENBERGER, M., and B. McGEAN 1993. REFERENCES Upland Philippine Communities: Gardians of the Final Forest Frontiers. Research Network Report No. 4. Berkeley: Center for Southeast Asia Studies. TURNER, M. and D. HULME 1997. Governance, Administration and Development: Making the State Works. Basingstoke: Macmillan Publications. PORTER, G. and D. GANAPIN, Jr. 1988. Resources, Population, and the Philippines Future. World Resource Institute Paper No.4. Washington D.C.: World Resource Institute. Provincial Environment and Natural Resource Office UNDP 1997. Reconceptualising Governance. United National Development Programme Discussion Paper 2. New York: UNDP Publications. UTTING, P. 2000. An Overview of the Potential and Pitfalls of Participatory Conservation. In: P Utting, ed. Forest Policy and Politics in the Philippines. The Dynamics of Participatory Conservation Pp. 171-215. Quezon City: Ateneo de Manila University Press and United Nations Research Institute for Social Development. i PENRO, Nueva Vizcaya 2004. Compilation of CBFM Progress Report in the Province. Bayombong: PENRO. Provincial Environment and Natural Resource Office PENRO, Quirino 2004. Compilation of CBFM Progress Report in the Province. Diffun: PENRO. VITUG, M.D. 2000. Forest Policy and National Politics. In: Forest Policy and Politics in the Philippines. The Dynamics of Participatory Conservation. P Utting, ed. Pp. 11-39. Manila: Ateneo de Manila University Press and United Nations Research Institute for Social Development. PULHIN, J. M. 2003. Community Based Forest Management in the Philippines: Retrospect and Prospects. In: Inoue, M. and H.Isozaki, eds.. People and Forest- Policy and Local Reality in Southeast Asia, the Russian Far East and Japan. London: Kluwer Academic Publishers. RMPFD 2003. Revised Master Plan of Forestry Development. Manila: Forest Management Bureau and Food and Agriculture Organisation of the United Nations. WARNER, K. 2000. Forestry and Sustainable Livelihoods. 51. Unasylva. FAO Publications. RIBOT, J. C. 2002. Democratic Decentralisation of Natural Resources: Institutionalising Popular Participation. Cape Town: World Resources Institute. WORLD BANK 1997. Building Institutions for Capable Public Sector. New York: Oxford University Press.
https://openalex.org/W1224277973	https://zenodo.org/records/5553821/files/IJCHNV4N3A7-Domi%CC%81nguez.pdf	English	null	Impact of School-Based Interventions on Children’s Healthful Nutritional Outcomes (2009-2013): Implications for Future Research	International journal of child health and nutrition	2,015	cc-by	10,798	Address correspondence to this author at the Department of Psychology and Anthropology, Faculty of Nursing and Ocuppational Therapy, University of Extremadura Avda. de la Universidad s/n, 10003 Cáceres, Spain; Tel: 927257000/Ext. 51241; E-mail: prohlfsdomnguez@yahoo.es International Journal of Child Health and Nutrition, 2015, 4, 180-193 International Journal of Child Health and Nutrition, 2015, 4, 180-193 180 Impact of School-Based Interventions on Children’s Healthful Nutritional Outcomes (2009-2013): Implications for Future Paloma Rohlfs Domínguez Department of Psychology and Anthropology, Faculty of Nursing and Ocuppational Therapy, University of Extremadura, Cáceres, Spain Abstract: Objective: The objective was to review the literature on school-based interventions during compulsory education (published in 2009-2013) that may be effective in increasing healthful nutritional outcomes in children. Specific strategies and their limitations will be identified. Methods: A qualitative systematic analysis of seventeen school-based interventions applied to six-eleven year-old children, and published in peer-reviewed journals from 2009-2013 was carried out. Results: These studies taken as a whole used a variety of fourteen strategies, such as provision of nutritional and gardening education; repeated exposure to vegetables (V); peer and adult modelling; chefs going into schools to teach healthy nutritional issues. These studies showed four types of limitations: limitations derived from the experimental designs and experimental procedures used and limitations affecting participant samples and data collection. Fifteen recommendations for overcoming the targeted research limitations were identified, such as applying the intervention during longer time intervals; examining the effectiveness of these interventions in different ethnicities and socioeconomic groups; including larger participant samples; using randomized designs. Finally, six new working hypotheses to guide future studies are proposed here. For example, a small number of V offered might lead to insufficient opportunities to learn to like the flavour of V; schools’ previous experience with these interventions might distort children’s post- intervention data related to their nutritional outcomes; providing children with nutritional and gardening education might increase their fruit (F) selection, consumption and preferences and application of evaluative conditioning under more appropriate experimental conditions might increase child V consumption. Conclusion: seventeen school-based interventions were included. These studies taken as a whole used a variety of strategies (fourteen), and showed four types of limitations. Fifteen recommendations for overcoming the targeted research limitations as well as six new hypotheses are proposed here. Future research in this field should focus on overcoming its research limitations, such as the ones highlighted here, and building new working hypotheses, such as those proposed here. Keywords: Children, vegetables, fruits, consumption, schools. INTRODUCTION and modify the establishment of stable food preference patterns and nutritional habits [14-18]. It is well known that early vegetable consumption -a healthy nutritional habit - counteracts childhood obesity, one of the most worrying public health problems throughout the world [1], as well as health problems in adulthood, such as obesity [2], hypertension, heart disease and cancer [3-5]. Children’s intake rates remain, however, lower than national recommendations [6, 7], an average of 3 portions daily in Europe [7]. In Spain, primary education is compulsory for 6-12- year-old children [19], while children from birth until the age of 6 years may voluntarily attend schools specially designed for their early age (kindergarten) [20]. It has been estimated that at least 20% of the children who attend a Spanish school (either private or public) have lunch (the main meal in Spain) each day in school during the 8-9 month academic period [21]. Other countries, such as the United Kingdom, Ireland, Argentina, Sweden, and United States of America also provide children with food in the school dining rooms [22-26]. Therefore, the fact that many children receive at least one meal at school is not only exclusive to Spain, but is a worldwide social phenomenon. Children’s patterns of food intake are also linked to their food preferences [8, 9] and their liking for specific foods [10], those early preferences lasting until adolescence and even young adulthood [11-13]. However, these early likings and preferences for foods are not immutable. Indeed, early experiences (i.e. during childhood) with edibles, including V, may induce Because the school is an environment that provides many children with edibles, it may thereby shape childhood food preferences and eating habits [27-30]. © 2015 Lifescience Global Based on this brief overview, the following questions were considered: Which school-based Based on this brief overview, the following questions were considered: Which school-based E-ISSN: 1929-4247/15 Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 1 Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal 181 interventions during the compulsory education period may be effective in increasing healthful nutritional outcomes in children? Nutritional outcomes are considered here as being healthy when the involved edibles were fruits (F), V, or F and V (FV). Criteria for Considering Studies A literature search of published studies on food consumption by children in school settings was carried out. More specifically, studies were included if they dealt with several particular healthy nutritional outcomes (Table 1). Moreover, studies were included if they were school-based, that is, if they were clearly carried out, at least partially, in any physical space of the target school buildings, such as classrooms, dining halls or cafeterias. The focus of the present work concerns 6-11 year-old children exclusively, an age- range when children are typically required to attend compulsory education at schools worldwide [19]. Younger children, in contrast, may attend school in many European countries, such as Spain [20] and Germany [31] as well as the USA [32], but in a voluntary way, depending on parents' wishes. Children older than 11 years are approaching adolescence [33, 34], and the focus is childhood. A wide range of study designs were included in order to ensure the inclusion of a vast spectrum of intervention strategies that improved children's healthy nutritional outcomes. Indeed, randomized and non-randomized, controlled and non-controlled trials and school-based interventions with and without follow-up evaluations were included. Furthermore, this work includes only those studies that were published in peer-reviewed journals between 2009 and August 2013. Previous related work [35] focused on similar school-based interventions published between 2000 and 2008. Table 2: Inclusion and Exclusion Criteria Inclusion criteria 1. Intervention: school-based. 2. Targeted edibles: F, V or FV. 3. Setting: compulsory education (6-11 year old). 4. Outcomes: Table 1. 5. Years of publication: from 2009 to August 2013. 6. Study design: randomized and non-randomized, controlled and non-controlled trials and school-based interventions with and without follow-up examinations. Exclusion criteria 1. Not school-based intervention. 2. Not focused on FV. 3. Targeted children having a specific medical problem. 4. Children aged outside the age range 6-11 years. Inclusion criteria 1. Intervention: school-based. 2. Targeted edibles: F, V or FV. 3. Setting: compulsory education (6-11 year old). 4. Outcomes: Table 1. 5. Years of publication: from 2009 to August 2013. 6. Study design: randomized and non-randomized, controlled and non-controlled trials and school-based interventions with and without follow-up examinations. Exclusion criteria 1. Not school-based intervention. 2. Not focused on FV. 3. Targeted children having a specific medical problem. 4. Children aged outside the age range 6-11 years. Inclusion criteria 1. Intervention: school-based. 2. Targeted edibles: F, V or FV. 3. Setting: compulsory education (6-11 year old). 4. Outcomes: Table 1. 5. Years of publication: from 2009 to August 2013. 6. Study design: randomized and non-randomized, controlled and non-controlled trials and school-based interventions with and without follow-up examinations. Exclusion criteria 1. Not school-based intervention. 2. Not focused on FV. 3. Targeted children having a specific medical problem. 4. Children aged outside the age range 6-11 years. Search Strategy The search strategy involved Academic Search Complete, PubMed and ScienceDirect as the databases to identify published studies. Only studies that met all of the following criteria were included: population (6-11 year-old and thus school-aged children); interventions (school-based interventions, school-based nutrition programs, or school-based nutritional interventions); school food environment (school lunches, school meals, canteen, cafeteria, or food services); targeted edibles (FV); nutritional outcomes. INTRODUCTION What limitations are found in these school-based interventions? How can these limitations be overcome in future research efforts to develop viable strategies for promoting healthy nutritional outcomes in children? In an attempt to answer these questions, pertinent research on school-based interventions published from 2009 to 2013 is reviewed. This work begins with a description of the criteria for considering studies and the corresponding search strategy, along with a synthesized comparison of the study findings. This is followed by a discussion of the implications of the results of these studies for future research in this field (recommendations for overcoming identified research limitations and new working hypotheses). Indeed, this work aims to complement that earlier research. Studies were excluded if they were not school-based, if they targeted pupils showing medical specific problems and/or children older or younger than the above-mentioned targeted age range (Table 2). Table 1: Healthy Nutritional Outcomes Included 1. FV-related nutritional knowledge. 2. Preferences for FV. 6. FV consumption. 3. Liking for FV. 4. Attitudes toward FV. 5. FV-related abilities, such as FV identification. 6. Willingness to try FV. Table 1: Healthy Nutritional Outcomes Included Table 1: Healthy Nutritional Outcomes Included Synthesized Comparison of Study Findings A total of seventeen studies that met the inclusion criteria were included. A systematic analysis and descriptive review of these studies, rather than a International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Paloma Rohlfs Domínguez 182 , , , g Table 3: Characteristics of the Studies Included in this Review Reference Objectives Explicit Hypothesis Study Design, Follow-up Participants (Groups, Age and sample size) 1. [36] To increase children's knowledge of healthy nutritional habits; psychosocial variables. Associated with eating FV. And preferences for FV. Children of the intervention group would achieve these objectives in a more extent than the control group. Randomized trial. Controlled trial. No follow- up. One intervention group. One control group. 9-11 years n = 255. 2. [37] To examine how slicing apples and oranges affect children's selection and consumption of fruit. Non-randomized trial. Non-controlled trial. No follow- up. One intervention group. No control group. Kindergarten-9-10 years. n = 491. 3. [38] To examine the impact of a school garden on children's FV knowledge, preference and consumption. Children of the intervention group would be more likely to choose and eat vegetables during the lunch time after the intervention, in comparison with both control groups. Non-randomized trial. Controlled trial. No follow- up. One intervention group: Nutrition education and gardening. Two control groups: 1) Only nutrition education. 2) No intervention. 7-8 years. n = 115. 4. [39] To examine if repeated tastings of vegetables would increase children's liking for vegetables. To determine the number of tastings required for changing children's disliking for vegetables to liking them or liking them a lot. Repeated exposure to vegetables would increase children's liking for vegetables. Non-randomized trial. Non-controlled trial. No follow- up. Three intervention groups: 1) Children who did not like vegetables prior to the intervention. 2) Children who liked them prior to the intervention. 3) Children who liked them a lot prior to the intervention. No control group. 9-11 years. n = 360. 5. [40] To examine the effect of a comprehensive school intervention on nutritional knowledge, attitudes and behaviours. In comparison to the other children, the children most exposed to the intervention would show: 1) Greater increases in nutritional knowledge. 2) Positive changes in attitudes toward healthy eating behaviours, including preferences for FV. 3) More consumption of FV at the school and outside of the school. Non-randomized trial. Non-controlled trial. No follow- up. Synthesized Comparison of Study Findings Three intervention groups: 1) High-degree intervention development. 2) Medium-degree intervention development. 3) Low-degree intervention development. No control group. 9-11 years. n = 327. 6. [41] To improve the school environment and child nutrition by youth partnerships. Child peer leaders' implication in the whole intervention process (planning, implementation and evaluation of results) would improve their dietary intake, in comparison to the control group. Non-randomized trial. Controlled trial. No follow- up. Two intervention groups, which received a nutritional education program by the researchers and peers: 1) These peers implemented a FV snack stand in classrooms. 2) These peers implemented the access to the teachers' salad bar. One control group, which received only the standard classroom nutrition education without peers' implication. 9-10 years. Table 3: Characteristics of the Studies Included in this Review Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 183 183 (Table 3). Continued. Reference Objectives Explicit Hypothesis Study Design, Follow-up Participants (Groups, Age and sample size) 7. [42] To increase children’s liking for FV by offering them repeated opportunities to taste these items. Non-randomized trial. Non- controlled trial. Follow-up: at 4 th and 10 th post intervention months. Two intervention groups: 1) Children who disliked the targeted FV before the intervention. 2) Children who liked the targeted FV before the intervention. No control group. 6-9 years. n = 379. 8. [43] To examine the impact of a multi-component, theory-based intervention in children’s FV consumption, preferences, knowledge and body mass index. Children exposed to the intervention would show an increased FV knowledge, preferences and lunch time consumption as well as lower BMIs, in comparison to the control group. Randomized trial. Controlled trial. Follow-up: at the first-3th post- intervention years. One intervention group. These children were exposed to the intervention. One control group. These children were not exposed to the intervention. Mean age: 6.2 years. n = 149. 9. [44] To examine the effects of exposure to vegetables of different liking levels on children's liking for and intake of vegetables. 1) Exposure to vegetables would increase children's preferences for and intake of vegetables. 2) Changes in liking, derived from exposure to vegetables, would be transferred to similar non-exposed vegetables. 3) Children would show increases and decreases in liking for and intake of neutrally liked vegetables paired with liked and non-liked vegetables, respectively. Randomized trial. Controlled trial. Synthesized Comparison of Study Findings Follow-up: at the first-post- intervention day. Three intervention groups: 1) These children received a mixture of a neutrally liked vegetable and a liked vegetable. 2) These children received only a neutrally liked vegetable. 3) These children received a mixture of a neutrally liked vegetable and a disliked vegetable. One control group. These children did not receive any exposure intervention. 9-11 years. n = 345. 10. [45] To evaluate the effects of a multi-component nutrition education program on children's knowledge, attitudes and behaviours related to consumption of FV. Non-randomized trial. Controlled trial. No follow- up. Two intervention groups: 1) Children received an educational program. Teachers and parents were involved in the intervention as role models for behaviour change. 2) Children received an educational program, but teachers and parents were not involved in the intervention. One control group. These children did not receive the educational program. 8-11 years. n = 399. 11. [46] To examine the impact of an educational program together with repeated food exposures on children's ability to identify FV and willingness to try FV. Children who received the nutritional education program together with food exposures would be better at identifying and more willing to try FV, compared to other children. Non-randomized trial. Non-controlled trial. No follow- up. Two intervention groups: 1) Children who received the educational program plus repeated FV exposures. 2) Children who only received the educational program. No control group. 5-6 years 184 International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Paloma Rohlfs Domínguez 184 (Table 3). Continued. Reference Objectives Explicit Hypothesis Study Design, Follow-up Participants (Groups, Age and sample size) 12. [47] To examine the impact of the so called CHANGE intervention on children's diet quality, thus preventing children's unhealthy weight gain. Children exposed to the intervention would improve their diet quality, in comparison with other children. Randomized trial. Controlled trial. No follow- up. One intervention group. These children were exposed to the intervention. One control group. These children were not exposed to the intervention. 6-12 years. n = 432. 13. [48] To examine the effects of chefs going into schools to teach children to prepare and taste healthy food on children's food preparation and consumption as well as cooking confidence. Randomized trial. Controlled trial. No follow- up. One intervention group. These children were exposed to the intervention. One control group. These children were not exposed to the intervention. 9-11 years. Synthesized Comparison of Study Findings n = 169. 14. [49] To examine the effectiveness of the choice strategy on children's vegetable consumption. Children provided with choice would consume more vegetables than children not provided with choice. Children having choice availability continuously during the meal would show a higher vegetable intake than those who had choice availability only once. Randomized trial. Controlled trial. No follow- up. Two intervention groups: 1) Children chose the vegetable to ingest once before the meal. 2) Children chose the vegetable during the meal, whenever they made a bite. One control group. These children could not choose the vegetable. 4-6 years. n = 150. 15. [50] To examine the effectiveness of adult modelling (the teacher being the exposure model) on children's fruit intake. Randomized trial. Controlled trial. Follow-up: at the first post- intervention year. Two intervention groups: 1) Children were exposed only to an educational program. 2) Children were exposed only to adult modelling. One control group. These children were not exposed to any intervention. 9 years. n = 184. 16. [51] To examine the effectiveness of integrating animal cartoon characters into a comprehensive nutritional education program in children's FV consumption and physical activity. Non-randomized trial. Non-controlled trial. No follow-up result assessment. One intervention group. These children were exposed to the intervention. 8-11 years. n = 4,128. 17. [52] To examine the effectiveness of peer modelling combined with rewarding in children's FV. Non-randomized trial. Non-controlled trial. No follow-up result assessment. Three intervention groups: 1) Children who consumed no fruit at pre-intervention base line. 2) Children who consumed no vegetable at pre-intervention base line. 3) Children who consumed FV at pre-intervention base line. All these children were exposed to the intervention. No control group. 6-11 years. n = 253. Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 185 Impact of School-Based Interventions on Children’s Healthful Nutritional 185 (Table 3). Continued. Country, City, School physical space Procedures Results (they are significant) Limitations USA; Southeast Louisiana. Classrooms of different public schools. Provision of nutritional knowledge aimed to learn the physiology of digestion and the importance of consuming healthy foods, such as FV. Modification of children's social environment leading to encourage to consume FV. In comparison to control children, children of the intervention group showed an increase in their healthy nutritional knowledge, and the psychosocial variables associated with eating FV were improved. Synthesized Comparison of Study Findings However, their preferences for FV did not change. The program had a relatively short, 12- week duration. This may be insufficient time for changing children's food preferences. Follow-up assessment of the program was not examined, so long-term effects of the program are unknown. The majority of children were black and resided in low-income households, so results cannot be generalized to other socioeconomic groups and ethnicities. USA; Rural Kentucky. One school cafeteria. The kitchen staff was asked to serve whole and sliced apples and oranges in the usual manner. On day 1, children could choose the sliced fruits among other food options. On day 2, children could choose the whole fruits among other food options. Slicing fruit encouraged children's fruit selection and consumption, only in the case of oranges. The greatest slicing effect was observed among the younger children. There were no independent subject and food samples, so different factors from slicing may have (also) influenced the results. Results cannot be generalized to other populations. USA; Southeastern. Classrooms and garden of one school. Provision of nutritional and gardening education. The first hypothesis was fulfilled. However, the intervention group and the first control group showed, with no significant differences between both groups, greater improvements in nutrition knowledge than the second control group. There was no impact on children’s fruit-related knowledge, preferences and consumption. The study did not use a randomized design. The sample size was not as large as desirable. The study did not include a follow-up assessment to determine long-term results. Participants were predominantly white, second-grade population, and results should not be generalized to other populations. USA; South-eastern Louisiana. The cafeterias and classrooms of four low-income public schools. Provision of a vegetable tasting program combined with a nutritional education program. Liking for the majority of the targeted vegetables was increased in the first group of children. No changes in liking for the vegetables were observed in the other two groups. Children were 9-11 year old and attending low-income schools of South-eastern Louisiana. The 80% of them were black, so results should not be generalized to other populations. Questionnaires were used to collect information on children, and social desirability might have biased the results. There was no control group. A random design was not used. Lower rate of participation of children of the first group. USA; California. Classrooms; dining halls and gardens of different schools. Synthesized Comparison of Study Findings A combined provision of nutritional lessons, cooking and garden classes, as well as exposure to FV. There was no difference in nutritional knowledge nor attitudes toward healthy eating behaviours by intervention degree. The most increases in preferences for FV were found in the children of the high-degree intervention development. These children also showed significant increases in FV consumption, but only at the school, while children of the low-degree intervention development showed a decrease in this variable Nor control nor random design was used. Some schools had advantage over other schools in implementing the intervention because of their greater experience in this program. The youngest children had difficulties in providing researchers the information required. Some of the information was collected only in working days, weekends having been omitted. The intervention was not applied at home The intervention was not applied at home. 186 International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Paloma Rohlfs Domínguez 186 (Table 3). Continued. Country, City, School physical space Procedures Results (they are significant) Limitations USA; rural East Tennesee. Classrooms of different schools. Provision of taste-related FV experiences by child peers and/or adults. The first intervention group showed an increased fruit consumption. The second intervention group showed a reduced energy intake. The second intervention group showed a reduced fruit and vegetable intake, due to problems related to the measurement of dietary consumption. USA; Southeast Louisiana. Cafeteria of low-income schools. Repeated exposure to FV during 8 weeks. Children of the first group showed an improvement of their liking for FV after the intervention and maintained this improvement at both follow-up assessments. There were some gender and grade differences at each assessment time. The number of exposures required for observing these increases in liking for FV were two and three, respectively. Children of the second group maintained their liking status at each assessment time, and there were no gender or grade differences. Participants were first, 2th and 3th graders from low-income schools, so results should not be generalized to other populations. Social desirability could have influenced children’s responses. There was no control group. USA; North-eastern. Classrooms, lunchrooms of different schools and children’s homes. Provision of the following components for promoting FV consumption: 1) Loudspeaker announcements. 2) Instructional material (DVD). 3) Contingent rewarding on a bite of FV. 4) Take-home activity books. The sample size was not as large as desirable, which may affect results’ generalization. Synthesized Comparison of Study Findings In comparison to the control group, the intervention group increased the FV consumption, although this effect declined across time. Preferences for FV and BMIs did not change between groups. There were persistent increases on children’s FV knowledge. The sample size was not as large as desirable, which may affect results’ generalization. Denmark; Copenhagen. Classrooms. Repeated exposure to vegetables in the context of an evaluative conditioning. In comparison to the control group, liking for most vegetables decreased after the intervention, but tended to recover somewhat during the follow-up. Vegetable intake decreased after the intervention. Intake quantity varied across the type of vegetable servings. Indeed, children consumed more of neutrally liked vegetables when these items were paired with a liked vegetable than when served alone or together with a disliked vegetable. Most of the children were Caucasian and middle class, so results should not be generalized to other populations. All classes received similar, but alternating serving orders of vegetables at liking test days, thus leading to confounding between serving order and intervention group. There could have been peer influences affecting the data. There could have been some overestimations of the intake data, in the case of children who dropped their vegetables on the floor. USA; Los Angeles. Classrooms. Adult modelling. Nutrition education sessions. Interactive activities. Neither of the 2 intervention groups showed an increased FV consumption. Neither of the 2 intervention groups showed an increased availability of FV at home. Only children of the first intervention group showed increases in knowledge, attitudes and beliefs toward vegetables. The parent/teacher influence on children's FV attitudes increased only in the first group. Participant groups represented only a few of some specific ethnic groups, so results should not be generalized to other populations. The small size of the firs intervention group was to small to observe changes in FV consumption. There was no follow-up result assessment. There was no intervention on the school food environment, this resulting in no behavioural change in vegetable consumption. All classes received similar, but alternating serving orders of vegetables at liking test days, thus leading to confounding between serving order and intervention group. There could have been some overestimations of the intake data, in the case of children who dropped their vegetables on the floor. Participant groups represented only a few of some specific ethnic groups, so results should not be generalized to other populations. Synthesized Comparison of Study Findings The small size of the firs intervention group was to small to observe changes in FV consumption. There was no follow-up result assessment, so it is not known, whether the results found are sustained in the long-term. There was no follow-up result assessment. There was no intervention on the school food environment, this resulting in no behavioural change in vegetable consumption. Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 187 (Table 3). Continued. Country, City, School physical space Procedures Results (they are significant) Limitations USA; Williamsburg James City. The classrooms of two different schools. An educational program, which included interactivities about healthy eating and physical activity. Repeated exposure to FV. All children were better able to identify FV. Only the children of the first intervention group were more willing to try FV. There were no changes in children's identification abilities or willingness to try vegetables. Children may have had insufficient opportunities to learn to like the flavour of the vegetables, due to the small number of vegetables offered. There was no follow-up result assessment, so effects of the intervention over the long-term are unknown. USA; rural areas of California, Kentucky, Mississippi and South Carolina. Classrooms and cafeterias of different schools; children's homes. Changes in the foods offered in the cafeteria: more FV and whole grains and less added-sugar and saturated-fat products. Educational program based on the social- cognitive theory. Parent and community outreach components. In comparison to the control group, children of the intervention group showed an increased consumption of vegetables and combined FV, as well as a trend to consume more fruits. They also showed a reduced average daily dietary glycaemic index. There were no differences in children’s consumption of whole grains. Results are not generalizable to other populations. Many children who agreed to participate in the study failed to complete a second survey at the end of the intervention. England; Est London, West London, North West England (near Manchester and Liverpool) and Midlands. Classrooms of different schools. Instructional lessons. Sensory experiences focused on taste. Practical cooking sessions with a chef. In comparison to the control group, the intervention group showed gains in skills and confidence to prepare and ask for the ingredients to be purchased for use in home. These children also showed an increased vegetable consumption. There was no follow-up result assessment, so it is not known, whether the results found are sustained in the long-term. Spain; Granada. Dining halls of different schools. Provision of choice availability at the lunch time. Children of both intervention groups ate more vegetables than the control group. No control group was used. Only one school participated in the study, which may affect the generalization of results. The socioeconomic status or ethnicity in schools with more cultural diversity may impact the results. No follow-up assessment was carried out to check the stability of results in the long-term. There was no follow-up result assessment. There were no differences between both choice conditions. There was no follow-up assessment, so it is not known, whether the results found are sustained in the long-term. Children's motivation (liking or preference) for the targeted vegetables during and after the meal was not measured. Children's attentional focus during the meal was neither measured. Greece; Chipre. Classrooms. Provision of a nutritional educational program. Adult modelling. In comparison to the control group, both intervention groups exhibited a higher fruit intake. At 1-year follow-up, that increase remained only for the children of the 2th intervention group. The misreported food information: food record during weekends was omitted. Parents reported information for their children, but they may not know exactly what their children ate outside the school. The results were obtained from a single school district, so results are not generalizable to other populations. USA; Minessota. Classrroms and cafeteris. Children's exposure to animal cartoon characters. Provision of nutritional lessons. Addition of FV to the menus offered in the cafeterias. Self-reported consumption of FV was improved. The number of sessions attended and improvements in fruit consumption was weakly but yet significantly related. No control group was used. The survey used self-reported dichotomous questions. USA; Northen Utah. Cafeteria. Children watched videos in which heroic peers were eating FV, and received a reward for eating FV. Children's FV intake increased after the intervention. This effect was more discriminable among children of the first intervention group. No control group was used. Only one school participated in the study, which may affect the generalization of results. The socioeconomic status or ethnicity in schools with more cultural diversity may impact the results. No follow-up assessment was carried International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Paloma Rohlfs Domínguez 188 of the psychosocial variables associated with eating FV is also found, but there were no changes in preferences for FV [36]. These authors therefore recommend that future research should identify ways to increase children’s tasting and exposure to these healthy foods, in addition to those strategies, in order to change their preferences for FV. Following this research line, at least three studies have obtained positive results. Wang et al. [40] found that provision of nutritional lessons, combined with cooking and gardening classes, as well as exposure to FV, increases children's consumption of and preferences for FV. DISCUSSION AND IMPLICATIONS FOR FUTURE RESEARCH Based on the review of the included studies, the answers to the three questions formulated in the introductory section are presented below, along with several implications for future research. There was no follow-up result assessment. Nutritional knowledge or attitudes toward healthy eating are not affected by this intervention, however. The authors of this study suggest that the potential advantage of some of the schools over others in the implementation of the intervention (because of previous relevant experience) may have distorted children’s post-intervention data related to their knowledge and attitudes toward healthy eating. That is, this previous experience could have resulted in a ceiling effect. Therefore, future school-based interventions should also control this variable. Secondly, a school-based intervention that combined an educational program and repeated exposure to FV improves children's ability to identify FV as well as willingness to try FV [46]. These variables are likely to promote FV consumption in children, a hypothesis that should be tested in the future. Thirdly, exposing children to animal cartoon characters within a comprehensive nutritional education program consisting of the provision of nutritional lessons, plus addition of FV offered in the school cafeterias, improves children's self-reported FV and F consumption [51]. meta-analysis, was conducted because of the marked heterogeneity in the types of school-based interventions carried out. Furthermore, the studies used a wide range of methods and outcomes to assess effects on children's nutritional outcomes. The included studies were analyzed according to the three research questions formulated in the introductory section: which school-based interventions executed during that period may be effective in increasing these healthful nutritional outcomes in children? What limitations do these studies present? How would it be possible to overcome these limitations in future investigations of viable strategies for promoting child healthy nutritional outcomes? (Table 3). School-Based Interventions Executed During the Compulsory Education Period that may be Effective in Increasing Healthful Nutritional Outcomes in Children 4, No. 3 189 tastings [39, 42]. Therefore, it is suggested that future research should examine whether adding to the targeted V some sweetener or even a sweet F, that is, using the so-called evaluative conditioning experimental paradigm –within this paradigm, a neutral or even rejected stimulus is paired with an already liked stimulus- (see Gast, Gawronski and De Houwer [53] for a review) might increase children’s liking for V that is already liked (but see Olsen et al. [44] for a different point of view). Indeed, as it is have been pointed out in a previous section, these authors found no increases in liking for or intake of already liked V motivated by evaluative conditioning. Authors of this study attribute this result, either to children’s limited attention focused on the targeted V, due to peer influence, or to children’s V-related boredom sensation or the large size of V servings. Future research should examine evaluative conditioning related to child V consumption under more appropriate experimental conditions. demonstrated to increase children's V intake as well as their skills and confidence to prepare and ask for the ingredients to be purchased for use at home [48]. Finally, promotion of FV consumption by means of loudspeaker announcements, instructional material (DVD), contingent rewards, and take-home activity books has been found to increase children's FV consumption, although this effect declines across time (three months later) [43]. This kind of intervention has also demonstrated increases in children's FV knowledge, but does not change their preferences for FV, however [43]. These authors attribute this result to the fact that they did not make the FV more palatable. Therefore, the effectiveness of increased palatability of the FV served in enhancing children’s preferences for those food items should be tested in the future. As can be seen, the effectiveness of a school-based intervention in increasing healthful nutritional outcomes in child population relies on the specific strategy or strategies used. Across the included studies, fourteen strategies have been used (Table 4). In addition to the strategies that have been discussed so far in this section, there are four more that deserve readers’ attention. The first involves manipulation of the food environment at schools. For example, offering sliced F in the cafeteria has been shown to encourage children's F selection and consumption of oranges, especially among younger children, but does not impact consumption of apples [37]. School-Based Interventions Executed During the Compulsory Education Period that may be Effective in Increasing Healthful Nutritional Outcomes in Children Provision of loudspeaker announcements, instructional material (DVD), contingent rewarding on a bite of FV, and take-home activity books. 10. Provision of an educational program combined with repeated exposure to FV. 11. Offering more FV in the school cafeteria. 12. Chefs going into schools to teach healthy nutritional issues. 13. Provision of the opportunity to choose the V to ingest. 14. Exposure to animal cartoon characters within a comprehensive nutritional education program. 3. Provision of nutritional and gardening education. 9. Provision of loudspeaker announcements, instructional material (DVD), contingent rewarding on a bite of FV, and take-home activity books. 10. Provision of an educational program combined with repeated exposure to FV. 12. Chefs going into schools to teach healthy nutritional issues. 14. Exposure to animal cartoon characters within a comprehensive nutritional education program. School-Based Interventions Executed During the Compulsory Education Period that may be Effective in Increasing Healthful Nutritional Outcomes in Children Therefore, future research should develop additional age- and F-specific strategies to encourage child F consumption. Similarly, offering children more FV and whole grains with less added-sugar and saturated-fat products in the school cafeteria has been found to increase children's V and FV consumption while reducing dietary glycaemic index [47]. Furtheremore, providing children with the opportunity to choose the V to ingest once before the meal or whenever they take a bite during the meal has been demonstrated to increase their V intake [49]. The second strategy refers to others’ influence. Provision of a FV snack stand in classrooms by peers increased children's F consumption [41]. Furtheremore, peer modeling has been observed to increase children's FV intake [52], and adult modeling also increased children's knowledge, attitudes and beliefs toward FV, although it did not increase their FV consumption nor availability of FV at children's home [45]. Such modelling also increases children's F consumption, with this effect remaining across time (one year later) [50]. The third strategy is associated with the influence of chefs. School programs in which chefs teach children to prepare and taste healthy food has been Table 4: Essential Strategies Used by School-Based Interventions Identified 1. Provision of nutritional knowledge combined with modification of children's social environment. 2. Slicing F. 3. Provision of nutritional and gardening education. 4. Repeated exposure to V. 5. Provision of nutritional lessons, combined with cooking and gardening classes, and exposure to FV. 6. Provision of a FV snack stand in classrooms by peers. 7. Peer modelling. 8. Adult modelling. 9. Provision of loudspeaker announcements, instructional material (DVD), contingent rewarding on a bite of FV, and take-home activity books. 10. Provision of an educational program combined with repeated exposure to FV. 11. Offering more FV in the school cafeteria. 12. Chefs going into schools to teach healthy nutritional issues. 13. Provision of the opportunity to choose the V to ingest. 14. Exposure to animal cartoon characters within a comprehensive nutritional education program. 1. Provision of nutritional knowledge combined with modification of children's social environment. 2. Slicing F. 3. Provision of nutritional and gardening education. 4. Repeated exposure to V. 5. Provision of nutritional lessons, combined with cooking and gardening classes, and exposure to FV. 6. Provision of a FV snack stand in classrooms by peers. 7. Peer modelling. 8. Adult modelling. 9. School-Based Interventions Executed During the Compulsory Education Period that may be Effective in Increasing Healthful Nutritional Outcomes in Children School-based interventions focused, at least partially, on providing children with nutritional education programs are effective in increasing healthful nutritional outcomes. Indeed, it has been demonstrated that children provided with nutritional and gardening education are more likely to choose and eat a variety of V (i.e. carrots, broccoli, cabbage and zucchini) during lunch time. This strategy also increases their preferences for these items. Their nutritional knowledge also improves if they are exposed to this combination of strategies, but to the same extent as when children only receive nutritional education [38]. Furthermore, this type of intervention does not impact children’s selection, consumption or preferences for F, when only one F (i.e. blueberry) is offered [38]. Therefore, these authors recommend that future research should examine the same intervention, but using a variety of F. Providing children with nutritional knowledge, such as the physiology of digestion and the need for consuming healthy foods, using a language adapted to their age, together with modification of the social environment by, for instance, encouraging children to participate in school assemblies where the message to consume FV is delivered, also result in an increase in healthy nutritional knowledge. Improvement Other effective school-based interventions are those focused on repeated exposure to FV as the only strategy applied. Repeated exposure increases liking for V and FV in children who did not like the targeted items before this repeated taste-related experience [39, 42]. This result was constant across time (four and ten months later) and subject to gender and grade variables [42]. In the case of F, fewer exposures –two times- are required for observing an increase in liking for these items, in comparison to the number of exposures required to increase liking for V –three times- [42]. The authors attribute this effect to children’s innate liking and rejection for the sweet and bitter tastes of F and V, respectively. In contrast, repeated exposure does not change liking for V and FV in children who already liked V before the repeated s on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 18 Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 n’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. Limitations of these Studies and ways to Overcome them in Future Research [38], Lakkakula et al. [39], Lakkakula et al. [42], Olsen et al. [44], Prelip et al. [45], Cohen et al. [50], Perikou et al. [53], and Wengreen et al. [52]. Consequently, the effectiveness of these interventions should be examined in different ethnic and socioeconomic groups in the future. The second most common limitation of this type is related to the participant sample size. This was not as large as desirable in several studies, such as those by Parmer et al. [38], Lakkakula et al. [39], Hoffman et al. [43], Prelip et al. [45], and Cohen et al. [47]. Finally, previous experience of some schools with these interventions may have resulted in advantages over other schools in implementing the intervention, for example, in the study by Wang et al. [40]. Hence, this variable should be controlled in future school-based interventions. 2. Limitations derived from the experimental procedures used. Within such limitations, Wang et al. [40] highlight that their intervention was not applied at home, thus the resulting increases in FV consumption observed among some students concern only foods eaten during school hours, and cannot be generalized to foods consumed at home. None of the studies described here targeted the home food environment, so this limitation also affects them. Prelip et al. [45] highlight, in turn, that their intervention did not target the school food intervention, which may explain the lack of changes in children's V consumption. Future studies should combine both home and school food environments. Thatchildren might have had insufficient opportunities to learn to like the flavor of the V during the intervention that included only 4 V offerings, is another limitation hypothesized by Schindler et al. [46]. 4. Limitations concerning data collection. In some studies, questionnaires were used to collect information on children (i.e. Lakkakula et al. [39]); thus social desirability might have biased the results [39]. Moreover, in the study by Leines et al. [51], the survey used self-reported dichotomous questions, which limits children's provision of information. As Wang et al. [40] have argued, young children have difficulties in providing researchers with required information because of their still limited language capacities. For these reasons, development of methods of data collection adapted to children's age and language are warranted. In some studies (i.e. Wang et al. [40]; Perikkou et al. Limitations of these Studies and ways to Overcome them in Future Research Future research should therefore examine the effectiveness of these interventions in the long-term by adding at least one follow-up assesment at any post- intervention month or even year. A second limitation relates to the lack of control groups with which to compare the results of the experimental groups. This limitation is found in the studies by Swanson et al. [37], Lakkakula et al. [39], Leines et al. [51], Wengreen et al. [52], and Wang et al. [40]. These comparisons are essential for ascertaining whether the effectiveness of a school-based intervention may be exclusively attributed to manipulation of the independent variable, in this case, the type of strategy used for improving children’s healthy nutritional outcomes. Consequently, a better examination of the efectivenness of these school-based interventions by adding control groups is warranted. The third limitation involves the lack of randomized experimental designs. This limitation exists in the studies by Parmer et al. [38], Lakkakula et al. [39], and Wang et al. [40]. Randomized experimental designs also ensure attribution of the effectiveness of a school-based intervention to the strategy used. To better examine the effectiveness of these school-based interventions randomized designs are thus necessary. Finally, short durations of the interventions may limit their effectiveness. Tuuri et al. [36] argue that the relatively short duration of their program - 12-weeks - was insufficient to change children's outcomes. It is therefore recommended to test their intervention in the future, but with a longer duration. Hence, this hypothesis should be tested in the future. Olsen et al. [44], on their part, affirm that all participants received similar, but alternating servings of V at liking test days, thus leading to confounding between serving order and intervention group, which has to be avoided in future studies. Finally, Rohlfs Domínguez et al. [49] underline that they did not assess children's motivation (liking or preference) for the targeted stimuli, nor attentional focus during and after their intervention, and therefore endorse the need to assess these variables in the future, to determine why children eat more V under choice conditions. 3. Limitations affecting participant samples. The most common of this type of limitation concerns the limited generalization of the results due to the fact that participants were mostly of one ethnicity (i.e. white or black) or socioeconomic group (i.e. low-income families). This limitation affects the interventions by Tuuri, et al. [36], Swanson et al. [37], Parmer et al. Limitations of these Studies and ways to Overcome them in Future Research Limitations of these studies can be placed into four categories. 1. Limitations derived from the experimental designs used. The most common of this type of limitation International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 Paloma Rohlfs Domínguez 190 concerns the lack of follow-up assesments to check whether the intervention remains effective in the long- term. This limitation affects the studies by Tuuri, et al. [36], Prelip et al. [45], Schindler et al. [46], Caraher et al. [48], Rohlfs Domínguez et al. [49] and Wengreen et al. [52]. Future research should therefore examine the effectiveness of these interventions in the long-term by adding at least one follow-up assesment at any post- intervention month or even year. A second limitation relates to the lack of control groups with which to compare the results of the experimental groups. This limitation is found in the studies by Swanson et al. [37], Lakkakula et al. [39], Leines et al. [51], Wengreen et al. [52], and Wang et al. [40]. These comparisons are essential for ascertaining whether the effectiveness of a school-based intervention may be exclusively attributed to manipulation of the independent variable, in this case, the type of strategy used for improving children’s healthy nutritional outcomes. Consequently, a better examination of the efectivenness of these school-based interventions by adding control groups is warranted. The third limitation involves the lack of randomized experimental designs. This limitation exists in the studies by Parmer et al. [38], Lakkakula et al. [39], and Wang et al. [40]. Randomized experimental designs also ensure attribution of the effectiveness of a school-based intervention to the strategy used. To better examine the effectiveness of these school-based interventions randomized designs are thus necessary. Finally, short durations of the interventions may limit their effectiveness. Tuuri et al. [36] argue that the relatively short duration of their program - 12-weeks - was insufficient to change children's outcomes. It is therefore recommended to test their intervention in the future, but with a longer duration. concerns the lack of follow-up assesments to check whether the intervention remains effective in the long- term. This limitation affects the studies by Tuuri, et al. [36], Prelip et al. [45], Schindler et al. [46], Caraher et al. [48], Rohlfs Domínguez et al. [49] and Wengreen et al. [52]. CONCLUDING REMARKS future studies. Results reported by Olsen et al. [44] could have reflected peer influences and the fact that some children dropped the targeted items on the floor. Finally, Perikkou et al. [50] affirm that in their study parents reported information for their children, but they may not have known exactly what their children ate outside the school. Therefore, peer influences, parents' ignorance regarding what their children eat outside the school and data loss due to children's actions associated with dropping the targeted stimuli should be avoided or compensated in future school-based interventions. Ways to overcome these limitations are summarized in Table 5. The present review aimed to identify school-based interventions executed during the compulsory education period that may be effective in increasing healthful nutritional outcomes in children, along with their associated limitations and implications for future research. Seventeen different school-based interventions that are effective in increasing children's nutritional outcomes during this period were identified. Taken as a whole, these school-based interventions vary according to the specific strategy used to promote healthy nutritional outcomes in children and evidence four types of limitations. Table 5: Strategies for Overcoming the Research Limitations of School-Based Interventions Identified Table 5: Strategies for Overcoming the Research Limitations of School-Based Interventions Identified I recommend fifteen ways to overcome the limitations described above, and present six new hypotheses to be assessed in the future. 1. Add at least one post-intervention follow-up assessment. 2. Add control groups. 3. Use randomized designs. 4. Apply the intervention during longer time intervals. 5. Include the home and school food environments. 6. Offer children a higher number of V. 7. Assess children's motivation (liking or preference) for the targeted stimuli and attentional focus during and after the intervention. 8. Examine the effectiveness of these interventions in different ethnicities and socioeconomic groups. 9. Include larger participant samples. 10. Control schools’ previous experience with interventions. 11. Use data-collection methods suited to children. 12. Collect information related to children’s nutritional outcomes also in weekends. 13. Control peer influences on children’s nutritional outcomes. 14. Avoid children's actions associated with dropping the targeted stimuli on the floor. 15. Avoid parents’ ignorance regarding what do their children eat outside the school. ACKNOWLEDGEMENTS 14. Avoid children's actions associated with dropping the targeted stimuli on the floor. 15. Avoid parents’ ignorance regarding what do their children eat outside the school. This work was developed through the efforts from Dr. Richard H Porter in making interesting suggestions and refining the English language of the text. Based on this discussion, I propose several new working hypotheses (Table 6). CONCLUDING REMARKS The principal contribution of this review is to enhance awareness within the scientific community of the need to conduct further research on strategies that promote children’s healthy nutritional outcomes as well as to overcome research-related limitations, even if this is not possible currently. 3. Use randomized designs. 4. Apply the intervention during longer time intervals. 5. Include the home and school food environments. 6. Offer children a higher number of V 7. Assess children's motivation (liking or preference) for the targeted stimuli and attentional focus during and after the intervention. 8. Examine the effectiveness of these interventions in different ethnicities and socioeconomic groups. Finally, the suggestions for the future presented here are probably not viable currently, but they may inspire other researchers to find solutions in the future for the obstacles that hinder viability of these suggestions currently. 9. Include larger participant samples. 10. Control schools’ previous experience with interventions. 11. Use data-collection methods suited to children. 12. Collect information related to children’s nutritional outcomes also in weekends. There is no conflict of interest to declare. There is no conflict of interest to declare. Research 1. The provision to children of nutritional and gardening education might increase their F selection, consumption and preferences. 2. Schools’ previous experience with these interventions might distort children’s post-intervention data related to their nutritional outcomes. 3. Application of evaluative conditioning under more appropriate experimental conditions might increase child V consumption. 4. Development of age- and F-specific strategies might encourage child F consumption. 5. Increasing the palatability of the FV served might increase children’s preferences for FV. 6. A small number of V offered might lead to insufficient opportunities to learn to like the flavour of V. Limitations of these Studies and ways to Overcome them in Future Research [50]), information on children's nutritional outcomes was collected only on working days, hence information should also be collected on weekends in Impact of School-Based Interventions on Children’s Healthful Nutritional International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 1 International Journal of Child Health and Nutrition, 2015, Vol. 4, No. 3 1 Impact of School-Based Interventions on Children’s Healthful Nutritional 191 CONFLICT OF INTEREST Table 6: New Hypotheses Derived from the Targeted School-Based Interventions for Future Research Table 6: New Hypotheses Derived from the Targeted School-Based Interventions for Future Research There is no conflict of interest to declare. 1. 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Prev Med 2011; 52(5): 370-375. http://dx.doi.org/10.1016/j.ypmed.2011.02.013 Published on 01-09-2015 Received on 15-06-2015 Accepted on 17-08-2015 http://dx.doi.org/10.6000/1929-4247.2015.04.03.7 http://dx.doi.org/10.6000/1929-4247.2015.04.03.7
https://openalex.org/W4391783390	https://link.springer.com/content/pdf/10.1007/s11356-024-32028-3.pdf	English	null	Experimental strategy for the preparation of adsorbent materials from torrefied palm kernel shell oriented to CO2 capture	Environmental science and pollution research international	2,024	cc-by	14,883	* Marlon Cordoba‑Ramirez mfcordoba@uniguajira.edu.co Abstract In this study, an experimental strategy to obtain biochar and activated carbon from torrefied palm kernel shell as an efficient material for CO2 removal was evaluated. Biochar was obtained by slow pyrolysis of palm kernel shell at different tempera- tures (350 °C, 550 °C, and 700 °C) and previously torrefied palm kernel shell at different temperatures (220 °C, 250 °C, and 280 °C). Subsequently, activated carbons were prepared by physical activation with CO2 from previously obtained biochar samples. The CO2 adsorption capacity was measured using TGA. The experimental results showed that there is a correlation between the change in the O/C and H/C ratios and the functional groups –OH and C=O observed via FTIR in the obtained char, indicating that both dehydration and deoxygenation reactions occur during torrefaction; this favors the deoxygenation reactions and makes them faster through CO2 liberation during the pyrolysis process. The microporous surface area shows a significant increase with higher pyrolysis temperatures, as a product of the continuous carbonization reactions, allowing more active sites for CO2 removal. Pyrolysis temperature is a key factor in CO2 adsorption capacity, leading to a CO2 adsorp- tion capacity of up to 75 mg/gCO2 for biochar obtained at 700 °C from non-torrefied palm kernel shell (Char700). Activated carbon obtained from torrefied palm kernel shell at 280 °C (T280-CHAR700-AC) exhibited the highest CO2 adsorption capacity (101.9 mg/gCO2). Oxygen-containing functional groups have a direct impact on CO2 adsorption performance due to electron interactions between CO2 and these functional groups. These findings could provide a new experimental approach for obtaining optimal adsorbent materials exclusively derived from thermochemical conversion processes. Environmental Science and Pollution Research (2024) 31:18765–18784 https://doi.org/10.1007/s11356-024-32028-3 Environmental Science and Pollution Research (2024) 31:18765–18784 https://doi.org/10.1007/s11356-024-32028-3 RESEARCH ARTICLE Experimental strategy for the preparation of adsorbent materials from torrefied palm kernel shell oriented to CO2 capture Marlon Cordoba‑Ramirez1,2 · Farid Chejne2 · Jader Alean1 · Carlos A. Gómez2 · África Navarro‑Gil3 · Javier Ábrego3 · Gloria Gea3 Received: 11 September 2023 / Accepted: 12 January 2024 / Published online: 13 February 2024 © The Author(s) 2024 3 Thermochemical Processes Group (GPT), Aragon Institute for Engineering Research (I3A), Universidad de Zaragoza, Edificio I+D, C/Mariano Esquillor s/n, 50018 Zaragoza, Spain Keywords Torrefaction · Pyrolysis · Activated carbon · Biochar · Porous structure · CO2 adsorption Introduction Growing concern about replacing fossil fuels and conven- tional polluting energy sources has generated interest in exploring new alternatives from renewable sources. Conse- quently, there has been a surge in the research and develop- ment of technologies aimed at mitigating pollutants such as CO2, CH4, N2O, and others, which contribute to atmos- pheric pollution and climate change. Rising global energy demand has been a significant contributor to the increased concentration of CO2 in the atmosphere. Consequently, vari- ous capture and storage technologies have been proposed as viable alternatives to reduce these emissions and prevent their release into the atmosphere. Responsible Editor: Tito Roberto Cadaval Jr Responsible Editor: Tito Roberto Cadaval Jr However, it is unclear what influence the activating agent (NaOH) could have on the increase in surface area, which is initially due to the increase in torrefaction temperature. Chen et al. (2017b) studied the influence of torrefaction on the physicochemi- cal characteristics of char during the pyrolysis of rice straw washed with pure water and unwashed. The results show that higher torrefaction temperatures result in higher solid yields after pyrolysis. This is mainly due to the increase in the fraction of fixed carbon that occurs because the torrefaction temperature increases, thus causing severe carbonization of some macromolecules with increasing temperature during the process. Similarly, Zheng et al. (2013) proposed a simpli- fied mechanism showing the effect of torrefaction on rapid cellulose pyrolysis. During torrefaction, the cellulose is depolymerized to form active cellulose, which successively undergoes polymerization and crosslinking reactions that lead to the formation of char. After a more severe pyrolysis, the crosslinking and the obtained char result in more solid structures, causing greater production of active carbon sites, which makes biochar prone to react with the molecules of the gasifying agent during activation (Wang et al. 2018). They also found, as did Zhang et al. (2016), that the surface area decreases with increasing torrefaction temperature after the pyrolysis process; this phenomenon can be attributed to changes in the volatile material content between the virgin and torrefied biomasses. The results of the proximate analy- sis indicate a lower volatile material fraction at higher tor- refaction temperatures. Although the reported literature has studied in detail the impact of torrefaction on biochar and activated carbon properties and composition that make it a promising pretreatment, there is still a lack of information related to the performance of these materials obtained from torrefied biomass in CO2 capture applications, which could be beneficiated with the structural changes promoted by the Different studies have focused their attention on the use of carbon materials from lignocellulosic wastes in the removal of these gases at different concentrations (Sharma et al. 2021; Lai et al. 2021; Fernandes et al. 2021; Ro et al. 2021; Aghel et al. 2022), considering important parameters such as the porosity developed, the functional groups present on the surface, and even additives that improve the performance during the process. Ma et al. (2021) conducted a compara- tive study of the performance of biochar samples for both CO2 and H2S removal in biogas streams. Responsible Editor: Tito Roberto Cadaval Jr 2019).f activated carbon have garnered significant attention for sev- eral reasons, including their abundance of renewable raw materials, making them particularly appealing (Li et al. 2008; Zhang et al. 2017; Cheng and Li 2018; Ranguin et al. 2021), and their surface and textural properties, such as high surface areas, diverse pore sizes, and surface chem- istry (Sethupathi et al. 2017; Gasquet et al. 2020; Scapin et al. 2021; Zhu et al. 2021). These materials can be obtained from agro-industrial lignocellulosic biomass residues, which can be transformed by thermochemical processes such as torrefaction and slow pyrolysis. Several agricultural wastes such as coffee husk (Mukherjee et al. 2022), coffee grounds (Laksaci et al. 2017; Chen et al. 2021), rice husk and rice straw (Chen et al. 2017a; Zhang et al. 2020; Gao et al. 2021), coconut shell (Arena et al. 2016; Gongxiang et al. 2022; Igh- alo et al. 2023), and palm kernel shell (Uemura et al. 2015; Md Zaini and Hassan 2018; Adilla Rashidi et al. 2021) have been proposed as precursors for carbon materials obtained by pyrolysis and torrefaction. Among these, palm kernel shell has gained special attention due to its availability (1.6 million ton/year of palm residues in Colombia) (Marrugo et al. 2016), and its physicochemical properties such as high lignin content, high carbon content, and high volatile matter content (Marrugo 2015), which make it suitable for thermo- chemical conversion through torrefaction and pyrolysis to obtain solid carbon materials (Vega et al. 2019).f Despite the existing knowledge about the benefits of tor- refaction to obtain liquids and gasses, little attention has been paid to the changes in biochar, including its porous structure, chemical composition, and performance in adsorp- tion applications and to obtain activated carbon. Zhang et al. (2016) investigated the effect of torrefaction on the yield and quality of pyrolyzed biochar with applications in the produc- tion of activated carbon. These authors propose the route “torrefaction–pyrolysis–activation (chemical)” to evaluate the yields of char, liquids, and gasses as well as the textural properties of activated carbon. They found that torrefaction improved the properties of the precursor (rice husk) as a fuel and had significant effects on the fixed carbon content of char obtained from pyrolysis, which is reflected in the final characteristics of activated carbon. Responsible Editor: Tito Roberto Cadaval Jr 1 Mechanical Engineering Program – DESTACAR Research Group, Faculty of Engineering, Universidad de La Guajira, km 3+354 via Maicao, 440001 Riohacha, Colombia 2 Department of Processes and Energy – Applied Thermodynamics and Alternative Energies Research Group, Faculty of Mines, Universidad Nacional de Colombia Sede Medellín, Cra. 80 No 65 – 223, 050034 Medellín, Colombia Porous adsorbent materials have emerged as a highly promising solution for removing pollutants in gas and aque- ous phases (El-Metwaly et al. 2022; Al-Hazmi et al. 2022; Almahri et al. 2023a). Among these materials, biochar and (012 3456789) Environmental Science and Pollution Research (2024) 31:18765–18784 18766 heating value, and biomass reactivity (Granados et al. 2022). In turn, it is possible to obtain bio-oils with a higher con- tent of phenolic compounds derived from lignin (Park et al. 2013; Pelaez-Samaniego et al. 2014).i activated carbon have garnered significant attention for sev- eral reasons, including their abundance of renewable raw materials, making them particularly appealing (Li et al. 2008; Zhang et al. 2017; Cheng and Li 2018; Ranguin et al. 2021), and their surface and textural properties, such as high surface areas, diverse pore sizes, and surface chem- istry (Sethupathi et al. 2017; Gasquet et al. 2020; Scapin et al. 2021; Zhu et al. 2021). These materials can be obtained from agro-industrial lignocellulosic biomass residues, which can be transformed by thermochemical processes such as torrefaction and slow pyrolysis. Several agricultural wastes such as coffee husk (Mukherjee et al. 2022), coffee grounds (Laksaci et al. 2017; Chen et al. 2021), rice husk and rice straw (Chen et al. 2017a; Zhang et al. 2020; Gao et al. 2021), coconut shell (Arena et al. 2016; Gongxiang et al. 2022; Igh- alo et al. 2023), and palm kernel shell (Uemura et al. 2015; Md Zaini and Hassan 2018; Adilla Rashidi et al. 2021) have been proposed as precursors for carbon materials obtained by pyrolysis and torrefaction. Among these, palm kernel shell has gained special attention due to its availability (1.6 million ton/year of palm residues in Colombia) (Marrugo et al. 2016), and its physicochemical properties such as high lignin content, high carbon content, and high volatile matter content (Marrugo 2015), which make it suitable for thermo- chemical conversion through torrefaction and pyrolysis to obtain solid carbon materials (Vega et al. Responsible Editor: Tito Roberto Cadaval Jr Therefore, a detailed study of biochar and activated carbon obtained from torrefied palm kernel shells for CO2 capture is necessary. This study aims to assess the impact of torrefaction as a pretreatment on palm kernel shells to produce biochar and activated carbon, which serve as efficient materials for CO2 capture. This research encompasses a compilation of experi- mental findings that examine alterations in thermal stability through thermogravimetric analysis, elemental composition, and porous structure via N2 and CO2 adsorption. In addi- tion, functional groups were analyzed using FTIR spectros- copy for both raw and torrefied palm kernel shells and their respective biochar and activated carbon. The acquired results offer a comprehensive understanding of the thermochemical conversion effects on palm kernel shells, encompassing tor- refaction and slow pyrolysis at various temperatures and the combination of these processes. These findings reveal cor- relations between the elemental and chemical compositions of functional groups, highlighting significant influences on deoxygenation reactions during torrefaction. Such reactions may alter potential active sites on char and activated carbon samples, consequently affecting the performance of CO2 capture. Furthermore, this study provides a crucial compara- tive analysis of porous structural changes across a spectrum of torrefaction and pyrolysis temperatures, extending to both char and activated carbon samples. The results derived from this experimental approach also open new avenues for opti- mizing the production process of carbonaceous adsorbent materials (biochar and activated carbon) designed for CO2 capture. For the torrefaction and pyrolysis experiments, a heat- ing rate of 10 °C/min was used. A nitrogen flow of 100 mL/min STP was used in all experiments with a residence time of 1 h. At the end of each process, samples were cooled by removing the metal spoon from the oven but keeping it within an excess section of the quartz tube with a continuous flow of nitrogen for 2 h until completely cooled, to avoid spontaneous combustion. p Subsequently, activated carbon was obtained from the biochar samples. The activated carbon was obtained from physical activation in CO2 atmosphere at 850 °C. For this, the same experimental device was used, programing the heating of the oven at 850 °C with a heating rate of 10°C/ min in N2 atmosphere with the sample placed in the excess section of the quartz tube. Responsible Editor: Tito Roberto Cadaval Jr This study pre- sented relevant results regarding the effect of N-containing functional groups on the surface of biochar samples, indicat- ing their beneficial effect on solid yield, porous structure, and CO2 retention.i To improve the performance of biomass and the final product of pyrolysis, some pretreatments are usually required before its conversion. Currently, pretreatment of biomass includes impregnation with acids and bases (Ahmed et al. 2016), impregnation with metals (Cai et al. 2016; Cho et al. 2017; He et al. 2018), and thermochemical processes such as torrefaction (Xu et al. 2018; Zeng et al. 2019; Sibiya et al. 2021). This process consists of partial pyrolysis of the biomass carried out in a short range of temperatures between 200 and 300 °C in an inert environment to remove mois- ture and some volatiles contained in the biomass (Nhuch- hen et al. 2014). Some advantages of torrefaction include improved thermal stability, equilibrium moisture absorption, Environmental Science and Pollution Research (2024) 31:18765–18784 18767 palm kernel shell preparation, temperatures of 220 °C, 250 °C, and 280 °C were selected. These samples were labeled as T220, T250, and T280, indicating that they were obtained through torrefaction at the proposed tem- peratures. Biochar prepared from raw palm kernel shell was obtained at temperatures of 350 °C, 550 °C, and 700 °C; these samples were labeled Char350, Char550, and Char700, indicating that these samples were obtained at the proposed temperatures. The specified temperatures (for torrefaction and pyrolysis process) were chosen with precision, considering previous reports affirming sig- nificant structural changes within the biomass at these temperature ranges, primarily driven by transformative reactions involving its major constituents—hemicellu- lose, cellulose, and lignin (Jia and Lua 2008a; Yang et al. 2016; Granados Morales 2017). To analyze the effect of previous torrefaction, torrefied palm kernel shells were also pyrolyzed at a temperature of 700 °C, considering the properties and porous structure of biochar obtained at this temperatures from original palm kernel shell. The samples obtained from pyrolysis of torrefied palm kernel shells were labeled as T220-Char700, T250-Char700, and T280-Char700, indicating that they are biochar obtained at 700 °C from torrefied palm kernel shell at 220 °C, 250 °C, and 700 °C. torrefaction process. In addition, the reported literature on this is limited to a few agricultural wastes (rice husk and rice straw). Responsible Editor: Tito Roberto Cadaval Jr After reaching the required tem- perature, the gas was changed from N2 to CO2, and biochar was introduced into the heating zone to start the activation process. After reaching the required temperature, the gas was changed from N2 to CO2 and biochar was introduced for 6 h to perform the activation process; the CO2 flow was set at 100 mL/min. At the end of the process, the sample is cooled in a nitrogen atmosphere to prevent spontaneous combustion. Activated carbon obtained from each biochar sample can be identified with the sample label followed by AC (for example, Char700-AC indicates that it is the activated carbon prepared from biochar obtained from pyrolysis at 700 °C). Char and activated carbon preparation Palm kernel shell obtained from Norte de Santander (Colom- bia) was used. The material was dried in a furnace at 110 °C for 24 h to remove moisture. The dry biomass was grinded using a homemade hammer mill, reducing the particle size up to 1– 2 mm. Palm kernel shells were labeled RawPKS. Torrefied palm kernel shell and biochar were prepared at different temperatures in a fixed-bed horizontal oven. For each experiment, 10 g of palm kernel was loaded into a metal spoon and subsequently placed in the center of the quartz tube used as reactor. Prior to the test, the inside of the quartz tube was purged with a constant flow of 100 mL/min STP of nitrogen for 10 min to remove all the remaining air and oxygen. Finally, the oven was heated to the desired temperature as indicated below. For torrefied Environmental Science and Pollution Research (2024) 31:18765–18784 18768 atmospheric pressure. For the adsorption measurements, a degasification process was performed to eliminate any possible physisorbed CO2 and moisture; for this process, approximately 80 mg of sample was heated at 150 °C and a heating rate of 10 °C/min in a N2 atmosphere for 1 h. Then, the temperature was lowered to 25 °C using a cool- ing rate of – 10 °C/min. When the reactor reached this temperature, the sample was exposed to various CO2/N2 mixtures, with a CO2 volume fraction ranging from 5 to 83%. A 20 mL/min STP of N2 was used as a protective gas flow in the TGA apparatus. The samples were exposed to each concentration of CO2 for 1 h to allow equilibrium between the gas phase and the adsorbent. Each experiment had a duration of 10 h and was performed in duplicate, considering the long adsorption times; for each sample, a total process of 20 h was employed. The CO2 adsorption capacity (mg CO2/g char) was calculated from the mass gain of the sample during exposure to different CO2 con- centrations using Eq. (1): where where Qadsorbed: quantity of CO2 adsorbed at different concentrations.i Qadsorbed: quantity of CO2 adsorbed at different concentrations.i mf,conc: final mass of the solid at each concentration.i i mdesg: mass of the solid after the degasification process (considered initial mass of the solid before the adsorption process).l Once adsorption tests were performed, CO2 flow was replaced by pure N2 and the temperature in the reactor was increased to 150 °C; this led to CO2 desorption in case the adsorption process is reversible. Mass loss occurring when the temperature is increased indicates the reversibility of the adsorption process. Morphological analysis of the samples was performed using FE-SEM microscopy technique. To this end, a Carl Zeiss® Merlin FE-SEM microscope was used by applying an accelerated voltage of 5 kV. Before analysis, the samples were impregnated with a gold coating to ensure good con- ductivity of the atoms on the surface. The chemical composition of the surface of the biochar samples was characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) using an Agilent Cary 600 spectrometer with a resolution of 4 cm−1 in the wavenumber range between 4000 and 400 cm−1 (mid-IR region). These tests were performed in triplicate for each char sample. The absorbance results are presented as the average of each test corresponding to each char sample. Statistical analysis Analysis of variance (ANOVA) was performed to compare the variation of CO2 adsorption performance between the biochar and activated carbons obtained from the different treatments (from torrefied and non-torrefied palm kernel shell). Fisher’s least significant difference (LSD) was per- formed to identify the samples whose means were statisti- cally different. On the other hand, Student’s t-test for the difference of dependent means was performed to analyze the paired effects between biochar and activated carbons. This was done with the aim to identify the changes between the biochars and its subsequent activated carbons. The soft- ware Statgraphics® was used for the statistical analysis. Characterization The elemental analysis of the obtained biochar was per- formed according to the standard on an Exeter CE-440 Elemental Analyzer. The oxygen content was calculated by difference with the percentage of C, H, N, and ash content. f The thermal performance was evaluated by thermogravi- metric analysis (TGA) on a Linseis STA PT 1600 thermo- gravimetric scale. The experiments were performed in a N2 atmosphere with a continuous flow of 50 mL/min and a heating rate of 10 °C/min. The samples were heated between room temperature (approx. 20 °C) and 700 °C. The textural parameters of the samples were character- ized by N2 isotherms at 77 K and CO2 isotherms at 273 K in a surface area and porosity analyzer system (Micromerit- ics TrisTar II Plus). Prior to the adsorption measurements, the samples were degassed at 250 °C for 24 h to release the moisture contained in the pores. Nitrogen adsorp- tion isotherms were measured in a relative pressure range (p/p0) between 0.04 and 0.99. The BET surface area was calculated from the nitrogen adsorption isotherms using the Brunauer–Emmett–Teller equation (BET), assuming a sectional area of 0.162 nm for N2 molecule (Brunauer et al. 1938). The total pore volume was calculated from the amount of nitrogen adsorbed at p/p0 = 0.99 converted to its liquid volume. The mean pore diameter was calculated from the ratio 4000Vt/A(BET). The surface area and micropore vol- ume were calculated using Dubinin Raduskevich’s model. The pore size distribution was calculated using non-local density functional theory (NLDFT). where (1) Qadsorbed = mf,conc −mdesg mdesg ∗1000 [mgCO2 gsolid ] (1) where Elemental analysis The change in the elemental composition of the biochar samples obtained from palm kernel slow pyrolysis is presented in Fig. 1a. The raw material was initially com- posed of 48.08% C, 43.01% O, 5.36% H, and 0.87% N, with the sulfur content being undetectable. Carbon (C), oxygen (O), and hydrogen (H) are the major elemental constituents of biomass, and their ratios determine its fuel properties (Aninda Dhar et al. 2022). Higher O content leads to higher reactivity of biomass during thermochemi- cal conversion (Pérez et al. 2019), in contrast to hydrogen content, which is directly related to the energy content of biomass. On the other hand, a high carbon content of biomass leads to more char formation after the pyrolysis process (Vega et al. 2019). The presence of sulfur in lig- nocellulosic materials can result in the generation of toxic compounds during pyrolysis, such as environmentally haz- ardous sulfides (SOx), which can lead to the generation of greenhouse gasses and acid rain (Aninda Dhar et al. 2022). Thus, palm kernel makes it a promising and environmen- tally friendly raw material to produce carbonaceous mate- rials by pyrolysis. The data indicate a gradual increase in the carbon content in the biochar from 57.29 to 83.83% as the process temperature is increased from 220 to 700 °C because of the high degree of carbonization due to aroma- tization processes. Likewise, there is a gradual decrease in the oxygen content (43.01% to 8.31%) and the hydro- gen content (5.36% to 1.34%) due to the devolatilization processes at high temperatures and the breaking of weak g A decrease in the O/C (0.95 to 0.17) and H/C (1.33 to 0.19) ratios was observed as the slow pyrolysis tempera- ture increased. This confirms the loss of oxygenated and hydrogenated compounds during the process. This behav- ior allows a gradual increase in the aromaticity of the obtained biochar samples, thus highlighting the impor- tance of dehydration reactions (loss of H and O in the form of H2O), decarboxylation (loss of oxygenated compounds in the form of CO andCO2), and demethylation (loss of H in the form of CH3) (Suliman et al. 2016). The atomic ratios of the obtained biochar samples exhibit an interest- ing behavior: a decrease in the O/C ratio (0.10 to 0.05) in the previously torrefied samples, contrary to the increase in the H/C ratio (0.19 to 0.28) as the previous torrefaction temperature increases. Results To obtain an efficient carbonaceous material (biochar or activated carbon) with the capability to adsorb CO2, two distinct routes were evaluated. The first follows the con- ventional approach commonly reported in the literature (pyrolysis–activation) (Li et al. 2008; Pallarés et al. 2018; Abuelnoor et al. 2021). The second route involves the experimental strategy proposed in this study, incorporating an additional torrefaction step before the pyrolysis process (torrefaction–pyrolysis–activation). Characterization of the obtained biochar from each route was conducted before activation using elemental analysis, thermogravimetric analysis, surface area analysis, and FTIR. The resulting activated carbon was characterized by surface area and SEM analyses. An additional route was implemented, involving direct activation of biomass without preceding thermal processes (torrefaction, pyrolysis), aiming to com- pare the impact of thermochemical conversion of biomass before the activated carbon production process. Previous reports (da Silva Veiga et al. 2020) indicate that this behavior is due to the incorporation of nitrogen in more complex and heat-resistant structures that are unable to devolatilize easily with an increase in pyrolysis tempera- ture. On the other hand, the ash content increases slightly in all biochar samples as the temperature increases because of the presence of inorganic components (carbonates, silicates, sulfates, phosphates) and nutrients (Na, Mg, K, Ca) that do not devolatilize during pyrolysis and, consequently, remain in the biochar structure (da Silva Veiga et al. 2020).f On the other hand, the effect of previous torrefaction is also observed. Data indicate that the previous torrefac- tion did not generate significant changes in the elemental composition of the biochar, increasing by 5% (83.83% to 88.85%) to more severe torrefaction and slight decreases in the oxygen content (8.32% to 4.27%) directly related to the release of oxygenated compounds during the previous torre- faction. In contrast, the hydrogen content slightly increased, going from 1.34% in non-torrefied sample to 2.03% in the previously torrefied sample at 250 °C. This behavior was confirmed by the evolution of the O/C and H/C ratios plotted on the Van Krevelen diagram of the biochar samples shown in Fig. 1b. CO2 adsorption test The adsorption capacity of the char and AC samples was determined using thermogravimetric analysis (TGA) in a Netzsch STA 449 Jupiter thermobalance following the procedure proposed by Gil-Lalaguna et al. (2022a). The adsorption tests were performed at 298 K (25 °C) and Environmental Science and Pollution Research (2024) 31:18765–18784 18769 bonds in the solid structure (Al-Wabel et al. 2013). The nitrogen content shows a slight increase up to 280 °C (typ- ical torrefaction range) of approximately 0.87%–2.04%. Elemental analysis Figure 2 shows the TG and DTG curves corresponding to the virgin and torrefied palm kernel subjected to slow pyrolysis at 700 °C and a heating rate of 10 °C/min. Three zones can be conversion of some carbohydrates to aromatic C–C and C–H bonds. conversion of some carbohydrates to aromatic C–C and C–H bonds. (Dai et al. 2018a) have reported that during the torrefac- tion of lignin, the release of CH4 and CH3OH through demethoxylation reactions is common, thus causing some degradation of oxygenated and hydrogenated compounds. The content of C–C and C–H bonds in the lignin structure can be favored as the release reactions of oxygenated com- pounds are stronger. Ben and Ragauskas (2012) attributed this behavior to several factors, including the cleavage of lignin ether bonds during the thermal process, which could re-condense in the form of aromatic C–C bonds and the (Dai et al. 2018a) have reported that during the torrefac- tion of lignin, the release of CH4 and CH3OH through demethoxylation reactions is common, thus causing some degradation of oxygenated and hydrogenated compounds. The content of C–C and C–H bonds in the lignin structure can be favored as the release reactions of oxygenated com- pounds are stronger. Ben and Ragauskas (2012) attributed this behavior to several factors, including the cleavage of lignin ether bonds during the thermal process, which could re-condense in the form of aromatic C–C bonds and the Elemental analysis There was a linear correlation between the changes in the O/C and H/C atomic ratios, presenting a slope ( ΔH∕C / ΔO∕C ) of 0.99. This value indi- cates that the release of compounds such as CO2 and CO derived from decarbonylation and decarboxylation reac- tions is dominant, and consequently, the release of oxygen- ated compounds is faster through these routes than through pure dehydration reactions. This behavior is attributable to the degradation suffered by hemicellulose and cellulose at these temperatures (Chen et al. 2018). Furthermore, despite its high thermal stability, the lignin fraction under- goes certain changes during torrefaction. Previous studies Environmental Science and Pollution Research (2024) 31:18765–18784 18770 18770 Environmental Science and Pollution Research (2024) 31:18765–18784 (Dai et al. 2018a) have reported that during the torrefac- tion of lignin, the release of CH4 and CH3OH through demethoxylation reactions is common, thus causing some degradation of oxygenated and hydrogenated compounds. The content of C–C and C–H bonds in the lignin structure can be favored as the release reactions of oxygenated com- conversion of some carbohydrates to aromatic C–C and C–H bonds. Thermal stability of the samples during pyrolysis The thermal stability of the torrefied palm kernel was meas- Fig. 1 a Elemental analysis of biochar samples obtained from raw and torrefied palm kernel shell. b Van Krevelen diagram Fig. 1 a Elemental analysis of biochar samples obtained from raw and torrefied palm kernel shell. b Van Krevelen diagram Fig. 1 a Elemental analysis of biochar samples obtained from raw and torrefied palm kernel shell. b Van Krevelen diagram (Dai et al. 2018a) have reported that during the torrefac- tion of lignin, the release of CH4 and CH3OH through demethoxylation reactions is common, thus causing some degradation of oxygenated and hydrogenated compounds. The content of C–C and C–H bonds in the lignin structure can be favored as the release reactions of oxygenated com- pounds are stronger. Ben and Ragauskas (2012) attributed this behavior to several factors, including the cleavage of lignin ether bonds during the thermal process, which could re-condense in the form of aromatic C–C bonds and the conversion of some carbohydrates to aromatic C–C and C–H bonds. Thermal stability of the samples during pyrolysis The thermal stability of the torrefied palm kernel was meas- ured by thermogravimetric analysis (TGA). Environmental Science and Pollution Research (2024) 31:18765–18784 18771 Fig. 2 Results of thermogravi- metric analysis of raw and torrefied palm kernel shell. A Mass loss curve (TG) and B DTG curve identified during the palm kernel heating process: a region shoulder-shaped peak is associated with the low decompo- Fig. 2 Results of thermogravi- metric analysis of raw and torrefied palm kernel shell. A Mass loss curve (TG) and B DTG curve identified during the palm kernel heating process: a region between 100 and 105 °C, where residual moisture is elimi- nated from the sample and some extractives. Two charac- teristic peaks occur in the range between 190 and 280 °C and 310–380 °C, where the greatest mass loss occurs due to the release of a large part of the volatiles contained in the biomass. This can be attributed mainly to the breakdown of hemicellulose and cellulose, respectively (Ma et al. 2015). Around 400 °C, there is a shoulder with a slower loss of mass, mainly attributed to the slow degradation of lignin, more severe carbonization process, and decomposition of high molecular weight volatiles (Ma et al. 2017). Figure 2a shows the TG curve obtained from virgin and torrefied palm kernel subjected to slow pyrolysis. As a result, the mass changes during the pyrolysis of the torrefied biochar present significant differences compared to not torrefied kernel. In the torrefied palm kernel shell, the weight loss exhibited a different behavior compared with the raw palm kernel shell, demonstrating enhanced thermal stability as a product of the torrefaction process. This phenomenon can be explained by the poor thermal stability of some functional groups of hemicellulose, such as hydroxyls and side chains, and sugars related to the cellulose structure, which are sensitive to high temperatures (Wang et al. 2016, 2020). During this process, it is possible to increase the solid yield at higher torrefac- tion temperatures because of the enrichment of lignin in the biomass, which is attributed to the increase in the content of insoluble lignin and some alkali metals (Pelaez-Samaniego et al. 2014; Zheng et al. 2015; Cao et al. 2016). shoulder-shaped peak is associated with the low decompo- sition of lignin at these temperatures. At more severe tor- refaction temperatures, only the lignin content was partially affected; thus, the complete degradation of hemicellulose and cellulose in the samples was confirmed. Environmental Science and Pollution Research (2024) 31:18765–18784 As the torrefac- tion temperature increases, hydrogen bonds in the cellulose structure of biomass are broken and the structure becomes more complicated; additionally, as the temperature increases, benzene ring units of lignin are further polymerized, increas- ing the aromaticity of torrefied biomass (Khairy et al. 2023). Thermal stability of the samples during pyrolysis The thermal stability of the torrefied palm kernel was meas- ured by thermogravimetric analysis (TGA). Figure 2 shows the TG and DTG curves corresponding to the virgin and torrefied palm kernel subjected to slow pyrolysis at 700 °C and a heating rate of 10 °C/min. Three zones can be Environmental Science and Pollution Research (2024) 31:18765–18784 Porous structure of biochar Textural parameters such as specific surface area, pore volume, and pore size distribution are determinants for evaluating the porosity of solid materials such as biochar and activated carbon. For this purpose, N2 and CO2 adsorp- tion tests were performed on the obtained samples. Fig- ure 3a shows the changes in the micropore surface area and micropore volume of the biochar samples obtained at temperatures between 220 and 700 °C. For the biochar samples, characterization by N2 adsorption was not per- formed because of the excessively long equilibration times required to start the measurements. This is typical of sam- ples that exhibit high microporosity and have micropores smaller than 1 nm (Suliman et al. 2016). During the first stage (220–280 °C), in which torrefaction typically occurs, the microporous area slowly increases to 250 °C because the palm kernel volatiles begin to be released, leading to the production of new porous structures. When the pyroly- sis temperature is increased to 350 °C, there is a consider- able increase in the area and volume of the micropores. During this stage, dehydration, depolymerization, glyco- side cleavage, decarboxylation, and de-branching reactions of cellulose and hemicellulose structures are performed. Likewise, by the continuous degradation of cellulose together with lignin to a lesser extent and the gas-solid interactions during devolatilization, the formation of more Figure 2b shows the disappearance of the first peak in the DTG curve in samples subjected to prior torrefac- tion, confirming that hemicellulose is the main compound affected by thermal degradation. This behavior agrees with previous studies (Barontini et al. 2021), which reported that this peak decreases in intensity at light torrefaction tem- peratures and completely disappears at more severe tem- peratures. As expected, the second decomposition peak is gradually reduced as the torrefaction temperature increases because of the partial degradation of hemicellulose, which is higher at high temperatures (close to 30 °C). The third Environmental Science and Pollution Research (2024) 31:18765–18784 18772 18772 ( ) fused aromatic structures joined with one or two aromatic rings is promoted. These bonds contain methyl, methylene, d f i l ( h l 2015 to depolymerization of the molten volatiles (Jia and Lua 2008b). At these temperatures, some divisions occur in the h l h l d f i l l Fig. Porous structure of biochar The results indicate that torrefaction at low temperatures (up to 250 °C) decreases the surface area and volume of micropores in the biochar samples; likewise, at more severe temperatures, val- ues similar to those obtained for the palm kernel without prior torrefaction are achieved. Some authors (Zhang et al. 2016) have indicated that as the pre-torrefaction tempera- ture increases, the surface area and pore volume decrease because there is a lower number of volatiles in the structure of the torrefied solid and a lower release of these during pyrolysis. Therefore, a lower pore opening. Crosslinking reactions during torrefaction are responsible for changes in the internal structure of the biochar. In contrast, Fig. 4 shows that the development of surface area and volume of micropo- res is favored as the volatile material content decreases and the fixed carbon content in the biochar sample increases. This behavior confirms the changes suffered in the devola- tilization of the previously torrefied palm kernel, resulting in the exposure of non-degraded compounds that delay the formation of fixed carbon in the char structure. On the other hand, the effect of previous torrefaction is presented. The activated carbon obtained from the char torrefied at 220 °C has a type I isotherm, typical of purely microporous materials; likewise, the other samples pre- sent a mixture of type I and type IV isotherms, typical of materials with micropores and mesopores, and a hyster- esis typical of condensation processes during the desorp- tion of gas in the analysis. As seen in Fig. 4b, the pore size distribution of the activated carbons changes with the previous torrefaction temperature. The activated car- bon obtained from the char torrefied at 220 °C presents a distribution curve with a tendency to have smaller pores, whereas the other curves present peaks between 4 and 5 nm. This confirms the increase in mesoporosity in these materials, following the trend of total pore volume due to pore enlargement. Contrary to what was obtained in the char samples, an increase in the surface area was observed with an increase in the torrefaction temperature to 250 °C, and it decreased at higher temperatures. It has been reported that, during torrefaction, intermediate lignin liq- uids are formed and function as binders of solid particles, which may explain the decrease in surface area during more severe torrefaction (Pelaez-Samaniego et al. 2014). Several authors (Li et al. 2018; He et al. Porous structure of biochar When the pyrolysis temperature is raised to 700 °C, the char reaches the maximum value of surface area and volume of micropores, respectively (685.88 m2/g and 0.274 m3/g), owing to the release of the high molecular weight volatiles by the high temperatures, as well as the destruction of certain aliphatic groups such as alkyl and ether groups. Moreover, the exposure of more groups of aromatic rings bound to lignin at these temperatures pro- motes a considerable increase in the specific surface area (Tomczyk et al. 2020).f pyrolysis (Char350, Char550, and Char700) were selected. Their textural properties were evaluated using N2 adsorp- tion isotherms at 77 K and CO2 at 273 K. Figure 6a shows the adsorption isotherms of activated carbon obtained from palm kernel and virgin palm kernel slow pyrolysis biochar. The activated carbons obtained exhibit a mixture of type I and type IV adsorption isotherms, typical of materials with micropores and mesopores. This can be confirmed in Fig. 6b from the pore size distribution of the obtained activated car- bons, which ranges between 2 and 6 nm. It is possible to highlight the presence of mesopores with a greater volume for the sample obtained without prior pyrolysis. Moreover, as the material is subjected to consecutive pyrolysis, there is a progressive decrease on the peaks from 2 nm, and the micropores tend to increase. This agrees with Fig. 5a and b and demonstrates that the continuous release of volatile material during the palm kernel pyrolysis process favors the release of previously plugged micropores, which are sub- sequently opened by the gasifying agent. In turn, we found that an increase in the slow pyrolysis temperature of the palm kernel up to 700 °C resulted in a significant increase in the BET surface area and total pore volume of the result- ing activated carbon (2353 m2/g and 1.291 m3/g, respec- tively). This is a result of the formation of more complete carbonaceous structures and the total release of the high- and low-molecular-weight volatile material contained in the solid structure. The latter is a product of the high pyrolysis temperatures reflected in the formation of an initial porous structure (Bouchelta et al. 2012).f The effect of previous torrefaction on the textural param- eters of the biochar samples was also observed. Porous structure of biochar 3 a Changes in micropore surface area (black lines) and micropore volume (red lines) measured with CO2 at 273 K of biochar obtained from raw and torrefied palm kernel shell. b Pore size distribution (micropo- rous zone) measured with CO2 at 273 K of biochar obtained from raw and torrefied palm kernel shell ges in micropore lack lines) and ume (red lines) CO2 at 273 K of ed from raw and kernel shell. b bution (micropo- asured with CO2 ochar obtained orrefied palm fused aromatic structures joined with one or two aromatic rings is promoted. These bonds contain methyl, methylene, and oxygen functional groups (Thommes et al. 2015; Yang et al. 2016). At 550 °C, a slight decrease in the surface area and micropore volume was observed. This can be attributed to the sintering and softening reactions of high-molecu- lar-weight volatiles that result in a blockage of pores due to depolymerization of the molten volatiles (Jia and Lua 2008b). At these temperatures, some divisions occur in the methyl, methylene, and oxygen functional groups, result- ing in a greater opportunity to be in contact with the fused aromatic rings at lower temperatures. Then, they go from having aromatic structures of one or two rings to aromatic structures of three to five rings more ordered (Yang et al. to depolymerization of the molten volatiles (Jia and Lua 2008b). At these temperatures, some divisions occur in the methyl, methylene, and oxygen functional groups, result- ing in a greater opportunity to be in contact with the fused aromatic rings at lower temperatures. Then, they go from having aromatic structures of one or two rings to aromatic structures of three to five rings more ordered (Yang et al. fused aromatic structures joined with one or two aromatic rings is promoted. These bonds contain methyl, methylene, and oxygen functional groups (Thommes et al. 2015; Yang et al. 2016). At 550 °C, a slight decrease in the surface area and micropore volume was observed. This can be attributed to the sintering and softening reactions of high-molecu- lar-weight volatiles that result in a blockage of pores due 18773 Environmental Science and Pollution Research (2024) 31:18765–18784 2016). Porous structure of biochar 2019) have found i Figure 3b presents the pore size distribution of the bio- char obtained from raw and torrefied palm kernel shell. It is possible to identify two distribution peaks in each sample: the first peak shows pores less than 0.8 nm, and the second peak shows pores between 1.2 and 1.4 nm. It evidences the marked microporosity of the material obtained. The sample obtained from the torrefied char at 280 °C shows a much higher volume than the torrefied samples at a lower tem- perature in the area between 1.2 and 1.4 nm. The opposite happens in the area comprising pores less than 0.8 nm. Like- wise, smaller pores are formed on the order of 0.4 nm (ultra- micropores) in the same sample. This behavior confirms that the complete devolatilization of the biochar structure promotes the release of micropores and, consequently, an increase in the specific surface area. Porous structure of activated carbon Subsequently, activated carbon was obtained from the bio- char samples. To evaluate the effect of slow pyrolysis tem- perature on the final characteristics of the activated carbons, the samples with the best textural properties during slow 18774 Environmental Science and Pollution Research (2024) 31:18765–18784 that torrefaction as a pretreatment reduces reactivity dur- ing gasification due to the degradation, polycondensation, and carbonization of most of the hemicellulose and frac- tions of the lignin during torrefaction, which are also due to the physical alteration that the solid undergoes during Surface morphology (SEM analysis) To observe the morphological changes on the surface of the biochar, Fig. 5 presents the SEM micrographs of the bi h l bt i d t 220 °C 250 °C 280 °C 350 Fig. 4 a BET surface area (red bars) and total pore volume (blue bars) measured with N2 at 77 K of activated carbons obtained from raw and torrefied palm kernel shell biochar. b Pore size distribution (2 – 20 nm) measured with N2 at 77 K of activated carbons obtained from raw and torrefied palm kernel shell’s biochar Environmental Science and Pollution Research (2024) 31:18765–18784 18774 that torrefaction as a pretreatment reduces reactivity dur- ing gasification due to the degradation, polycondensation, and carbonization of most of the hemicellulose and frac- tions of the lignin during torrefaction, which are also due to the physical alteration that the solid undergoes during torrefaction. Surface morphology (SEM analysis) To observe the morphological changes on the surface of the biochar, Fig. 5 presents the SEM micrographs of the biochar samples obtained at 220 °C, 250 °C, 280 °C, 350 °C, 550 °C, and 700 °C. that torrefaction as a pretreatment reduces reactivity dur- ing gasification due to the degradation, polycondensation, and carbonization of most of the hemicellulose and frac- tions of the lignin during torrefaction, which are also due to the physical alteration that the solid undergoes during torrefaction. Surface morphology (SEM analysis) To observe the morphological changes on the surface of the biochar, Fig. 5 presents the SEM micrographs of the biochar samples obtained at 220 °C, 250 °C, 280 °C, 350 °C, 550 °C, and 700 °C. Porous structure of activated carbon 6 SEM micrographs of activated carbon obtained from Char350, Char550, and Char700 samples the change in the morphology of the activated carbons’ porous structure. According to the obtained textural param- eters, higher pyrolysis temperatures allow the generation of biochars with a more defined porous structure that serve as a platform for the access of CO2 molecules to more sites within the network. Therefore, ensuring complete devola- tilization and restructuring of the solid before the activa- tion process is relevant to the process. When the pyrolysis temperature is lower (350 °C), the resulting activated carbon maintains a slightly smoother and continuous surface and exhibits less accessible pore formation. 280 °C, the structural damage is more noticeable; there is a continuous appearance of new pores that are not easily observed at lower temperatures; however, part of the origi- nal structure remains unchanged. At higher temperatures (greater than 350 °C), the collapse of the biochar surface is more marked as the process progresses. At these tempera- tures (350–550 °C), the most severe devolatilization pro- cesses occur because of the complete degradation of the cel- lulose contained in the biomass, enabling the appearance of new porous structures clearly visible on the surface. At 700 °C, where higher micropore surface areas are obtained, we observe a smoother surface with the formation of new pores inside the channels. This confirms that the devolatilization and reorganization of the structure have been completed.i Porous structure of activated carbon that torrefaction as a pretreatment reduces reactivity dur- ing gasification due to the degradation, polycondensation, and carbonization of most of the hemicellulose and frac- tions of the lignin during torrefaction, which are also due to the physical alteration that the solid undergoes during torrefaction. Surface morphology (SEM analysis) To observe the morphological changes on the surface of the biochar, Fig. 5 presents the SEM micrographs of the biochar samples obtained at 220 °C, 250 °C, 280 °C, 350 °C, 550 °C, and 700 °C. Environmental Science and Pollution Research (2024) 31:18765–18784 18775 18775 Environmental Science and Pollution Research (2024) 31:18765 18784 Fig. 5 Morphological changes in biochar obtained from palm kernel shell at different torrefaction and pyrolysis temperatures Fig. 5 Morphological changes in biochar obtained from palm kernel shell at different torrefaction and pyrolysis temperatures Fig. 5 Morphological changes in biochar obtained from palm kernel shell at different torrefaction and pyrolysis temperatures Fig. 5 Morphological changes in biochar obtained from palm kernel shell at different torrefaction and pyrolysis temperatures In general, physical changes in the physical structure of the biochar are evident as the process temperature increases. The images show the continuous degradation of the solid material as an effect of the continuous structural changes that occur in the biochar as the pyrolysis process moves on at higher temperatures (Yang et al. 2016). It is interesting to observe honeycomb-like structures composed of canals and nets in most of the obtained biochar samples, which are pre- served at high process temperatures (550 and 700 °C). These structures consist of heterogeneous veins, lateral pits, and channels that originate from the cellular tissue structure of the precursor material (palm kernel) (Suliman et al. 2016). However, there are marked differences in two stages of the process: up to 280 °C, the structural damage to the surface of the solid is less marked; in the samples obtained at 220 °C and 250 °C, it is possible to see that a large part of the structure of the cell walls belonging to the original biomass is maintained. In addition, the appearance of pores on the surface can be observed. This may be due to the devola- tilization of unstable polymers bound to hemicellulose. At Environmental Science and Pollution Research (2024) 31:18765–18784 18776 Fig. 6 SEM micrographs of activated carbon obtained from Char350, Char550, and Char700 samples Fig. FTIR analysis The evolution of functional groups was evaluated by FTIR analysis and is presented in Fig. 7. It is possible to highlight five characteristic functional groups in the biochar sam- ples: the first band—one of the most notorious—is located A significant increase in the porosity of all samples is evident in general and indicates that physical activation with CO2 significantly increased the porous structure (see Fig. 6). A clear effect of the previous pyrolysis can be noticed in Fig. 7 FTIR spectra of biochar obtained from raw palm kernel shell (Char700) and torrefied palm kernel shell (T220- Char700, T250-Char700, and T280-Char700) Fig. 7 FTIR spectra of biochar obtained from raw palm kernel shell (Char700) and torrefied palm kernel shell (T220- Char700, T250-Char700, and T280-Char700) Fig. 7 FTIR spectra of biochar obtained from raw palm kernel shell (Char700) and torrefied palm kernel shell (T220- Char700, T250-Char700, and T280-Char700) Fig. 7 FTIR spectra of biochar obtained from raw palm kernel shell (Char700) and torrefied palm kernel shell (T220- Char700, T250-Char700, and T280-Char700) 18777 Environmental Science and Pollution Research (2024) 31:18765–18784 between the 3500 and 3300 cm−1 wavenumbers related to the stretching vibration of O–H groups and possibly linked to hydrogenated structures and water linked to the solid struc- ture (Orrego Restrepo 2021). The peak located between the 2900 and 2700 cm−1 wavenumbers is attributed to the stretching vibrations of C–H groups coming from the ali- phatic compounds –CH2 and alkanes-CH3 (Aninda Dhar et al. 2022). A weak peak occurs at wave number 2130 cm−1 and is attributed to C=O groups related to the CO2 released during pyrolysis (Orrego Restrepo 2021). The peak located between 1600 and 1400 cm−1 is attributed to the stretch- ing vibration of C=C and C=O groups corresponding to aromatic groups derived from lignin compounds. Likewise, a peak between 1200 and 1000 cm−1 caused by the stretch- ing vibration of C–O groups derived from phenols (Ma et al. 2017) and polysaccharides located on the cell wall of biomass. a trend contrary to that reported by several authors (Shang et al. 2012; Kanwal et al. 2019). They confirmed that as the severity of the torrefaction increases, mainly carboxylic groups associated with hemicellulose degrade and decrease these band. Previous reports (Chen et al. 2018; Dai et al. 2018b) indicate that during the degradation of hemicellu- lose in torrefaction, the deoxygenation reactions are faster and prevail over the dehydration reactions. FTIR analysis On the other hand, the intensity of the peak at 1200 cm−1 decreases with increasing torrefaction temperature, indicating the effect of deoxygenation and degradation of cell wall polysaccharides. The C=C functional groups related to aromatic compounds derived from lignin show a slight decrease with increasing torrefaction temperature, indicating partial degradation of lignin due to its high thermal stability. According to Khairy et al. (2023) decarbonylation and decarboxylation promote the reduction of C=O functional groups; additionally, as the torrefaction temperature increases, the reduction of C–C absorption bands can occur due to lignin heat breakdown and the formation of phenylpropane structural units of lignin in the torrefaction range of temperatures.i In general, as the pyrolysis temperature increases, the intensity of the functional groups decreases and –OH, phe- nolic groups, and—to a lesser extent—some aliphatic and aromatic compounds prevail. In the original biomass, most of the functional groups are weakly linked to the three main components (cellulose, hemicellulose, and lignin). Hence, when subjected to the thermal process, they are usually eliminated with those compounds, especially cellulose and hemicellulose, which undergo the greatest degradation dur- ing pyrolysis. This can be confirmed with the results obtained in the ele- mental analysis (see Fig. 1), which show a direct correlation among the presence of the –OH groups, the increase in the hydrogen–carbon ratio, and the decrease in the oxygen–car- bon ratio in the char. During the torrefaction process, carbo- hydrate breakdown was evident, resulting in a reduction of C=O and C–O molecular bonds and aliphatic bonds. This finding, in correlation with elemental analysis, confirms that the oxygenated groups are primarily degraded attributable to deoxidization impact of torrefaction (Khairy et al. 2023). The changes in functional groups induced by torrefaction before pyrolysis were also evaluated. It is interesting to note the increase in the intensity of the band corresponding to the –OH groups as the torrefaction temperature increases, Fig. 8 Relationship between the change in O/C and H/C ratio with peak intensity –OH (a, b) and the change in carbon and oxygen composition with C=O peak intensity (c, d) Fig. 8 Relationship between the change in O/C and H/C ratio with peak intensity –OH (a, b) and the change in carbon and oxygen composition with C=O peak intensity (c, d) Fig. FTIR analysis 8 Relationship between the change in O/C and H/C ratio with peak intensity –OH (a, b) and the change in carbon and oxygen composition with C=O peak intensity (c, d) Environmental Science and Pollution Research (2024) 31:18765–18784 18778 Based on Fig. 8, it can be concluded that the increase in the OH groups and the decrease in the oxygen content and oxygen–carbon ratio in the torrefied product obtained at 280 °C are mainly due to the release of oxygenated compounds via CO and CO2 formation during the torrefaction process, which could influence the presence of new active sites on the char surface. at 700 °C (Char700); group D, corresponding to biochar obtained from torrefied palm kernel shell at 280 °C (T280- Char700); group E, activated carbon obtained from the Char350 sample; group F, corresponding to activated car- bons obtained from Char550, Char700, and T220-Char 700; and group G, corresponding to activated carbons obtained from T250-Char700 and T280-Char700. From statistical analysis, significant differences between the adsorption performance of biochar and activated car- bon samples can be observed. It is clear that the physically activated samples generally displayed higher CO2 retention capacities than the non-activated chars (increasing from 37.16 ± 0.335–72.146 ± 2.925 to 86.96 ± 0.17–102.2 ± 0.29 mg/g for activated carbons). This observation aligns with the findings from the textural properties analysis presented in Fig. 3, confirming that a well-developed porosity, featuring both micropores and mesopores, along with a high surface area, enhances the overall adsorption performance. Previous research (Gil-Lalaguna et al. 2022b) highlighted the signifi- cant role of micropores in the adsorption of CO2 by carbona- ceous materials. The micropore-filling mechanism proposed by Dubinin (1989) suggests that high microporosity is pri- marily responsible for CO2 retention. For the char samples, it could be observed that torrefaction has little effect on the CO2 uptake, which is also reduced for those previously tor- refied at low temperatures (220 and 250 °C with 61.036 ± 0.045 and 60.93 ± 0.05 mg/g, respectively). This behavior has a similar trend with the evolution of the microporous surface (see Fig. 3); samples with the highest microporous surface have the highest CO2 adsorption capacity; this can be confirmed with the pore size distribution (Fig. 3b), where a peak is observed at pore sizes < 1 nm. These results sug- gest that micropore filling is the main mechanism for CO2 adsorption in char samples (Gil-Lalaguna et al. FTIR analysis 2022b) and can be contrasted with Table S2, which shows that a multi- layer adsorption mechanism is observed for these samples, CO2 adsorption performance The CO2 adsorption performance of the samples was ev ated using thermogravimetry at 25 °C (298 K), a temper more representative of practical CO2 removal operatio gas streams, as compared to the commonly used 0 °C K) for surface property measurements. Figures 8 and 9 i trate the change in the CO2 adsorption capacity at a pa pressure p/p0 = 0.83 of biochar and activated carbon ples obtained from raw and torrefied palm kernel shel determine the statistical differences between CO2 adsorp capacity of adsorbent prepared, an ANOVA was perfor for biochar and activated carbon samples independently. ANOVA confirmed the existence of a statistical differ (p-value ≤ 0.05) among the samples; therefore, Fisher’s significant difference (LSD) was performed to identify samples whose means were statistically different. Stud t-test for the difference in dependent means was perfor to analyze the paired effects between biochar and activ carbons. Results presented from this analysis in Fig. 9 re that the samples can be classified into seven homogen groups with similar adsorption capacity: group A, responding to the Char350 sample, which has the lo adsorption capacity (37.167 ± 0.335 mg/g); group B responding to the biochar obtained at 550 °C (Char 3 and biochars obtained from torrefied palm kernel she 220 and 250 °C (T220-Char700 and T250-Char700); g C corresponding to biochar obtained from palm kernel Fig. 9 CO2 adsorption capacity measured at a p/p0 = 0.83 of (a) biochar obtained from raw and torrefied palm kernel shell and (b) activated carbon obtained from biochar samples. Columns with similar letters indicate that there are no significant differ- ences between the means of these groups CO2 adsorption performance Initially, the sam- ples exhibit similar behavior at low partial pressures (lower than 0.1); in this range of concentrations, all the samples (except for sample char350) exhibit a very similar amount of CO2 adsorbed revealing that, at low concentrations, CO2 is adsorbed faster and more efficiently on the carbonaceous materials obtained regardless of their textural properties. As the CO2 concentration increases during the adsorption process, evident changes are observed in the behavior of each sample during the tests. The presented results clearly demonstrate that while the surface area of micropores exhib- its a slight decrease after the activation process, micropo- res smaller than 1 nm, which are primarily involved in the adsorption process, show significant development compared with those found in the biochar samples. This, coupled with the well-developed mesopores resulting from the activa- tion process, enhances the availability of a greater number of active sites, thereby facilitating the retention of a larger amount of CO2. Table S1 shows the parameters obtained from Langmuir and Freundlich adsorption models of biochar and activated carbons obtained from raw palm kernel shell. The results obtained show that there is a good correlation between the experimental data and the two models; how- ever, Freundlich model presents the best adjustment (R2 > Fig. 10 Relationship between functional groups and textural properties of char samples obtained at different pyroly- sis temperatures and CO2 adsorption capacity. (a) C=O functional groups, (b) O–H functional groups, (c) micropore surface area and (d) micropore volume Adsorption isotherms presented in Fig. S1 provide further evidence supporting this behavior through comparison with pore size distribution analysis (0–2 nm) obtained from CO2 adsorption at 273 K using NLDFT models. Initially, the sam- ples exhibit similar behavior at low partial pressures (lower than 0.1); in this range of concentrations, all the samples (except for sample char350) exhibit a very similar amount of CO2 adsorbed revealing that, at low concentrations, CO2 is adsorbed faster and more efficiently on the carbonaceous materials obtained regardless of their textural properties. As the CO2 concentration increases during the adsorption process, evident changes are observed in the behavior of each sample during the tests. The presented results clearly demonstrate that while the surface area of micropores exhib- its a slight decrease after the activation process, micropo- res smaller than 1 nm, which are primarily involved in the adsorption process, show significant development compared with those found in the biochar samples. CO2 adsorption performance The CO2 adsorption performance of the samples was evalu- ated using thermogravimetry at 25 °C (298 K), a temperature more representative of practical CO2 removal operations in gas streams, as compared to the commonly used 0 °C (273 K) for surface property measurements. Figures 8 and 9 illus- trate the change in the CO2 adsorption capacity at a partial pressure p/p0 = 0.83 of biochar and activated carbon sam- ples obtained from raw and torrefied palm kernel shell. To determine the statistical differences between CO2 adsorption capacity of adsorbent prepared, an ANOVA was performed for biochar and activated carbon samples independently. The ANOVA confirmed the existence of a statistical difference (p-value ≤ 0.05) among the samples; therefore, Fisher’s least significant difference (LSD) was performed to identify the samples whose means were statistically different. Student’s t-test for the difference in dependent means was performed to analyze the paired effects between biochar and activated carbons. Results presented from this analysis in Fig. 9 reveal that the samples can be classified into seven homogeneous groups with similar adsorption capacity: group A, cor- responding to the Char350 sample, which has the lowest adsorption capacity (37.167 ± 0.335 mg/g); group B cor- responding to the biochar obtained at 550 °C (Char 350), and biochars obtained from torrefied palm kernel shell at 220 and 250 °C (T220-Char700 and T250-Char700); group C corresponding to biochar obtained from palm kernel shell Fig. 9 CO2 adsorption capacity measured at a p/p0 = 0.83 of (a) biochar obtained from raw and torrefied palm kernel shell and (b) activated carbon obtained from biochar samples. Columns with similar letters indicate that there are no significant differ- ences between the means of these groups Environmental Science and Pollution Research (2024) 31:18765–18784 18779 considering that the Freundlich model has the best fit with the experimental results obtained on CO2 adsorption iso- therms at 298 K. 0.99). In agreement with these results, a multilayer adsorp- tion mechanism is prevalent on both biochar and activated carbon samples because of the heterogeneous surfaces of these samples (Nasri et al. 2014). considering that the Freundlich model has the best fit with the experimental results obtained on CO2 adsorption iso- therms at 298 K. Adsorption isotherms presented in Fig. S1 provide further evidence supporting this behavior through comparison with pore size distribution analysis (0–2 nm) obtained from CO2 adsorption at 273 K using NLDFT models. CO2 adsorption performance This dynamic translates into enhanced adsorption capacity (Khosrowshahi et al. 2022). Previous research conducted by Chen et al. (2016) suggests that the CO2 adsorption capacity at 25 °C does not directly correlate with the textural proper- ties of the activated carbon; these authors suggest that there are synergistic effects between the textural properties of the solid and its chemical composition, with better performance of those with intermediate surface areas and nitrogen con- tent. This aligns with a previous study presented by Ding and Liu (2020), which suggest that as the presence of oxy- gen-containing functional groups increases, CO2 adsorption becomes more effective because of the presence of more active sites. reported that the CO2 adsorption performance of activated carbons obtained from palm kernel shell is dependent on a combination of physicochemical parameters. According to this study, activated carbons with intermediate properties (carbon content, BET surface area, and pore volume) pre- sented the best adsorption capacity, indicating synergistic effects between textural properties and elemental composi- tion. Based on these previous studies, it can be interpreted that there are synergistic effects between the textural param- eters and the physicochemical composition of the samples obtained, positively influenced in the activated carbon samples obtained, observing an improved CO2 adsorption capacity. The reusability of the adsorbent is an important factor in determining the feasibility of the material in practical appli- cations. Table 1 summarizes the CO2 uptake results obtained at p/po = 0.83 for chars and activated carbon after a second desorption–adsorption cycle. In general, a slight loss of CO2 adsorption capacity (less than 1%) was observed for most of the samples; Char700 and T280-Char700 exhibited a loss of adsorption capacity of 7.79% and 9.95%, respectively. These preliminary results suggest that the CO2 adsorption in the evaluated samples is reversible. Aljohani et al. (2023) sug- gested that the reversibility of an adsorbent could be reduced due to blockage of adsorption sites during each adsorption cycle. On the other hand, the structural stability of an adsor- bent material can benefit the adsorption performance dur- ing repetitive adsorption–desorption cycles (Almahri et al. 2023b). However, more work is needed to confirm these A particular trend was observed for char samples obtained from torrefied palm kernel shells (Fig. 11). On the basis of the obtained results, the dominant effect of the developed surface area in the samples is confirmed, substantiating that an increase in available pores enhances CO2 adsorption. CO2 adsorption performance This, coupled with the well-developed mesopores resulting from the activa- tion process, enhances the availability of a greater number of active sites, thereby facilitating the retention of a larger amount of CO2. Table S1 shows the parameters obtained from Langmuir and Freundlich adsorption models of biochar and activated carbons obtained from raw palm kernel shell. The results obtained show that there is a good correlation between the experimental data and the two models; how- ever, Freundlich model presents the best adjustment (R2 > p ( ) A different trend was observed for the activated carbon samples; although there were no significant differences in adsorption capacity, the previously torrefied samples had a higher adsorption capacity than the char samples (102.2 mg/g for AC-T280Char700 sample). No direct correlation was observed between the adsorption capacity and the BET surface area of the activated carbons; however, the evolution of the total pore volume was similar to the CO2 adsorp- tion behavior of the previously torrefied samples. Figure 10 shows the relationship between the adsorption capacity and the abundance of O–H and C=O groups, for biochar samples obtained under different slow pyrolysis temperatures. An evident reduction in CO2 adsorption capacity is observed as the prevalence of O–H phenolic functional groups increases, in contrast to the evident augmentation observed as the abundance of C=O carboxyl functional groups increases. This phenomenon could be explained by alterations in the surface charge associated with these groups. These oxygen- ated groups confer negative charges on the surface, inducing a modification in the electronegativity of the solid. Conse- quently, the heightened abundance of carboxyl groups on the surface enhances surface polarity because of their propensity to accept electrons from adjacent atoms. Consequently, an elevated capacity to interact with CO2 molecules, charac- terized by covalent bonds, ensues. Upon contact with the surface, these CO2 molecules engage in electron exchange Fig. 10 Relationship between functional groups and textural properties of char samples obtained at different pyroly- sis temperatures and CO2 adsorption capacity. (a) C=O functional groups, (b) O–H functional groups, (c) micropore surface area and (d) micropore volume Fig. 10 Relationship between functional groups and textural properties of char samples obtained at different pyroly- sis temperatures and CO2 adsorption capacity. (a) C=O functional groups, (b) O–H functional groups, (c) micropore surface area and (d) micropore volume Environmental Science and Pollution Research (2024) 31:18765–18784 18780 with the C=O groups, resulting in quadrupolar momentum. CO2 adsorption performance Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s11356-024-32028-3. results. The apparent reversibility of the process supports the idea that the contribution of physical adsorption through micropore filling plays an important role in the adsorption mechanism instead of chemical adsorption enhanced by the precipitation of CO2 with minerals on the solid surface (Gil-Lalaguna et al. 2022a). Further analysis is required to confirm this possibility. Acknowledgements The authors thank the General Service of Support to Investigation—SAI—of University of Zaragoza for their collabora- tion with the FE-SEM analysis. Author contributions All authors contributed to the study conception and design in the following form: Marlon Cordoba-Ramirez: concep- tualization, methodology, investigation, experimental campaign, and writing—original draft. Farid Chejne: conceptualization, supervision, and writing—review and editing. Jader Alean: methodology and writ- ing—review and editing. Carlos A. Gomez: methodology and writ- ing—review and editing. Javier Abrego: methodology. Gloria Gea: methodology. Africa Navarro-Gil: methodology and experimentation. All authors read and approved the final manuscript. Conclusions Funding Open Access funding provided by Colombia Consortium This work was supported by the project “Transformation Strategy of the Colombian Energy Sector in Horizon 2030” funded by the call 788 for scientific ecosystems of Colciencias, contract number FP44842- 210-2018 and the network “Alliance for Biomass and Sustainability Research (ABISURE), Universidad Nacional de Colombia,” Hermes code 53024 for their support to conduct this study. The author Marlon Cordoba-Ramirez was supported by the Universidad de La Guajira to conduct this research by “contrato 008 de comisión de estudios.” This study focused on the preparation of biochar and acti- vated carbon from torrefied palm kernel shell for CO2 capture applications. Experimental routes of pyrolysis- activation and torrefaction-pyrolysis-activation were con- ducted to explore the effects of torrefaction on the phys- icochemical properties of the prepared solid materials and their adsorption performance. It is crucial to highlight that the pyrolysis temperature proved to be a decisive factor in the development of micropores in the obtained biochar; the thermal processes of devolatilization and degrada- tion of cellulose and hemicellulose-derived compounds are more pronounced at higher temperatures. On the other hand, one of the most significant findings was the pro- nounced influence of torrefaction on chemical reactions, particularly decarboxylation and decarbonylation, result- ing in a higher release of CO and CO2 compounds com- pared with dehydration reactions. In addition, torrefaction was observed to promote an increase in the lignin content in the resulting solid, favoring the presence of C–C and C–H bonds on the solid surface. As a result, the activated carbon obtained from torrefied palm kernel shell biochar Data availability The datasets generated during the current study are not publicly available due to confidentiality requirements, but are avail- able from the corresponding author on request. CO2 adsorption performance Conversely, a minimal influence of the oxygenated func- tional groups present on the surface is observed, leading to the conclusion that while the surface chemistry of the oxygenated groups indeed promotes interaction with CO2, the physical gas-solid interactions hold greater relevance for the retention of this gas. In contrast, Sanz-Pérez et al. (2015) found that the CO2 uptake capacity at 1 bar does not seem to correlate with textural properties; however, the presence of functional groups and active sites can enhance the adsorp- tion capacity of sorbent materials. Rashidi and Yusup (2019) Fig. 11 Relationship between functional groups, micropore surface area, and CO2 adsorp- tion capacity of char obtained from torrefied palm kernel shell. (a) O–H functional groups, (b) C=O functional groups and (c) micropore surface area ween pore sorp- ined el shell. ps, (b) nd (c) Fig. 11 Relationship between functional groups, micropore surface area, and CO2 adsorp- tion capacity of char obtained from torrefied palm kernel shell. (a) O–H functional groups, (b) C=O functional groups and (c) micropore surface area Environmental Science and Pollution Research (2024) 31:18765–18784 18781 Table 1 Performance of biochar and activated carbon samples in the cyclic adsorption–desorption test (mg CO2/gchar adsorbed at p/p0 = 0.83) exhibits an enhanced adsorption capacity (102.2 ± 0.29 mg/g) compared with activated carbon from untreated palm kernel shell (89.83 ± 0.46 mg/g), possibly due to the emergence of new active sites interacting with CO2. Table 1 Performance of biochar and activated carbon samples in the cyclic adsorption–desorption test (mg CO2/gchar adsorbed at p/p0 = 0.83) Sample Cycle #1 Cycle #2 Char350 37.48 36.85 Char550 59.51 60.18 Char700 75.07 69.22 T220-Char700 61.08 60.99 T250-Char700 60.88 60.98 T280-Char700 70.05 63.08 Char350-AC 86.79 87.13 Char550-AC 89.63 88.93 Char700-AC 90.29 89.37 T220-Char700-AC 90.46 87.97 T250-Char700-AC 102.61 101.1 T280-Char700-AC 101.91 102.49 In summary, these results provide a profound under- standing of the chemical changes that occur during tor- refaction and their direct impact on the composition and porosity of biochar. These findings not only contribute to the fundamental knowledge of thermochemical processes but also offer valuable insights for practical applications, such as efficient CO2 capture in various industries. 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https://openalex.org/W4313830786	https://www.researchsquare.com/article/rs-1885399/latest.pdf	English	null	High Dose Intravenous Anakinra Treatment is Safe and Effective in Severe and Critical COVID-19 Patients: A Propensity Score Matched Study in a Single Center	Research Square (Research Square)	2,023	cc-by	8,939	High Dose Intravenous Anakinra Treatment is Safe and Effective in Severe and Critical COVID-19 Patients: A Propensity Score Matched Study in a Single Center High Dose Intravenous Anakinra Treatment is Safe and Effective in Severe and Critical COVID-19 Patients: A Propensity Score Matched Study in a Single Center MURAT BEKTAS (  murat.b88@hotmail.com ) Aksaray University https://orcid.org/0000-0002-1788-3837 Servet Yüce Istanbul University Istanbul Faculty of Medicine: Istanbul Universitesi Istanbul Tip Fakultesi Mustafa Ay Aksaray University: Aksaray Universitesi Muhammed Hamdi Uyar Aksaray University: Aksaray Universitesi Mustafa Erkut Önder Aksaray University: Aksaray Universitesi Muhammed İkbal Kılıç Aksaray University: Aksaray Universitesi Research Article Keywords: COVID-19, anakinra, cytokine storm, hyperinflammation, propensity score Posted Date: January 9th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-1885399/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Inflammopharmacology on January 27th, 2023. See the published version at https://doi.org/10.1007/s10787-023-01138-8. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Inflammopharmacology on January 27th, 2023. See the published version at https://doi.org/10.1007/s10787-023-01138-8. Page 1/23 Page 1/23 Abstract Background: In COVID-19 severe disease course such as need of intensive care unit (ICU) as well as development of mortality is mainly due to cytokine storm. In this study, we aimed to evaluate the high dose intravenous anakinra treatment response and outcome in patients with severe and critically ill COVID-19 compared to standard of care. Method : This retrospective observational study was carried out at a tertiary referral center. The study population consisted of two groups as follows; the patients receiving high dose intravenous anakinra (anakinra group) between 01.09.2021 and 01.02.2022 and the patients treated with standard of care (SoC, control group) as historical control group who were hospitalized between 01.07.2021 and 01.09.2021. Results:After the propensity score 1:1 matching 79 patients in anakinra and 79 patients in SoC matched and included into the analysis. Mean±SD patient age was 67.4±16.7 and 67.1±16.3 years in anakinra and SoC group, respectively (p=0.9). Male gender was 38 (48.7 %) in anakinra and 36 (46.2 %) SoC (p=0.8). Overall, ICU admission was in 14.1 % (n=11) and 30.8 % (n=24) (p=0.013; OR: 6.2), intubation in 12.8 % (n=10) and 16.7 % (n=13) patients (p=0.5), 14.1 % (n=11) and 32.1 % (n=25) patients died in anakinra and control group, respectively (p=0.008; OR: 7.1) Conclusion : In our study mortality was lower in patients receiving anakinra compared to SoC. Intravenous high dose anakinra is safe and effective treatment in patients with severe and critical COVID- 19. Background Coronavirus 2019 (COVID-19) is a life-threatening disease that is caused by SARS-Cov-2 (WHO, 2020). Disease spectrum of COVID-19 are ranged from asymptomatic to severe pneumoniae leading acute respiratory failure and even death (Lai, Shih, Ko, Tang, & Hsueh, 2020). Hyperinflammatory state (cytokine storm) developed by excessive release of several pro-inflammatory cytokines such as interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (Hu, Huang, & Yin, 2021). In COVID-19 severe disease course such as need of intensive care unit (ICU) as well as mortality is mainly due to development of cytokine storm (Tang, Yin, Hu, & Mei, 2020). COVID-19 associated cytokine storm develops with decreasing of type 1 interferon production via inhibition toll-like receptor-3 (TLR-3) signaling by virus accessory proteins and therefore virus can escape the immune response. Defective clearance of SARS-CoV-2 and virus-host interaction induces the overproduction of immune mediators that leads to increase in secretion of chemokines and cytokines including IL-1, IL‐6, IL‐8, IL‐21 and TNF in infected cells (Seyed Hosseini et al., 2020). There is also another pathway into the development of COVID-19 associated cytokine storm. SARS-CoV-2 activates an intracellular multiprotein complex that is called ‘inflammasome’ after binding TLRs into the development of cytokine storm in severe COVID-19 (López-Reyes et al., 2020). Inflammasomes have essential role in Page 2/23 Page 2/23 the host defense against microorganisms including viruses that are present in multiple innate immune cells such as neutrophils, macrophages and dendritic cells. Activation of inflammasomes is leading to the cleavage of pro-IL-1β to produce active IL-1β (Vora, Lieberman, & Wu, 2021). Anakinra, an IL-1 receptor antagonist, is commonly used in the treatment of autoinflammatory diseases such as hereditary periodic fever syndromes (HPFS), gout and adult onset still disease (AODS) (Saag et al., 2021) (Atas et al., 2021) (Hong et al., 2014). Safety and efficacy of anakinra was established in several hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) secondary to various diseases including malignancies, viruses and rheumatological conditions (Bami et al., 2020) (Gilboa, Bornstein, Ben-Zvi, & Grossman, 2020). Recently anakinra also become an emerging treatment option against to COVID-19 associated cytokine storm (Bozzi et al., 2021). In this study, we aimed to evaluate the high dose intravenous anakinra treatment response and outcome in patients with severe and critical COVID-19 compared to standard of care. Patients and data: This retrospective observational study was carried out at a tertiary referral center in Turkey. All patients had positive polymerase chain reaction (PCR) and delta variant (B.1.617.2) proven by variant analysis as well as typical computer tomography (CT) findings in addition to clinical signs and symptoms. Patients with negative PCR results and/or variants rather than delta and/or inconsistent CT findings with COVID- 19 were excluded from the study. The study population consisted of two groups as follows; the patients receiving high dose intravenous anakinra (anakinra group) added to background therapy between 01.09.2021 and 01.02.2022 and the patients treated with standard of care (SoC, control group) as historical control group who were hospitalized between 01.07.2021 and 01.09.2021. COVID-19 disease severity was evaluated according to National Institute of Health (NIH) severity scale and only severe (NIH score 3; patients with SpO2 < 94% on room air at sea level, PaO2/FiO2 < 300 mm Hg, a respiratory rate > 30 breaths/min, or lung infiltrates > 50%) and critically ill (NIH score 4; patients have acute respiratory distress syndrome, septic shock that may represent virus-induced distributive shock, cardiac dysfunction, an exaggerated inflammatory response, and/or exacerbation of underlying comorbidities, as well as requirement of high flow nasal oxygen therapy [HFNO] or invasive mechanical ventilation [IMV]) patients in the ward were included into the study ("COVID-19 Treatment Guidelines Panel. Coronavirus Disease 2019 (COVID-19) Treatment Guidelines.," 2022). Individual written patient consent and local ethic committee approval was obtained for this study (date/number: 24.02.2022, 2022/04–09). Laboratory evaluation Statistical analysis In our study, 22.0 version (IBM, Armonk, NY, USA) of SPSS (Statistical Package for the Social Sciences) program was used for statistical analysis of data. Descriptive statistics, discrete and continuous numerical variables were expressed as mean, ± standard deviation or median (minimum-maximum). Categorical variables were expressed as number of cases (%). Cross table statistics were used to compare categorical variables (Chi-Square, Fisher’ exact test). Normally distributed parametric data were compared with Student's t-test and non-parametric data that did not meet normal distribution were compared with Mann Whitney U and Kruskal Wallis tests. Correlation analysis was performed by Pearson or Spearman method according to normality distribution. Kaplan-Meier and log-rank methods were used for survival analysis. Multivariate analysis was performed by using logistic regression. p < 0.05 value was considered statistically significant. Treatment protocol and outcome All patients received background corticosteroid therapy with 80 mg/day methylprednisolone (or its equivalent) and enoxaparin 0.4 mg/day at the admission and consecutive days (SoC). Anakinra was started in patients who did not respond to steroid therapy at least two days or concomitantly with steroids in patients with higher risk and/or critical illness at admission. Average starting dose of anakinra was 400 mg/day intravenously and increased gradually to maximum 1600 mg/day if necessary (10 mg/kg/day). Anakinra dose adjustment was performed by the same rheumatologist (MB) according to daily clinical and laboratory findings. Severe infection was defined as development of opportunistic infection, intravenous antibiotics, sepsis or requirement of intensive care unit (ICU) admission or development of death due to secondary infection. Laboratory evaluation Page 3/23 Page 3/23 Laboratory values such as hemogram, liver enzymes, C-reactive protein (CRP), ferritin, D-dimer, lactate dehydrogenase (LDH) at admission; the peak levels of CRP, ferritin, D-dimer and LDH levels were recorded Inflammatory state of the patients was evaluated and derived based on COVID hyperinflammatory syndrome score (cHIS) and it was calculated according to combination of neutrophil and lymphocyte counts at the admission and the peak levels of CRP, ferritin, D-dimer and LDH during to the follow-up (Webb et al., 2020). The item of fever was removed due to presence of lower frequency (<%10) of the patients in both arms. Therefore, maximum score of the new version of cHIS score was 5 points (modified cHIS [mcHIS] score) were calculated in both groups. Propensity score matching The first step in Propensity Score Matching (PSM) is to identify the covariates from which to calculate propensity scores (PS). These variables, which can explain the output variable, should be included in the propensity score creation process (Fan & Nowell, 2011). Age, gender, baseline NLR, ferritin, CRP, D-dimer, LDH levels and NIH COVID disease scores of the patients were determined as the variables to be matched. The PS matching was done as 1:1 with nearest neighbor method. Caliper value was 0.2. When matching, we performed this analysis by assigning values according to the averages of the parameters with missing data. PSM was performed with SPSS package program 28.0.1 using R package program Page 4/23 Page 4/23 and an auxiliary plugin (PS matching 3.0 SPE). Dotplot of standardized mean differences for all covariates before and after PS matching was showed in Fig. 1. Jitter plots for trend scores and lineplot of standardized differences were described in supplemental Figs. 1 and 2, respectively. and an auxiliary plugin (PS matching 3.0 SPE). Dotplot of standardized mean differences for all covariates before and after PS matching was showed in Fig. 1. Jitter plots for trend scores and lineplot of standardized differences were described in supplemental Figs. 1 and 2, respectively. Initial analysis with study group before PS Matching Data of 148 patients in anakinra and 114 patients in control group were analyzed. Flow-chart of the study design and participants was showed in Fig. 2. Mean ± standard deviation (SD) patient age was 66.8 ± 17 and 63.1 ± 17 years in anakinra and SoC group, respectively (p = 0.09). Male gender was 78 (52.7%) in anakinra and 45 (39.5%) SoC (p = 0.033; Odds ratio [OR]: 4.5). Median (IQR) duration of hospitalization were 11 (12) days and 9 (7.3) days in anakinra and SoC group, respectively (p = 0.02). Diabetes mellitus (DM) was in 28% (n = 41) and 34.2% (n = 39), hypertension (HT) in 58.7% (n = 84) and 56% (n = 64), coronary heart disease (CHD) in 27% (n = 19) and 24% (n = 21), heart failure (HF) in 12.6% (n = 18) and 20% (n = 23), chronic kidney failure (CKD) in 21% (n = 31) and 5.3 (n = 6), chronic obstructive pulmonary disease (COPD) in 16% (n = 23) and 16.7% (n = 19), dementia in 12.8% (n = 15) and 1.8% (n = 2), malignancy in 11% (n = 16) and 7% (n = 8), immunosuppressive usage in 12.3% (n = 18) and 1.8 (n = 2) in anakinra and control group, respectively. Baseline clinical and laboratory findings of participants in anakinra and control group before and after PSM were described in Table 1. Initial analysis with study group before PS Matching Mean mcHIS score was 3.4 ± 1.2 and 2.64 ± 1.5 in patients receiving anakinra and SoC, respectively (p = < 0.001). Overall, pneumothorax was developed in 2.2 (n = 3) and 0, myocardial infarction (MI) in 2.3% (n = 3) and 5.3 (n = 6), pulmonary embolism (PE) in 3% (n = 4) and 9.6% (n = 11), ICU admission was in 40.5% (n = 60) and 22% (n = 25), intubation was in 36.5% (n = 54) and 11.4% (n = 13) patients, 37.8% (n = 56) and 23.7% (n = 27) patients died in anakinra and control group, respectively (Table 2). Fifty-seven (38.5%) and 68 (59.6%) patients had severe, 91 (61.5%) and 46 (40.4%) had critical disease in anakinra and control group, respectively (p = 0.001; OR: 11.5). Mean mcHIS score was 3.4 ± 1.2 and 2.64 ± 1.5 in patients receiving anakinra and SoC, respectively (p = < 0.001). Overall, pneumothorax was developed in 2.2 (n = 3) and 0, myocardial infarction (MI) in 2.3% (n = 3) and 5.3 (n = 6), pulmonary embolism (PE) in 3% (n = 4) and 9.6% (n = 11), ICU admission was in 40.5% (n = 60) and 22% (n = 25), intubation was in 36.5% (n = 54) and 11.4% (n = 13) patients, 37.8% (n = 56) and 23.7% (n = 27) patients died in anakinra and control group, respectively (Table 2). Initial analysis with study group before PS Matching Page 5/23 Table 1 Table 1 Table 1 Baseline clinical and laboratory features of patients receiving standard of care (SoC) and Anakinra before and after propensity score (PS) matching Before PS matching After PS matching Variables Anakinra (n = 148) SoC (n = 114) p value (OR) Anakinra (n = 78) SoC (n = 78) p value (OR) Age (years) (mean ± SD) 66.8 ± 17 63.1 ± 17 0.09 67.4 ± 16.7 67.1 ± 16.3 0.9 Gender, male (n, %) 78 (52.7) 45 (39.5) 0.033 (4.5) 38 (48.7) 36 (46.2) 0.8 Duration of hospitalization (days) (median, IQR) 11 (12) 9 (7.3) 0.02 7.5 (9) 11 (8) 0.01 Comorbidities (n, %) Diabetes mellitus 41/146 (28.1) 39 (34.2) 0.3 18 (23) 31 (39.7) 0.025 (5) Hypertension 84/143 (58.7) 64 (56) 0.7 30 (61.5) 50 (64) 0.7 Coronary heart disease 27/143 (19) 24 (21) 0.7 18 (23) 20 (25.6) 0.7 Heart failure 18/143 (12.6) 23 (20) 0.1 14 (18) 20 (25.6) 0.24 Chronic renal failure 31 (21) 6 (5.3) < 0.001 (13.06) 15 (19) 6 (7.7) 0.035 (4.5) Chronic obstructive lung disease 23/144 (16) 19 (16.7) 0.9 14 (18) 15 (19) 0.8 Dementia 15/117 (12.8) 2 (1.8) 0.001 (10.4) 3/61 (5) 2 (2.6) 0.5 Malignancy 16/146 (11) 8 (7) 0.3 9 (11.5) 6 (7.7) 0.4 Immunosuppressive usage 18/146 (12.3) 2 (1.8) 0.001 (10.08) 5 (6.5) 2 (2.6) 0.2 Disease severity (n, %) NIH score 3 (severe) 57 (38.5) 68 (59.6) 0.001 (11.5) 48 (61.5) 44 (56.4) 0.5 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Baseline clinical and laboratory features of patients receiving standard of care (SoC) and Anakinra before and after propensity score (PS) matching PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Page 6/23 Page 6/23 Before PS matching After PS matching NIH score 4 (critical) 91 (61.5) 46 (40.4) 30 (38.5) 34 (43.6) mcHIS score (mean ± SD) 3.4 ± 1.2 2.64 ± 1.5 < 0.001 2.9 ± 1 3.1 ± 1.3 0.2 Laboratory results Neutrophil to lymphocyte ratio (median, IQR) 7 (7.6) 4.34 (4.4) < 0.001 6.6 (7.7) 6.4 (7.9) 0.6 Hemoglobin (mean ± SD) 13.2 ± 2.2 13 ± 2 0.9 13.5 ± 2.2 13.2 ± 2.1 0.4 Platelets (mean ± SD) 206.4 ± 79.5 199 ± 74 0.5 213 ± 77 195 ± 70 0.13 C-reactive protein (mg/L) (median, IQR) Baseline 119.5 (110) 101 (99) 0.2 112 (151.5) 121 (71.3) 0.3 Peak level 152.5 (124) 126 (88) 0.004 147 (174) 169 (70) 0.5 Ferritin (pg/mL) (median, IQR) Baseline 393 (616) 393 (205) 0.3 118 (777) 393 (35.6) 0.1 Peak level 716.5 (1023) 396.7 (627) < 0.001 737 (841) 694 (913) 0.3 D-dimer (pg/mL) (median, IQR) Baseline 1.25 (1.2) 2.2 (1) 0.001 1.6 (1.2) 2.2 (0.9) 0.044 Peak level 4.3 (14.8) 2.25 (5.2) < 0.001 14.6 (28) 10.8 (28) 0.6 Lactate dehydrogenase (U/L) (median, IQR) Baseline 398.5 (209) 312 (30) 0.045 329.5 (361) 448.4 (66.4) 0.037 Peak level 569.5 (285) 420.5 (191) < 0.001 604 (659) 598 (478) 0.02 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Page 7/23 Page 7/23 Before PS matching After PS matching ALT (U/L) (median, IQR) 22 (19) 21.8 (19) 0.9 17 (21.3) 15 (18) 0.4 AST (U/L) (median, IQR) 39 (33) 38 (19.3) 0.9 32 (50) 42 (25) 0.6 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Fifty-seven (38.5%) and 68 (59.6%) patients had severe, 91 (61.5%) and 46 (40.4%) had critical disease in anakinra and control group, respectively (p = 0.001; OR: 11.5). Initial analysis with study group before PS Matching Table 2 Outcomes of patients receiving SoC and Anakinra before and after PS matching Before PS matching After PS matching Variables (n, %) Anakinra (n = 148) SoC (n = 114) p value (OR) Anakinra (n = 78) SoC (n = 78) p value (OR) Pneumothorax 3/134 (2.2) 0 0.25* 2/73 (2.7) 0 0.5* Myocardial infarction 3/132 (2.3) 6 (5.3) 0.3 2/72 (2.8) 2/56 (3.6) 1 Pulmonary embolism 4/134 (3) 11 (9.6) 0.034 (4.8)* 3/73 (4.1) 7 (9) 0.3* Intensive care unit 60 (40.5) 25 (22) 0.001 (10.2) 11 (14.1) 24 (30.8) 0.013 (6.2) Intubation 54 (36.5) 13 (11.4) < 0.001 (21.3) 10 (12.8) 13 (16.7) 0.5 Mortality 56 (37.8) 27 (23.7) 0.015 (5.96) 11 (14.1) 25 (32.1) 0.008 (7.1) PS: Propensity score, SoC: Standard of care, OR: Odds ratio PS: Propensity score, SoC: Standard of care, OR: Odds ratio In univariable analysis, higher patient age (p < 0.001 and p = 0.002), higher frequency of critical illness (p < 0.001; OR: 52.3 and p < 0.001; OR: 51.3), higher mcHIS score (p < 0.001 and p < 0.001), higher peak levels of CRP (p < 0.001 and p < 0.001), ferritin (p < 0.001 and p < 0.001), d-dimer (p < 0.001 and p < 0.001) and LDH (p < 0.001 and p < 0.001) were observed in patients had mortality in SoC and anakinra groups, Page 8/23 Page 8/23 respectively. Higher frequency of male gender (p = 0.004; OR: 4.8), coronary artery disease (p = 0.001; OR: 11.6) and ALT levels (p = 0.009) in SoC; higher frequency of dementia (p = 0.002; OR: 9.8), higher NLR (p < 0.001), baseline CRP (p = 0.004) and d-dimer (p = 0.016) levels were observed in anakinra group in patients had mortality (Table 3). respectively. Higher frequency of male gender (p = 0.004; OR: 4.8), coronary artery disease (p = 0.001; OR: 11.6) and ALT levels (p = 0.009) in SoC; higher frequency of dementia (p = 0.002; OR: 9.8), higher NLR (p < 0.001), baseline CRP (p = 0.004) and d-dimer (p = 0.016) levels were observed in anakinra group in patients had mortality (Table 3). respectively. Initial analysis with study group before PS Matching Higher frequency of male gender (p = 0.004; OR: 4.8), coronary artery disease (p = 0.001; OR: 11.6) and ALT levels (p = 0.009) in SoC; higher frequency of dementia (p = 0.002; OR: 9.8), higher NLR (p < 0.001), baseline CRP (p = 0.004) and d-dimer (p = 0.016) levels were observed in anakinra group in patients had mortality (Table 3). respectively. Higher frequency of male gender (p = 0.004; OR: 4.8), coronary artery disease (p = 0.001; OR: 11.6) and ALT levels (p = 0.009) in SoC; higher frequency of dementia (p = 0.002; OR: 9.8), higher NLR (p < 0.001), baseline CRP (p = 0.004) and d-dimer (p = 0.016) levels were observed in anakinra group in patients had mortality (Table 3). Analysis of study group after PS Matching Analysis of study group after PS Matching After PS procedure 79 patients in anakinra and 79 patients in SoC matched and included into the analysis. Mean ± SD patient age was 67.4 ± 16.7 and 67.1 ± 16.3 years in anakinra and SoC group, respectively (p = 0.9). Male gender was 38 (48.7%) in anakinra and 36 (46.2%) SoC (p = 0.8). Median (IQR) duration of hospitalization were 7.5 (9) days and 11 (8) days in anakinra and SoC group, respectively (p = 0.01). Frequency of DM was in 23% (n = 18) and 39.7% (n = 31), HT in 61.5% (n = 30) and 64% (n = 50), CHD in 23% (n = 18) and 25.6% (n = 20), HF in 18% (n = 14) and 25.6% (n = 20), CKD in 19% (n = 15) and 7.7 (n = 6), COPD in 18% (n = 14) and 19% (n = 15), dementia in 5% (n = 3) and 2.6% (n = 2), malignancy in 11.5% (n = 9) and 7.7% (n = 6), immunosuppressive usage in 6.5% (n = 5) and 2.6 (n = 2) in anakinra and control group, respectively. The differences were only significant in patients had DM (p = 0.025; OR: 5) and CRF (p = 0.035; OR: 4.5) and those had not among comorbidities after PSM (Table 1). Forty-eight (61.5%) and 44 (56.4%) patients had severe, 30 (38.5%) and 34 patients (43.6%) had critical disease in anakinra and control group, respectively (p = 0.5). Mean mcHIS score was 2.9 ± 1 and 3.1 ± 1.3 in patients receiving anakinra and SoC, respectively (p = 0.2). Overall, pneumothorax was developed in 2.7 (n = 2) and 0 (p = 0.5), myocardial infarction (MI) in 2.8% (n = 2) and 3.6 (n = 2) (p = 1), pulmonary embolism (PE) in 4.1% (n = 3) and 9% (n = 7) (p = 0.3), ICU admission was in 14.1% (n = 11) and 30.8% (n = 24) (p = 0.013; OR: 6.2), intubation in 12.8% (n = 10) and 16.7% (n = 13) patients (p = 0.5), 14.1% (n = 11) and 32.1% (n = 25) patients died in anakinra and control group, respectively (p = 0.008; OR: 7.1) (Table 2). Initial analysis with study group before PS Matching Page 9/23 Table 3 Univariable analysis of mortality in patients receiving SoC and Anakinra before PS matching Univariable analysis of SoC group before PS matching Univariable analysis of Anakinra group before PS matching Variables Alive (n = 87) Deceased (n = 27) p value Alive (n = 92) Deceased (n = 56) p value Age (years) (mean ± SD) 59.7 ± 16 73.9 ± 15.5 < 0.001 63.4 ± 18 72.3 ± 13.4 0.002 Gender, male (n, %) 28 (32) 17 (63) 0.004 (8.2) 44 (47.8) 34 (60.7) 0.1 Comorbidities (n, %) Diabetes mellitus 31 (35.6) 8 (29.6) 0.6 23 (25) 18/54 (33.3) 0.3 Hypertension 42 (52) 19 (70.4) 0.088 52 (56.5) 32/51 (63) 0.5 Coronary heart disease 12 (13.8) 12 (44.4) 0.001 (11.6) 15 (16.3) 12/51 (23.5) 0.3 Heart failure 15 (17.2) 8 (29.6) 0.2 11 (12) 7/51 (13.7) 0.8 Chronic renal failure 4 (4.6) 2 (7.4) 0.6 16 (17.4) 15 (26.8) 0.2 Chronic obstructive lung disease 13 (15) 6 (22) 0.4 14 (15.2) 9/52 (17.3) 0.7 Dementia 1 (1) 1 (3.7) 0.4 5/80 (6.3) 10/37 (27) 0.002 (9.8) Malignancy 3 (3.4) 5 (18.5) 0.018 (7.2) 9 (9.8) 7/54 (13) 0.6 Immunosuppressive usage 2 (2.3) 0 0.4 10/91 (11) 8/55 (14.5) 0.5 Disease severity (n, %) NIH score 3 (severe) 68 (78.2) 0 < 0.001 (52.3) 56 (61) 1 (1.8) < 0.001 (51.3) NIH score 4 (critical) 19 (21.8) 27 (100) 36 (39) 55 (98.2) PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Page 10/23 Page 10/23 Univariable analysis of SoC group before PS matching Univariable analysis of Anakinra group before PS matching mcHIS score (mean ± SD) 2.2 ± 1.4 4.2 ± 0.9 < 0.001 3 ± 1 4.1 ± 1.1 < 0.001 Laboratory results Neutrophil to lymphocyte ratio (median, IQR) 4.4 (3.9) 4.14 (9.4) 0.9 5.7 (5.1) 9.4 (8.4) < 0.001 Hemoglobin (mean ± SD) 13.3 ± 2.2 12.9 ± 1.2 0.4 13.4 ± 2.2 13 ± 2.3 0.4 Platelets (mean ± SD) 201 ± 72 192 ± 81 0.6 215 ± 77 192 ± 82 0.08 C-reactive protein (mg/L) (median, IQR) Baseline 98 (97) 117 (130) 0.3 104 (109) 136 (125) 0.004 Peak level 100 (90) 171 (77) < 0.001 136 (92) 206 (132) < 0.001 Ferritin (pg/mL) (median, IQR) Baseline 288 (425) 434 (600) 0.3 330 (543) 533 (767) 0.06 Peak level 426 (502) 930 (931) < 0.001 532 (613) 1188 (2386) < 0.001 D-dimer (pg/mL) (median, IQR) Baseline 0.8 (0.8) 1.44 (4.4) 0.064 1 (1.1) 1.5 (1.4) 0.016 Peak level 1.5 (2.6) 15.3 (30) < 0.001 2.7 (7.2) 12.4 (23.5) < 0.001 Lactate dehydrogenase (U/L) (median, IQR) Baseline 411 (241) 421 (210) 0.6 398 (186) 391 (254) 0.9 Peak level 368 (154) 655 (519) < 0.001 505 (201) 707 (527) < 0.001 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Page 11/23 Univariable analysis of SoC group before PS matching Univariable analysis of Anakinra group before PS matching ALT (U/L) (median, IQR) 22.5 (20) 19.5 (18) 0.009 24 (25) 21 (15) 0.3 AST (U/L) (median, IQR) 38 (22) 44 (41) 0.8 39 (31) 39.5 (33) 0.8 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Analysis of study group after PS Matching After PS procedure 79 patients in anakinra and 79 patients in SoC matched and included into the analysis. Initial analysis with study group before PS Matching Mean ± SD patient age was 67.4 ± 16.7 and 67.1 ± 16.3 years in anakinra and SoC group, respectively (p = 0.9). Male gender was 38 (48.7%) in anakinra and 36 (46.2%) SoC (p = 0.8). Median (IQR) duration of hospitalization were 7.5 (9) days and 11 (8) days in anakinra and SoC group, respectively (p = 0.01). Frequency of DM was in 23% (n = 18) and 39.7% (n = 31), HT in 61.5% (n = 30) and 64% (n = 50), CHD in 23% (n = 18) and 25.6% (n = 20), HF in 18% (n = 14) and 25.6% (n = 20), CKD in 19% (n = 15) and 7.7 (n = 6), COPD in 18% (n = 14) and 19% (n = 15), dementia in 5% (n = 3) and 2.6% (n = 2), malignancy in 11.5% (n = 9) and 7.7% (n = 6), immunosuppressive usage in 6.5% (n = 5) and 2.6 (n = 2) in anakinra and control group, respectively. The differences were only significant in patients had DM (p = 0.025; OR: 5) and CRF (p = 0.035; OR: 4.5) and those had not among comorbidities after PSM (Table 1). Univariable analysis of SoC group before PS matching Univariable analysis of Anakinra group before PS matching ALT (U/L) (median, IQR) 22.5 (20) 19.5 (18) 0.009 24 (25) 21 (15) 0.3 AST (U/L) (median, IQR) 38 (22) 44 (41) 0.8 39 (31) 39.5 (33) 0.8 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Analysis of study group after PS Matching Analysis of study group after PS Matching In univariable analysis, higher patient age (p = 0.005 and p < 0.001), higher frequency of critical illness (p < 0.001; OR: 47.6 and p < 0.001; OR: 14.9), higher mcHIS score (p = 0.005 and p = 0.036), higher peak levels of ferritin (p = 0.005 and p = 0.04) and LDH (p = 0.002 and p = 0.01) were observed in patients had mortality in SoC and anakinra groups, respectively. Higher frequency of CHD (p = 0.002; OR: 9.6), higher peak CRP (p = 0.006) and d-dimer levels (p < 0.001) in SoC; higher frequency of CRF (p = 0.005; OR: 10.3) were observed in anakinra group in patients had mortality (Table 4). In univariable analysis, higher patient age (p = 0.005 and p < 0.001), higher frequency of critical illness (p < 0.001; OR: 47.6 and p < 0.001; OR: 14.9), higher mcHIS score (p = 0.005 and p = 0.036), higher peak levels of ferritin (p = 0.005 and p = 0.04) and LDH (p = 0.002 and p = 0.01) were observed in patients had mortality in SoC and anakinra groups, respectively. Higher frequency of CHD (p = 0.002; OR: 9.6), higher peak CRP (p = 0.006) and d-dimer levels (p < 0.001) in SoC; higher frequency of CRF (p = 0.005; OR: 10.3) were observed in anakinra group in patients had mortality (Table 4). Analysis of study group after PS Matching Page 12/23 Table 4 h Univariable analysis of mortality in patients with SoC and Anakinra after PS matching Univariable analysis of SoC group after PS matching Univariable analysis of Anakinra group after PS matching Variables Alive (n = 53) Deceased (n = 25) p value Alive (n = 67) Deceased (n = 11) p value Age (years) (mean ± SD) 63.6 ± 15.4 74.4 ± 15.8 0.005 65.5 ± 17 79 ± 6.7 < 0.001 Gender, male (n, %) 21 (39.6) 15 (60) 0.09 31 (46.3) 7 (63.6) 0.3 Comorbidities (n, %) Diabetes mellitus 25 (45.3) 7 (28) 0.15 15 (22.4) 3 (27.3) 0.7 Hypertension 32 (60.4) 18 (72) 0.3 39 (58.2) 9 (81.8) 0.2 Coronary heart disease 8 (15) 12 (48) 0.002 (9.6) 13 (19.4) 5 (45.5) 0.1 Heart failure 12 (20.6) 8 (32) 0.4 10 (15) 4 (36.4) 0.1 Chronic renal failure 4 (7.5) 2 (8) 0.9 9 (13.4) 6 (54.5) 0.005 (10.3)* Chronic obstructive lung disease 9 (17) 6 (24) 0.5 10 (15) 4 (36.4) 0.1 Dementia 1 (2) 1 (4) 0.5 3/56 (5.4) 0 0.6 Malignancy 2 (3.8) 4 (16) 0.08 9 (13.4) 0 0.2 Immunosuppressive usage 2 (3.8) 0 1 5 (8) 0 0.3 Disease severity (n, %) NIH score 3 (severe) 44 (83) 0 < 0.001 (47.6) 47 (70) 1 (9) < 0.001 (14.9) NIH score 4 (critical) 9 (17) 25 (100) 20 (30) 10 (90) mcHIS score (mean ± SD) 2.85 ± 1.3 3.76 ± 1.3 0.005 2.78 ± 0.9 3.45 ± 1.3 0.036 Laboratory results PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Page 13/23 Page 13/23 Univariable analysis of SoC group after PS matching Univariable analysis of Anakinra group after PS matching Neutrophil to lymphocyte ratio (median, IQR) 5.64 (4.2) 6.4 (9.2) 0.2 5.6 (4.2) 7 (7.4) 0.1 Hemoglobin (mean ± SD) 13.25 ± 2.4 13 ± 1.3 0.3 13.5 ± 2.2 13 ± 2.6 0.5 Platelets (mean ± SD) 203 ± 76 177 ± 53 0.1 214 ± 76 204 ± 87 0.7 C-reactive protein (mg/L) (median, IQR) Baseline 110 (102) 121 (86) 0.3 99 (101) 121 (94) 0.15 Peak level 121.2 (87.4) 168.9 (88) 0.006 136 (82) 155 (167) 0.2 Ferritin (pg/mL) (median, IQR) Baseline 393 (144) 393 (87) 1 300 (280) 146 (853) 0.6 Peak level 523 (544) 572 (915) 0.005 398 (494) 789 (1108) 0.04 D-dimer (pg/mL) (median, IQR) Baseline 2.1 (1.5) 2.2 (0.94) 0.3 1 (1.3) 1.6 (2.3) 0.4 Peak level 1.8 (3) 10.8 (28) < 0.001 2.54 (4.6) 11 (27) 0.07 Lactate dehydrogenase (U/L) (median, IQR) Baseline 448.4 (29) 448.4 (107.4) 0.6 395 (178) 363 (330) 0.8 Peak level 401 (182.4) 557 (474.5) 0.002 480 (177) 642 (487) 0.01 ALT (U/L) (median, IQR) 20.3 (19) 19.5 (16.8) 0.1 21 (18) 18 (26) 0.7 AST (U/L) (median, IQR) 36 (20) 39 (21) 0.5 38 (22) 42 (36) 0.8 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase PS: Propensity score, SoC: Standard of care, OR: Odds ratio, SD: Standard deviation, IQR: Interquartile range, mcHIS: Modified Covid hyperinflammatory syndrome score, NIH: National Institute Health, ALT: Alanin aminotransferase, AST: Aspartate aminotransferase Page 14/23 Page 14/23 Higher patient age (OR: 1.048, p = 0.018 [95% confidence interval (CI): 1.008–1.09]), male gender (OR: 3.2, p = 0.048 [95% CI: 1.013–10.3]), critical disease (compared to severe) (OR: 141.9, p < 0.001 [95% CI: 16.8– 1198]), SoC treatment (compared to anakinra) (OR: 6.7, p = 0.002 [95% CI: 1.98–22.3]) were associated with higher mortality in multivariable analysis after PSM (Table 5). Analysis of study group after PS Matching Table 5 Multivariable analysis of mortality in patients receiving Anakinra after PS matching Variables B p value OR 95% CI Patient age 0.047 0.018 1.048 1.008–1.09 Gender (male) 1.17 0.048 3.2 1.013–10.3 Critical disease (compared to severe disease) 4.95 < 0.001 141.9 16.8–1198 SoC (compared to anakinra) 1.9 0.002 6.7 1.98–22.3 PS: Propensity score, SoC: Standard of care, OR: Odds ratio, CI: Confidence interval Log-rank: p = 0.14 survival analysis mortality rate was tended to be lower in patients with anakinra compared to SoC roup after PSM (Log-Rank: p = 0.14) (Fig. 3). In survival analysis mortality rate was tended to be lower in patients with anakinra compared to SoC group after PSM (Log-Rank: p = 0.14) (Fig. 3). In survival analysis mortality rate was tended to be lower in patients with anakinra compared to SoC group after PSM (Log-Rank: p = 0.14) (Fig. 3). Discussion In COVID-19 mortality rates differ according features of the study population (hospitalized vs outpatient patients or ward vs ICU), inflammatory burden and disease severity of patients. Several studies revealed higher mortality rate among severe and critically COVID-19 patients. In a retrospective study from Saudi Arabia with 352 critically ill patients, 28-day mortality was 32.1%. In this study, mortality was associated with older age, higher LDH and d-dimer levels (Alharthy et al., 2021). In a multicenter observational study from Mexico with 164 critically ill patients, mortality was 51.8% and higher age as well as CRP levels were also associated with higher mortality (Ñamendys-Silva et al., 2021). In Zhao et al. study, mortality was 2% and 47% among severe and critically ill patients, respectively (Zhao et al., 2021). Beyond the demographic features, there is a close association between poor outcome and higher inflammatory response. A hyperinflammatory score (cHIS score) reflecting cytokine storm was defined by Webb at al (Hu et al., 2021). In this study, mortality increased gradually with higher scores; those were 1%, 15% and 33% in patients had < 2, ≥2 and ≥ 3 scores, respectively. The fact that an average higher mcHIS score of current study population than originally study even removing item of fever reflects a higher inflammatory burden in our study compared to the previous one. Therefore, our study group had more severe disease and higher mortality risk at admission which was expected to have a higher mortality rate. In our study mortality rate of control group was consistent with previous results. Although mortality rate was higher in patients receiving anakinra compared to SoC before PS matching, that was lower in anakinra group than SoC after PS matching. This finding emphasizes the importance of making comparisons in the term of Page 15/23 Page 15/23 relevant treatment efficacies between similar disease groups according to patient characteristics and disease severity. Several risk factors for higher mortality were defined such as age and gender, presence of cancer or on immunosuppression in COVID-19 (Alwani et al., 2021) (Aboueshia et al., 2021). In addition to traditional risk factors some laboratory parameters such as elevated CRP, ferritin, d-dimer, LDH and IL-6 levels were associated poor outcomes including higher mortality (Terpos et al., 2020) (Hanff, Mohareb, Giri, Cohen, & Chirinos, 2020) (Feld, Tremblay, Thibaud, Kessler, & Naymagon, 2020). Discussion These laboratory values are also compatible with other reason of MAS beyond the COVID-19 and reflect higher inflammatory and/or prothrombotic state (Zou et al., 2020) (Gavand et al., 2017). Higher baseline and peak levels of NLR, CRP, ferritin, d-dimer and only peak levels of LDH were associated with mortality both in two groups before PS matching. These findings compatible with previous results. On the other hand, after PS matching only peak levels of CRP, ferritin, d-dimer and LDH levels remained higher in patients deceased compared to survivors. These results indicate that peak levels of such laboratory parameters are more relevant than those baseline levels into the development of mortality in patients with COVID-19 associated cytokine storm. These results were consistent in both treatment groups in our study and further studies are needed to clarify this issue. IL-1 has a driven role in several diseases such as HPFS, AODS and also several hyperinflammatory conditions such as MAS and HLH due to various immune related disorders, infections and malignancies (Gabay, Lamacchia, & Palmer, 2010) (Dinarello, 2011). Additionally, safety and efficacy of IL-1 inhibitors such as anakinra were established in many studies in cytokine storms due to various diseases (Giavridis et al., 2018) (Grom, Horne, & De Benedetti, 2016) (Mehta, Cron, Hartwell, Manson, & Tattersall, 2020). Furthermore, anakinra became one of the standard targeted therapies for the treatment of COVID-19- assciated cytokine storm during the pandemic (Kyriazopoulou et al., 2021). In a comparative study from Italy with 392 patients who consisted of 62 anakinra and 55 anti-IL-6 treatment, overall mortality was 25%, 32% in biologic-naïve, 14% in anakinra and 18% in tocilizumab receiving patients (Cavalli et al., 2021). In this study, higher CRP levels at baseline and decreasing levels of LDH with treatment predicted higher IL-1 and IL-6 inhibitor response and reduced mortality. In a prospective comparative study, mortality was lower in patients receiving anakinra compared to those with standard of care (29% vs 46%; p = 0.082) (Balkhair et al., 2021). These studies revealed higher efficacy of anakinra which were consistent with our results in patients with COVID-19. Intravenous and high dose anakinra is an emerging therapeutic option both in rheumatology and COVID- 19 practice. Intravenous administration of anakinra enables higher and fast maximum plasma concentration compared to subcutaneous administration (Mehta et al., 2020). Declarations Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out work described in this article. Conflicts of Interest: Authors declare no conflicts of interest and also financial relationships. Conflicts of Interest: Authors declare no conflicts of interest and also financial relationships. Availability of data and material: Datasets of the study are available from the corresponding author on reasonable request. Availability of data and material: Datasets of the study are available from the corresponding author on reasonable request. Code availability: Not applicable. Author contributions: M.B. designed and planned the study, M.H.U, M.E.Ö and M.A collected the samples from patients, M.B, S.Y and M.İ.K carried out the data evaluation and basic analysis. All authors contributed to the follow-up of the patients and interpretation of results. M.B. wrote the first draft. All authors provided critical feedback for the last version. Ethics approval: Local ethics committee approval and participant consent were obtained for this study (date and number: 24.02.2022 and 2022/04-09). Ethics approval: Local ethics committee approval and participant consent were obtained for this study (date and number: 24.02.2022 and 2022/04-09). Acknowledgements: Not applicable. Acknowledgements: Not applicable. Conclusion In our study mortality was lower in patients receiving anakinra compared to SoC. Our study also indicates that hyperinflammatory response is one of the most important risk factor for the development of mortality in patients with COVID-19. Intravenous high dose anakinra is safe and effective treatment in patients with severe and critical COVID-19. Discussion Recently there is sufficient evidence with high dose intravenous anakinra administration in various hyperinflammatory syndromes (Nigrovic et al., 2011) (Phadke, Rouster-Stevens, Giannopoulos, Chandrakasan, & Prahalad, 2021). High dose intravenous anakinra treatment was safe and effective in a preliminary retrospective study from Italy in patients with COVID-19 (Cavalli et al., 2020). In another prospective controlled study (ESCAPE open label study) with critically ill COVID-19 patients, high dose intravenous anakinra had lower mortality Page 16/23 Page 16/23 Page 16/23 rate than standard of care as well as tocilizumab treatment (Karakike et al., 2022). No increase in the frequency of severe infection as well other complications such as myocardial infarction in anakinra group compared to controls in our study indicates that high dose intravenous anakinra is safe in patients with COVID-19. Daily dose adjustment of anakinra may allow early intervention of the hyperinflammatory state according to daily clinical and laboratory parameters, as well as withdrawing the drug in case of infection and/or other potential complications. This study has some limitations. Retrospective design of the study was the main limitation. Having missing data is another limitation of the study. 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Clinical and laboratory features, treatment, and outcomes of macrophage activation syndrome in 80 children: a multi-center study in China. World J Pediatr, 16(1), 89–98. doi:10.1007/s12519-019-00256-0 37. Zou, L. X., Zhu, Y., Sun, L., Ma, H. H., Yang, S. R., Zeng, H. S.,. .. Lu, M. P. (2020). Clinical and laboratory features, treatment, and outcomes of macrophage activation syndrome in 80 children: a multi-center study in China. World J Pediatr, 16(1), 89–98. doi:10.1007/s12519-019-00256-0 Figures Figures Page 20/23 Figure 1 Figure 1 Dotplot of standardized mean differences for all covariates before and after PS matching Figure 1 Dotplot of standardized mean differences for all covariates before and after PS matching Dotplot of standardized mean differences for all covariates before and after PS matching Page 21/23 Page 21/23 Figure 2 Flow-chart of the study design and participants Page 22/23 Figure 2 Flow-chart of the study design and participants Figure 3 Comparison of mortality between patients receiving Anakinra and SoC in survival analysis Comparison of mortality between patients receiving Anakinra and SoC in survival analysis Supplementalfigures.docx Figure 2 Flow-chart of the study design and participants Page 22/23 Page 22/23 Fi 3 Figure 3 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. Supplementalfigures.docx Page 23/23
https://openalex.org/W3017004849	https://link.springer.com/content/pdf/10.1007/s12567-020-00310-y.pdf	English	null	Small satellite TT&C allocations below 1 GHz: outcome of ITU WRC-19	CEAS space journal	2,020	cc-by	4,465	Abstract As hundreds of new small satellites are being launched each year, the RF spectrum for satellite communication becomes increasingly occupied. The International Telecommunication Union recognized this problem at World Radiocommunica- tion Conference 2015 (WRC-15) and invited study groups to investigate the utilization of frequency allocations. The studies followed a three-step approach: first, the TT&C spectrum requirements of small satellites, being a new class of satellites, were assessed. Second, the utilization of existing TT&C frequency allocations and their potential to incorporate the future number of satellites was studied. Third, the study groups investigated new potential TT&C frequency allocations in the frequency ranges 150.05–174 MHz and 400.15–420 MHz. The studies were completed for WRC-19. This paper presents the results of the study groups. A study of the spectrum requirements of small satellites has been completed. The required spectrum for TT&C was estimated to be less than 2.5 MHz for downlink and less than 1 MHz for uplink. Consequently, the study groups conducted sharing studies in various bands which yield that no new allocations are suitable for small satellite TT&C on a co-channel sharing basis. However, regulatory measures are proposed that in the study groups’ view will satisfy the small satellite developers’ needs. The paper will summarize the regulatory measures taken after WRC-19 along with a personal appraisal of the author. Keywords Frequency coordination · ITU · WRC · Small satellites · Short duration missions · TT& Keywords Frequency coordination · ITU · WRC · Small satellites · Short duration missions · TT&C Martin von der Ohe1 Received: 20 December 2019 / Revised: 24 March 2020 / Accepted: 25 March 2020 / Published online: 15 April 2020 © The Author(s) 2020 CEAS Space Journal (2020) 12:565–571 https://doi.org/10.1007/s12567-020-00310-y CEAS Space Journal (2020) 12:565–571 https://doi.org/10.1007/s12567-020-00310-y ORIGINAL PAPER * Martin von der Ohe martin.vonderohe@tu‑berlin.de 2.1 Spectrum requirements of satellites with short‑duration missions for TT&C To analyze if existing frequency allocations for TT&C are sufficient to accommodate satellites with short-duration mis- sions, an investigation of the actual spectrum requirements of these satellites was required. The spectrum requirements were assessed based on the report that was published in 2015 [1]. A new report [4] was drafted as the new term “short-duration missions” needed to be included and some parameters had to be corrected or further specified. This report defines typical characteristics of short-duration mis- sions, e.g. a bandwidth of up to 25 kHz, orbits between 300 and 1000 km and satellite EIRP (equivalent isotropically radiated power) of not more than 3 dBW. It should be noted that these reports do not incorporate minimum (worst-case) values that would be needed for a positive link margin, but rather aim to include all kinds of small satellites that have been launched in the past. Accordingly, these values have not been optimized for sharing studies, which was later found to be not favorable for further analyses. The most utilized and at the same time, most crowded bands for short-duration missions have so far been bands below 1 GHz. In these bands, the satellites typically conduct their TT&C. Payload data downlink is usually transferred in higher bands (S band or X band). Therefore, the studies were focused on bands below 1 GHz that are available for satel- lite TT&C, which is defined as “Space Operation Service” (SOS) in ITU terminology. The following chapters will review the studies that have been conducted to investigate potential new allocations for small satellites. Chapter 2 summarizes the technical work that was done by the ITU study group. The author was part of this study group; however, it has to be noted that great parts of the study work have been done by other authors. Study group contributions are submitted to the ITU on behalf of national administrations; therefore, there are no publications available that allow reference to a certain author. The author of this paper, therefore, wants to express that the studies that are described in chapter 2 are only partly the original work of the author. These parts are identified accordingly. The rest of the chapter summarizes the study group work for which the references [4–6] are recommended for further reading. Chapter 3 describes the conclusions of the work of the study group, while chapter 4 presents the outcome of ITU WRC-19. 1 Introduction the suitability of existing allocations to the space opera- tion service in the frequency range below 1 GHz, taking into account the current use. As mentioned before, the characterizing properties of small satellites (mass, dimensions) do not have significance for frequency coordination and another characteristic had to be found that has relevance for coordination. This char- acteristic was found in the typically short mission lifetime which can be directly linked to the “period of validity” of ITU filing procedures. For this reason, ITU study groups refer to “satellites with short duration missions” rather than “small satellites” when this class of satellites is discussed. The mission lifetime (or period of validity) is defined as not more than 3 years. Obviously, this opens the floor for large satellite systems as well; however, it is not expected that large, expensive satellites will be deployed for a mission lifetime of only three years. If studies of the current TT&C allocations indicate that small satellite requirements cannot be met, they shall further study 3. sharing and compatibility studies to consider possible new allocations or an upgrade of the existing allocations for small satellite TT&C within the frequency ranges 150.05–174 MHz and 400.15–420 MHz. 1. the spectrum requirements of satellites with mission durations for TT&C. 2 Study work The work that was assigned to one of the ITU study groups (ITU-R Working Party 7B) was defined in ITU-R Resolution 659 (WRC-15) [7] as follows: The study group shall study between WRC-15 and WRC-19 2.1 Spectrum requirements of satellites with short‑duration missions for TT&C The spectrum needs were derived by simulating 300 sat- ellite–ground station pairs and their interference potential against each other. The number was set to 300 pairs as this value was expected to represent the number of co-existing short duration mission systems that will need to use spec- trum for TT&C below 1 GHz. The ground stations (Fig. 1) and the satellites were distributed based on the current dis- tribution. Two studies (one of which was conducted by the author [8]) yield that the required spectrum for the space-to- Earth direction (Downlink) is less than 2.5 MHz, while the required spectrum for the Earth-to-space direction (Uplink) is less than 1 MHz. The evaluation of existing SOS (TT&C) allocations was based on these estimates. The results are summarized in a report [5]. 1 Introduction frequency coordination were omitted or underestimated by the developers. Additionally, the new developers complained that the existing regulatory procedures are too complicated and time consuming for satellites with short development times. The ITU, therefore, decided at the WRC-12 to study the characteristics of picosatellites and nanosatellites and their current practice in filing satellites to the ITU. The stud- ies were concluded in 2015 with two reports on the charac- teristics [1] and current filing practice [2]. In these reports, it was identified that the characteristics that define small satellites (low mass, small dimensions, low power, etc.) are not relevant from a frequency coordination perspective and that the short development times are still long enough to properly file the systems to the ITU. As a result, it was stated that small satellites fit into the regulatory framework (the ITU Radio Regulations [3]) and that nothing needs to be changed. However, it was found that the bare number of new satellites, irrespective of their size and mass, poses potential harm to existing satellite systems in the respective frequency allocations. The ITU, therefore, decided at WRC- 15 to study the spectrum needs of small satellites and the After the Cubesat design standard was introduced in 1999 and first satellites of this new class have been launched in the subsequent years, small satellites have become increas- ingly popular in the past 5 years. Today not only universi- ties use small satellite platforms for education and technol- ogy demonstration but also commercial operators started to develop and deploy satellites with masses of typically less than 50 kg and reasonably short development times. Cur- rently hundreds of new satellites are launched into space per year. The increase of launches was recognized by the Inter- national Telecommunication Union (ITU) which is respon- sible for the coordination of the shared use of frequencies. As the first Cubesats were mainly launched by new entrants into the space sector, mandatory regulatory procedures like 1 Technische Universität Berlin, Department of Aeronautics and Astronautics, Chair of Space Technology, Marchstr. 12‑14, 10587 Berlin, Germany :(0123 1 3456789) 3 566 M. von der Ohe potential necessity of new frequency allocations for their TT&C (Tracking, telemetry & control). 2. the suitability of existing allocations to the space opera- tion service in the frequency range below 1 GHz, taking into account the current use. 2. 2.2 Suitability of existing SOS (TT&C) allocations below 1 GHz The existing frequency allocations that can be used for TT&C below 1 GHz are summarized in Table 1. The table 1 3 Small satellite TT&C allocations below 1 GHz: outcome of ITU WRC‑19 567 Fig. 1 Distribution of ground stations as defined in the study group work [8] Fig. 1 Distribution of ground stations as defined in the study group work [8] Table 1 Existing allocations to the Space Operation Service below 1 GHz Frequency band (MHz) Status Direction Allocated bandwidth (MHz) Special obligations/constraints 30.005–30.010 Primary N/A 0.005 Satellite identification 137–138 Primary Space-to-Earth 1.0 – 148–149.9 Primary Earth-to-space 1.9 RR No. 9.21 applies 267–272 Secondary Space-to-Earth 5 RR No. 9.21 applies, NATO band 272–273 Primary Space-to-Earth 1 NATO band 400.15–401 Secondary Space-to-Earth 0.85 401–402 Primary Space-to-Earth 1 449.75–450.25 Primary Earth-to-space 0.5 RR No. 9.21 Table 1 Existing allocations to the Space Operation Service below 1 GHz Article No. 9.21 states that “before an administration notifies to the [ITU] Bureau or brings into use a frequency assign- ment […], it shall effect coordination, as required, with other administrations […] for any station of a service for which the requirement to seek the agreement of other administrations is included in a footnote to the Table of Frequency Alloca- tions referring to this provision” [3]. This statement is as difficult to understand as it is dangerous for potential users of the band; all potential users must search agreement from other administrations before the band can be used. The other administrations can refuse agreement without a reason. Con- sequently, even if sharing would be technically feasible, the sharing can be prohibited, e.g. for political reasons. For this reason, ITU-R Resolution 659 states that “that the existing shows the allocated bands, the protection status, the direc- tion of the allocation, the bandwidth and if special obliga- tions apply. The status is either primary or secondary. Pri- mary allocations are protected against secondary services and only have to coordinate with other primary services. Accordingly, primary services are preferable. The direction depicts if the band can be used for uplink or downlink. The 30.005–30.010 MHz slot can be neglected as it is very small and not feasible for small satellite antenna sizes. The remaining allocations sum up to 2.4 MHz in the uplink and to 8.85 MHz for the downlink. 2.3 Potential new frequency allocations for SOS (TT&C) ITU-R Resolution 659 states that if the existing SOS alloca- tions are not sufficient to accommodate satellites with short- duration missions, other frequency ranges between 150.05 and 174 MHz as well as between 400.15 and 420 MHz shall be studied. Obviously, these bands have incumbent services that use the bands, either globally or regionally harmonized. Tables 2 and 3 summarize the incumbent services for each band portion. Capitalized service names refer to primary sta- tus while uncapitalized refers to secondary status. Footnotes in form 5.XXX add special obligations to many of the band portions but are not included in this paper. fi c) Method C was to use 137–138 MHz for the downlink and make available 148–149.9 MHz for the uplink by removing the footnote regarding Article No. 9.21 and with that remove the dependency on the goodwill of other administrations. While Method C was seen to be the most probable solu- tion by most members of the ITU study groups, it was high- lighted that the compatibility of 137–138 MHz with aero- nautical mobile (route) services in the adjacent band below 137 MHz was not yet studied. Therefore, strong opposition was expected at WRC-19. The reason why effects to adjacent bands where not studied is that 137–138 MHz already was an existing allocation for space operation service and as such the need for new compatibility studies was not seen. Sharing studies on each of the bands 150–174 MHz and 400.15–420 MHz have been done and the results are sober- ing. Almost all bands have been identified as not feasible for co-channel sharing. The sharing analyses have been based on characteristics of the incumbent services and their protection criteria as well as the short-duration missions’ parameters. A report of more than 150 pages [6] was writ- ten, and the results clearly state that co-channel sharing is not possible. Only in the band that is currently mainly used by radiosonde systems (403–406 MHz), the studies show two different results. Some administrations state that sharing with radiosondes would be possible under certain circum- stances and proposed a new allocation in these bands. Besides the studies on TT&C allocations for short-dura- tion missions, late in the study cycle a modification to the regulatory procedure to file short-duration missions was pro- posed. 3 Short‑duration missions at WRC‑19 The study group and a subsequent conference preparatory meeting (CPM) proposed three possible methods to solve the agenda item on short-duration missions at WRC-19: Irrespective of the parallel developments mentioned in the paragraph above, the studies on potential new allocations for TT&C yielded that, in general, the existing downlink allocations are sufficient, while uplink allocations are not feasible, given the existing constraints. a) Method A proposes to not change anything, since some administrations thought that changes are not needed for short duration missions. b) Methods B1 and B2 propose a new Earth-to-space allo- cation either between 403 and 404 MHz or 404 and 405 MHz. These bands are the before-mentioned radio- sonde bands and it was expected that this proposal will be difficult for many administrations. 2.2 Suitability of existing SOS (TT&C) allocations below 1 GHz In general, these numbers should be sufficient to incorporate short-duration missions; however, special obligations exist that make the use of the bands not possible or at least difficult. Radio Regulations 1 568 M. von der Ohe be sufficient, while the Earth-to-space bands are blocked for short-duration missions by Radio Regulations Article No. 9.21. As a simple solution, it was proposed to delete the reference to Article No. 9.21. Most study group participants agreed that this would solve the problem and render the need for new allocations unnecessary. allocations to the space operation service below 1 GHz, where No. 9.21 applies, are not suitable”. In the space-to- Earth direction, the portions 267–273 MHz are part of a NATO harmonized band and coordination needs agreement of military administrations, at least in NATO countries. At the same time as developers and operators of satel- lites with short-duration missions tried to identify potential new frequency allocations, other operators of “traditional” satellites sought to limit the use of certain bands around 400 MHz to limit the interference potential for data col- lecting systems operating in these bands. More details can be found in ITU-R Resolution 765 [9] and the final acts of WRC-19 which put power limits in these bands [10]. 2.3 Potential new frequency allocations for SOS (TT&C) In this proposal, it was suggested that the time that is needed to register a short-duration mission with the ITU can be significantly reduced, if the system is only short term and consists of less than ten satellites. This proposal was linked with the studies on potential new TT&C allocations during WRC-19. As a result, the following changes were agreed upon dur- ing the conference: 2.4 Upgrade of existing SOS allocations During the ITU study group meetings between 2015 and 2019, it was proposed to change the constraints of exist- ing TT&C bands instead of finding new allocations. 2.4 Upgrade of existing SOS allocations von der Ohe 570 570 M. von der Ohe Table 2 (continued) Allocation to services Region 1 Region 2 Region 3 162.0375–174 FIXED MOBILE except aeronautical mobile 5.226 5.229 162.0375–174 FIXED MOBILE 5.226 5.229 5.231 162.0375–174 FIXED MOBILE 5.226 5.229 5.231 Table 3 Frequency allocation table in the frequency range 400.15–420 MHz [3] Allocation to services 400.15–401 METEOROLOGICAL AIDS METEOROLOGICAL-SATELLITE (space-to-Earth) MOBILE-SATELLITE (space-to-Earth) 5.208A 5.208B 5.209 SPACE RESEARCH (space-to-Earth) 5.263 Space operation (space-to-Earth) 5.262 5.264 401–402 METEOROLOGICAL AIDS SPACE OPERATION (space-to-Earth) EARTH EXPLORATION-SATELLITE (Earth-to-space) METEOROLOGICAL-SATELLITE (Earth-to-space) Fixed Mobile except aeronautical mobile 402–403 METEOROLOGICAL AIDS EARTH EXPLORATION-SATELLITE (Earth-to-space) METEOROLOGICAL-SATELLITE (Earth-to-space) Fixed Mobile except aeronautical mobile 403–406 METEOROLOGICAL AIDS Fixed Mobile except aeronautical mobile 5.265 406–406.1 MOBILE-SATELLITE (Earth-to-space) 5.265 5.266 5.267 406.1–410 FIXED MOBILE except aeronautical mobile RADIO ASTRONOMY 5.149 5.265 410–420 FIXED MOBILE except aeronautical mobile SPACE RESEARCH (space-to-space) 5.268 other services. In this case, simplified filing procedures apply. other services. In this case, simplified filing procedures apply. 2.4 Upgrade of existing SOS allocations As stated above, the space-to-Earth bands were considered to • The frequency band 137.025–138 MHz shall be used for short-duration missions, if they do not cause harm- ful interference and if they do not claim protection from 1 3 Small satellite TT&C allocations below 1 GHz: o Table 2 Frequency allocation table in the fre Allocation to services Region 1 150.05–153 FIXED MOBILE except aeronautical mobile RADIO ASTRONOMY 5.149 153–154 FIXED MOBILE except aeronautical mobile (R) meteorological aids 154–156.4875 FIXED MOBILE except aeronautical mobile (R) 5.225A 5.226 156.4875–156.5625 MARITIME MOBILE (distress and calling via DSC) 5.111 5.226 5.227 156.5625–156.7625 FIXED MOBILE except aeronautical mobile (R) 5.226 156.7625–156.7875 MARITIME MOBILE Mobile-satellite (Earth-to-space) 5.111 5.226 5.228 156.7875–156.8125 MARITIME MOBILE (distress and calling) 5.111 5.226 156.8125–156.8375 MARITIME MOBILE Mobile-satellite (Earth-to-space) 5.111 5.226 5.228 156.8375–161.9375 FIXED MOBILE except aeronautical mobile 5.226 161.9375–161.9625 FIXED MOBILE except aeronautical mobile Maritime mobile-satellite (Earth-to-space) 5.228AA 5.226 161.9625–161.9875 FIXED MOBILE except aeronautical mobile Mobile-satellite (Earth-to-space) 5.228F 161.9875–162.0125 FIXED MOBILE except aeronautical mobile Maritime mobile-satellite (Earth-to-space) 5.228AA 5.226 5.229 162.0125–162.0375 FIXED MOBILE except aeronautical mobile Mobile-satellite (Earth-to-space) 5.228F 569 Small satellite TT&C allocations below 1 GHz: outcome of ITU WRC‑19 Table 2 Frequency allocation table in the frequency range 150.05–174.00 MHz [3] Allocation to services Region 1 Region 2 Region 3 150.05–153 FIXED MOBILE except aeronautical mobile RADIO ASTRONOMY 150.05–154 FIXED MOBILE 150.05–154 FIXED MOBILE 5.149 5.225 5.225 153–154 FIXED MOBILE except aeronautical mobile (R) meteorological aids 154–156.4875 FIXED MOBILE except aeronautical mobile (R) 5.225A 5.226 154–156.4875 FIXED MOBILE 5.226 154–156.4875 FIXED MOBILE 5.225A 5.226 156.4875–156.5625 MARITIME MOBILE (distress and calling via DSC) 5.111 5.226 5.227 156.4875–156.5625 MARITIME MOBILE (distress and calling via DSC) 5.111 5.226 5.227 156.4875–156.5625 MARITIME MOBILE (distress and calling via DSC) 5.111 5.226 5.227 156.5625–156.7625 FIXED MOBILE except aeronautical mobile (R) 5.226 156.5625–156.7625 FIXED MOBILE 5.226 156.5625–156.7625 FIXED MOBILE 5.226 156.7625–156.7875 MARITIME MOBILE Mobile-satellite (Earth-to-space) 5.111 5.226 5.228 156.7625–156.7875 MARITIME MOBILE MOBILE-SATELLITE (Earth-to-space) 5.111 5.226 5.228 156.7625–156.7875 MARITIME MOBILE Mobile-satellite (Earth-to-space) 5.111 5.226 5.228 156.7875–156.8125 MARITIME MOBILE (distress and calling) 5.111 5.226 156.7875–156.8125 MARITIME MOBILE (distress and calling) 5.111 5.226 156.7875–156.8125 MARITIME MOBILE (distress and calling) 5.111 5.226 156.8125–156.8375 MARITIME MOBILE Mobile-satellite (Earth-to-space) 5.111 5.226 5.228 156.8125–156.8375 MARITIME MOBILE MOBILE-SATELLITE (Earth-to-space) 5.111 5.226 5.228 156.8125–156.8375 MARITIME MOBILE Mobile-satellite (Earth-to-space) 5.111 5.226 5.228 156.8375–161.9375 FIXED MOBILE except aeronautical mobile 5.226 156.8375–161.9375 FIXED MOBILE 5.226 156.8375–161.9375 FIXED MOBILE 5.226 161.9375–161.9625 FIXED MOBILE except aeronautical mobile Maritime mobile-satellite (Earth-to-space) 5.228AA 5.226 161.9375–161.9625 FIXED MOBILE Maritime mobile-satellite (Earth-to-space) 5.228AA 5.226 161.9375–161.9625 FIXED MOBILE Maritime mobile-satellite (Earth-to-space) 5.228AA 5.226 161.9625–161.9875 FIXED MOBILE except aeronautical mobile Mobile-satellite (Earth-to-space) 5.228F 161.9625–161.9875 AERONAUTICAL MOBILE (OR) MARITIME MOBILE MOBILE-SATELLITE (Earth-to-space) 161.9625–161.9875 MARITIME MOBILE Aeronautical mobile (OR) 5.228E Mobile-satellite (Earth-to-space) 5.228F 161.9875–162.0125 FIXED MOBILE except aeronautical mobile 161.9875–162.0125 FIXED MOBILE 161.9875–162.0125 FIXED MOBILE Table 2 Frequency allocation table in the frequency range 150.05–174.00 MHz [3] 154–156.4875 FIXED MOBILE 5.226 156.4875–156.5625 MARITIME MOBILE (distress and calling via DSC) 5.111 5.226 5.227 156.5625–156.7625 FIXED MOBILE 5.226 156.7625–156.7875 MARITIME MOBILE MOBILE-SATELLITE (Earth-to-space) 5.111 5.226 5.228 156.7875–156.8125 MARITIME MOBILE (distress and calling) 5.111 5.226 156.8125–156.8375 MARITIME MOBILE MOBILE-SATELLITE (Earth-to-space) 5.111 5.226 5.228 156.8375–161.9375 FIXED MOBILE 5.226 161.9375–161.9625 FIXED MOBILE Maritime mobile-satellite (Earth-to-space) 5.228AA 5.226 161.9625–161.9875 1 M. other services. In this case, simplified filing procedures apply. References The main reason why new allocations have not been opened is because of the way that the compatibility stud- ies are conducted. When potential new allocations were discussed during WRC-15, it was defined that new alloca- tions are only possible if co-channel sharing is possible. This definition of sharing study does not incorporate the fact that some of the bands between 150.05–174 MHz and 400.14–420 MHz are not as heavily used as in recent years. Additionally, the recommendations that were applied to investigate sharing feasibility are at least debatable and, in many cases, outdated. It is believed that small satellites/ short-duration missions could have been accommodated in some UHF ranges, if studies could have been oriented at the realistic use instead of co-channel studies which are based on partly outdated recommendations. 1. ITU-R: Report ITU-R SA.2312-0, Characteristics, definitions and spectrum requirements of nanosatellites and picosatellites, as well as systems composed of such satellites, Geneva, Switzerland (2014) ( ) 2. ITU-R: Report ITU-R SA.2348-0, Current practice and procedures for notifying space networks currently applicable to nanosatellites and picosatellites, Geneva, Switzerland (2015) 3. ITU-R: ITU-R Radio Regulations, Geneva, Switzerland (2016) 4. ITU-R: Report ITU-R SA.2426: Technical characteristics for telemetry, tracking and command in the space operation service below 1 GHz for non-GSO satellites with short duration missions, Geneva, Switzerland (2018) 5. ITU-R: Report ITU-R SA.2425: Studies to accommodate spec- trum requirements in the space operation service for non-geosta- tionary satellites with short duration missions, Geneva, Switzer- land (2018) 6. ITU-R: Report ITU-R SA.2427: Studies on the suitability of exist- ing allocations to the space operation service below 1 GHz and additional sharing studies on possible new and/or upgraded alloca- tions, Geneva, Switzerland (2018) Given this personal judgment, it is debatable how future will change the regulatory landscape for small satellites. It is most likely that small satellite developers will move to higher bands in the absence of satisfying solutions in UHF. The few usable allocations will become more crowded and interferences are expected to increase. In preparation for the next WRC in 2023, no agenda items on new allocations below 1 GHz are scheduled. 7. ITU-R: ITU-R Resolution 659 (WRC-15), Studies to accommo- date requirements in the space operation service for non-geosta- tionary satellites with short duration mission, Geneva, Switzer- land, (2015) 8. M. Buscher, A. Balke, K. 4 Conclusion and outlook pp y • For most countries, Art. 9.21 was removed from the uplink band 148–149.9 MHz. Only for some countries (Armenia, Azerbaijan, Belarus, China, Korea (Rep. of), Cuba, Russian Federation, India, Iran (Islamic Repub- lic of), Japan, Kazakhstan, Malaysia, Uzbekistan, Kyrgyzstan, Thailand and Viet Nam) this was linked to power limits and a 1% duty cycle that shall not be exceeded at the country borders. pp y • For most countries, Art. 9.21 was removed from the uplink band 148–149.9 MHz. Only for some countries (Armenia, Azerbaijan, Belarus, China, Korea (Rep. of), Cuba, Russian Federation, India, Iran (Islamic Repub- lic of), Japan, Kazakhstan, Malaysia, Uzbekistan, Kyrgyzstan, Thailand and Viet Nam) this was linked to power limits and a 1% duty cycle that shall not be exceeded at the country borders. This chapter is intended to provide the author’s conclu- sions of the work and might diverge from other opinions. Although it could be claimed that with these changes new opportunities have opened for small satellites, the benefits in the author’s opinion are marginal. The only changes that have been made are in VHF bands. Even though losses are lower in VHF than in UHF, VHF bands are not desired by many developers because of the bad ratio between antenna size and satellite size. Moreover, there are already exist- ing TT&C downlink allocations in 400.15–402 MHz that might be more favorable than the 137–138 MHz allocation. 1 3 571 Small satellite TT&C allocations below 1 GHz: outcome of ITU WRC‑19 The 148–149.9 MHz uplink band has been opened for short-duration missions; however, it is disputable whether this band will be favored by small satellite developers. From a developer’s perspective, the results of WRC-19 for new TT&C allocations are not satisfying. New allocations in UHF would have been favored. The 148–149.9 MHz uplink band has been opened for short-duration missions; however, it is disputable whether this band will be favored by small satellite developers. From a developer’s perspective, the results of WRC-19 for new TT&C allocations are not satisfying. New allocations in UHF would have been favored. 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/. 10. ITU-R: WRC-19 Provisional Final Acts, Sharm El-Sheikh, Egypt (2019) References Brieß: Spectrum requirements for small satellite TT&C and regulatory status of small satellites, Proceed- ings of the 11th IAA Symposium on Small Satellites for Earth Observation, Berlin, Germany (2017) Acknowledgements Open Access funding provided by Projekt DEAL. This project was funded by the Federal Ministry for Economic Affairs and Energy (BMWi) under Grant no. 50 YB1635. The author would like to express his gratitude for the good and cooperative discussions and negotiations in the ITU study groups, which always stay on an objective, professional level even when opinions diverge. 9. ITU-R: ITU-R Resolution 765, Establishment of in-band power limits for earth stations operating in mobile-satellite the meteoro- logical service, the meteorological-satellite service and the Earth exploration-satellite service in the frequency bands 401–403 MHz and 399.9-400.05 MHz, Geneva, Switzerland (2015) 10. ITU-R: WRC-19 Provisional Final Acts, Sharm El-Sheikh, Egypt (2019) Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1 3 1 3 3
https://openalex.org/W2150868856	https://lipidworld.biomedcentral.com/counter/pdf/10.1186/1476-511X-10-73	English	null	Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane	Lipids in health and disease	2,011	cc-by	13,226	© 2011 Corsetto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane * Correspondence: angelamaria.rizzo@unimi.it Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Italy Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane Paola A Corsetto, Gigliola Montorfano, Stefania Zava, Ilaria E Jovenitti, Andrea Cremona, Bruno Berra and Angela M Rizzo* Abstract Background: PUFAs are important molecules for membrane order and function; they can modify inflammation- inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells. Methods: Two lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane. Results: We have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR. N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa. Conclusions: Our results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Antibodies The mouse monoclonal anti-Bcl2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the C20 goat polyclonal anti-procaspase-8 p18 antibody were used to study the n-3 PUFA induction of the apoptosis process. The 1005 rabbit polyclonal anti-EGFR antibody and the 11C2 mouse monoclonal anti-pEGFR antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to investigate the alterations of EGFR receptors after treat- ment with PUFAs. The monoclonal anti-actin (AC-40) antibody (Sigma-Aldrich, USA) was used to normalize gel loading. Nevertheless the mechanism by which n-3 PUFAs inhibit the growth of breast cancer cells is not well understood, but it has been suggested that these fatty acids might change the fluidity and structure of the cell membrane. In fact, changes in the structural characteris- tics of the plasma membrane in mammalian cells can modify the activity of proteins that function as ion chan- nels, transporters, receptors, signal transducers or enzymes [21-25]. Bound primary antibodies were visualized by second- ary horseradish peroxidase (HRP)-linked antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and immunoreactivity was assessed by chemilumines- cence (ECL, Amersham). In this study, we have investigated the impact of EPA, DHA and AA on breast cancer cell growth, on cell sig- nalling in apoptosis and on epidermal growth factor receptor (EGFR) activity. We hypothesize that the alteration of cellular cycle, of gene expression and the induction of apoptosis determined from n-3 PUFAs are also a consequence of membrane architecture modifica- tions. For these reasons we have analyzed PUFA incor- poration in breast cancer membrane and their PL- specific enrichment. PUFAs EPA (cis-5,8,11,14,17-eicosapentaenoic acid sodium salt), DHA (cis-4,7,10,13,16,19-docosahexaenoic acid sodium salt) and AA (arachidonic acid sodium salt) were pur- chased from Sigma-Aldrich, USA. The PUFAs were dis- solved in ethanol and stored at -80°C under nitrogen gas. Cell viability assay Th b f i The numbers of viable cells exposed to fatty acids were evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) colorimetric assay [26]. Initially, cells were seeded and cultured in 96-well plates for 48 h to allow adhesion to the plate and to reach 50- 60% confluence. After this period, the culture medium was changed to the experimental medium supplemented with EPA or DHA or AA then cultured for 72 h. We stu- died the effects of different concentrations of PUFAs (50-300 μM). The final concentration of ethanol (<1%) in the culture medium had no antiproliferative effect on any cell line tested; therefore, 10 μl of MTT stock solution (5 mg/ml in PBS, pH 7.5) was added to each well and incubated for 4 h as a control. Then 100 μl of solubilizing solution (10% SDS in 0.01M HCl) was added and incu- bated overnight. Plates were read at 540 nm in a plate reader. All reagents were purchased from Sigma-Aldrich, Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 2 of 16 process estradiol via cytoplasmic estrogen receptors. The MDA-MB-231 cells over-express EGFR. cells [10,11]. Similarly, in vitro treatment with EPA is reported to arrest the growth of K-562 human leukemic and many other cancer cells accompanied by down- regulation of cyclin expression in some instances [12-14]. These cell lines were maintained in DMEM (Gibco- BRL, Life Tecnologies Italia srl, Italy) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. In addition, recent studies of human breast cancer have shown that n-3 PUFAs up-regulate syndecan 1 (SDC-1), which has been shown to play a role in cell adhesion [15,16], inhibit matrix metalloproteinases [17] and decrease invasion of tumour cells. SDC-1 induces apoptosis in myeloma cells and some studies suggest a similar property in breast cancer cells [18,19]. The tran- scriptional pathway for the n-3 PUFA regulation of SDC-1 expression involves the nuclear hormone recep- tor peroxisome proliferator-activated receptor gamma (PPARg) [20]. Moreover n-3 PUFAs down-regulate the expression of HER2/neu, a well characterized oncogene that plays a key role in aetiology, progression and che- mosensitivity of various types of human cancer in which this oncogene is over-expressed. HER2/neu encodes transmembrane tyrosine kinase orphan receptor p185Her2/neu, which regulates biological functions includ- ing cellular proliferation, differentiation, motility and apoptosis [21]. Medium for treatments (MFT) was DMEM supple- mented with 10% FBS. Cells were grown at 37°C in a 5% CO2 atmosphere with 98% relative humidity. Background actions of n-3 PUFAs. The most prominent mechanism for the chemopreventive action of n-3 PUFAs is their suppressive effect on the production of arachidonic acid (AA)-derived prostanoids, particularly prostaglandin E2 (PGE2), which has been implicated in the immune response to inflammation, cell proliferation, differentia- tion, apoptosis, angiogenesis and metastasis [6]. Breast cancer is the most common cancer among women worldwide, with an estimated 1.4 million new breast can- cer cases only in 2008. Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and subsequent progression of breast can- cer [1-3]. The role that long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play in the aetiology of cancer has been highlighted by animal experiments and in vitro studies [4,5]. A number of mechanisms have been proposed for the anticancer The n-3 PUFAs might alter the growth of tumour cells by influencing cell replication, by interfering with compo- nents of the cell cycle or by increasing cell death either by way of necrosis or apoptosis [7,8]. For example, these fatty acids are involved in regulating the tumour p53 proapop- totic signal and superoxide dismutase (SOD) levels, telo- mere shorting and tumour angiogenesis [9]. In vitro treatment with DHA arrested cell-cycle progression in human-derived breast cancer and malignant melanoma * Correspondence: angelamaria.rizzo@unimi.it Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Italy Cell treatment Cell culture experiments were done with the MDA-MB- 231 and MCF-7 cell lines to determine the concentra- tions of EPA (230 μM), DHA (200 μM), required to inhibit growth by 20-30%, and AA (200 μM). Cells were seeded at 1.5 × 104 cells/cm2 for MDA-MB-231 and at 3 × 104 cells/cm2 for MCF-7 in 18 ml of medium con- taining 10% FBS and allowed to adhere for 48 h, then the medium was replaced with 18 ml of fresh medium (DMEM, 10% FBS) containing the experimental fatty acids and incubated for 72 h without changing the med- ium. Experiments included untreated cells that were not exposed to any exogenous fatty acids but to an equal content of ethanol during the incubation to serve as controls. After 72 h, cells were harvested using trypsin- EDTA and centrifuged at 900 rpm for 10 min. The supernatant was removed and the pellets were subjected to lipid analysis. After elution, the eluate was split with one part going to the detector and nine parts to a Gilson fraction col- lector model 201 to collect the different PL classes for GC analysis. Total fatty acids and PL fatty acids were determined as methylesters by gas chromatography (GC). The methyl esters were obtained by reaction with 3.33% (w/v) sodium methoxide in methanol and injected into an Agilent Technologies (6850 series II) gas chromatograph equipped with a flame ionization detector (FID) and a capillary column (AT Silar) (length 30 m, film thickness 0.25 μm). The carrier gas was helium, the injector tem- perature was 250°C, the detector temperature was 275°C, the oven temperature was set at 50°C for 20 min and then increased to 200°C at 10°C min-1 for 20 min. Cell lines and culture conditions Human breast cancer cell lines MDA-MB-231 (ER-nega- tive) and MCF-7 (ER-positive) were kindly provided by Dr P. Degan from the IST (Italian National Cancer Research Institute, Genoa Italy, Laboratory of Molecular Mutagenesis and DNA Repair). Both cell lines are derived from human mammary adenocarcinoma; the MCF7 line retains several characteristics of differen- tiated mammary epithelium, including the ability to Page 3 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 USA. Data points represent the mean of eight wells and the results are expressed as relative growth rate (RGR) in comparison to controls that were exposed to a concen- tration of ethanol equal to that in the samples exposed to fatty acids. time was 40 min/sample; 23 min analysis, 12 min to restore initial conditions and 5 min for re-equilibration. Eluent A was chloroform/methanol/water (80:19.5:0.5, by vol.) and eluent B was chloroform/methanol/water (60:34:6, by vol.) and the flow rate of the eluent was 1.0 ml/min. An evaporative light-scattering detector (ELSD) was used to detect and quantify the separated PL species. Western blot analysis for Bcl2, caspase-8, EGFR and pEGFR For western blot analysis, cells were harvested by scrap- ing in phosphate-buffered saline containing 0.4 mM Na3VO4. Cells were centrifuged and then suspended in 1.4 ml of lysis buffer (1% Triton X-100, 10 mM Tris buf- fer, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 75 mU/ml aproti- nin), kept on ice for 20 min and then disrupted by 10 strokes in a tight-fitting Dounce homogenizer. The cell lysate was centrifuged (5 min at 1300 g) and the supernatant was transferred to an eppendorf tube. Total protein was determined by the Lowry assay [27]. Control and treated cell lysates (100 μg protein/lane) were separated by SDS-PAGE (10% polyacrylamide gel), transferred to a polyvinylidene difluoride (PVDF) mem- brane and analysed by western blot with anti-Bcl2 (1:100), anti-caspase-8 (1:500) and b-actin (1:1800) anti- bodies. The PVDF membrane was blocked for 1 h in blocking buffer 5% (w/v) dried non-fat milk in Tris-buf- fered saline (T-TBS: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween®20) followed by incubation with an appropriate primary antibody in blocking buffer at room temperature for 2 h. The blots were washed with T-TBS and then incubated with the proper second- ary antibody in blocking buffer at room temperature for 1 h. The protein bands were visualized using ECL wes- tern blot detection reagents (PerkinElmer, USA). DHA and EPA induce apoptosis in breast cancer cells DHA and EPA induce apoptosis in breast cancer cells In order to delineate the possible mechanism(s) by which EPA and DHA induce apoptosis we examined the cytoplasmic levels of the Bcl2 protein. Figure 2A and 2B indicate that there was a slight reduction of Bcl2 level in MCF7 cells after treatment with 200 μM DHA, whereas treatment with 230 μM EPA determined the loss of sig- nal; the expression of Bcl2 is also decreased when MDA-MB-231 cells are treated with 230 μM EPA and the protein is not detectable after incubation with 200 μM DHA. The expression of procaspase-8 was determined by western blot analysis. In Figure 3A and 3B it is possible to observe a reduction of the proform of caspase-8 for both cell lines treated with EPA and DHA; the reduc- tion was statistically significant after DHA treatment in both cell lines, and also after EPA treatment of MCF7 cells. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 0 50 100 150 50 100 150 200 220 240 260 280 300 EPA (M) RGR (%) MDA MCF7 ** * 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA (M) RGR (%) * ** 0 50 100 150 50 100 150 200 250 300 AA (M) RGR (%) ** A B C Figure 1 Effects of PUFA on viability of breast cancer cells. The effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). * p < 0.05; p < 0.01 compared to control cells. blots were probed under the same conditions with primary antibody b-actin diluted 1:1800 to confirm equal protein loading. blots were probed under the same conditions with primary antibody b-actin diluted 1:1800 to confirm equal protein loading. 0 50 100 150 50 100 150 200 220 240 260 280 300 EPA (M) RGR (%) MDA MCF7 * A The relative intensities of band signals were deter- mined by digital scanning densitometry and b-Actin was used to normalize the results to protein content. Statistical analysis The data are presented as mean ± SD. Student’s unpaired t-test was used for comparisons between trea- ted and control cells and the level of statistical signifi- cance was set at P < 0.05 and P < 0.01. 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA (M) RGR (%) * ** ** B B Lipid composition analysis Cell lipids were extracted with three different chloro- form/methanol mixtures (1:1, 1:2 and 2:1, v/v) and parti- tioned with a theoretical upper phase (chloroform/ methanol/water, 47:48:1, by vol.) and then with water. The organic phase was dried and then suspended in chloroform/methanol (2:1, v/v) for the analysis of total and PL fatty acids. For the analysis of EGFR and p-EGFR, cells treated or not with DHA and EPA were cultured in MFTs supple- mented with 10 nM EGF (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C for 15 min of stimulation. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed as described above. Equal amounts of protein (100 μg/lane) from each treatment were sepa- rated by SDS-PAGE (10% polyacrylamide gel) and trans- ferred onto a PVDF membrane then blocked in blocking buffer at room temperature for 1 h. Primary antibodies to EGFR and p-EGFR were diluted 1:200 in blocking buffer at room temperature for 2 h and then with an appropriate secondary antibody at room temperature for 1 h. Parallel Purification of single PL moieties was achieved with an HPLC-ELSD system (Jasco, Japan) equipped with one pump, a SCL-10 Advp, a degasser module and a Rheo- dyne manual injector with 20 μl sample loop and a col- umn (length 250 mm, I.D 4.6 mm and film thickness 5 μm) packed with silica normal-phase LiChrospher Si 60 (LiChroCART 250-4, Merck, Darmstadt, Germany). The chromatographic separation was achieved with a linear binary gradient of 0% B to 100% B in 14 min and then 100% B for 9 min. The total chromatographic run Page 4 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 The effects of treatment with PUFAs on breast cancer cell growth * p < 0.05; p < 0.01 compared to control cells. From these experiments, we extrapolated the dose to be used in successive experiments to assess n-3 PUFA incorporation into cell membrane PLs: 230 μM for EPA, 200 μM for DHA, which correspond to 70~80% viability for both cell lines, and 200 μM for AA. The effects of treatment with PUFAs on breast cancer cell growth To evaluate the effects of PUFAs on breast cancer pro- liferation, cells were incubated for 3 days in medium supplemented with n-3 and n-6 PUFAs (EPA, DHA and AA). 0 50 100 150 50 100 150 200 250 300 AA (M) RGR (%) ** C C The effect on cell viability of MDA-MB-231 and MCF7 cells was assessed and quantified by the MTT assay. As shown in Figure 1, cells were treated with var- ious concentrations of n-3 and n-6 PUFAs in the range 50-300 μM. DHA and EPA induce a dose-dependent reduction of cell viability at concentrations > 200 μM (Figure 1A and 1B). Figure 1 Effects of PUFA on viability of breast cancer cells. The Figure 1 Effects of PUFA on viability of breast cancer cells. The effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). * p < 0.05; p < 0.01 compared to control cells. In contrast, AA (Figure 1C), the major n-6 PUFA, had no significant effect on MCF7 cell viability. The MCF7 cell line was more resistant than the MDA-MB-231 cell line to all treatments with PUFAs. effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). EPA and DHA alter the EGFR and pEGFR levels in MDA- MB-231 cells p < 0.01 compared to control cells; n = 3 Bcl2 MCF7 0 20 40 60 80 100 120 EPADHA CTR TREATMENT A MCF7 Bcl2 ȕ-actin CTR EPA CTR DHA CTR EPA CTR DHA Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT ProCASPASE 8 A MCF7 -actin CTR EPA CTR DHA B MDA-MB-231 CTR EPA CTR DHA B MDA-MB-231 CTR EPA CTR DHA Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT B MDA-MB-231 -actin ProCASPASE 8 -actin ProCASPASE 8 Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPADHA CTR TREATMENT B MDA-MB-231 CTR EPA CTR DHA Bcl2 ȕ-actin B MDA-MB-231 CTR EPA CTR DHA Bcl2 ȕ-actin B Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPADHA CTR TREATMENT Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment with DHA (200 μM) and EPA (230 μM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. B: MDA-MB-231 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. Semi-quantitative analysis performed by plate scanning. b-actin was used to normalize results of protein content. p < 0.01 compared to control cells; n = 3 Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment with DHA (200 μM) and EPA (230 μM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. B: MDA-MB-231 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. Semi-quantitative analysis performed by plate scanning. b-actin was used to normalize results of protein content. p < 0.01 compared to control cells; n = 3 Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment Figure 2 Effects of n-3 PUFA on Bcl2 expression. Both cell lines were treated with DHA (200 μM) or EPA (230 μM) for 72 h. EPA and DHA alter the EGFR and pEGFR levels in MDA- MB-231 cells Control and treated cell lysates are separated on 10% SDS-PAGE and transferred to PVDF membrane. The expression of the anti-apoptotic protein Bcl2 was assessed by western blot and semi-quantitative analysis performed by plate scanning. b actin was used to normalize results of protein content. A: MCF7 cells, B MDA-MB-231 cells. of EGF. EPA did not modify EGFR expression in breast cancer cells; while EGF stimulation significantly increase EGFR phosphorylation to about 140%; co-treatment with EPA/EGF significantly inhibit EGFR activation down to about 40% compared to control non stimulated cells (Figure 4A). As shown in Figure 4B, DHA significantly reduces the EGFR level (70%) compared to control cells and com- pletely inhibit EGFR activation in cells treated with DHA or DHA/EGF. mitogenic signals to downstream target signalling cas- cades that involve cell survival and proliferation, such as phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-acti- vated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) [29]. In human breast cancer cell lines, such as MDA-MB- 231, the EGFR level is elevated compared with that in other breast cancer cells, such as MCF7 [30]; for this reason, we have studied the effects of DHA and EPA on EGFR activity mainly in MDA-MB-231 cells. EPA and DHA alter the EGFR and pEGFR levels in MDA- MB-231 cells Furthermore, apoptosis involves the activation of pro- caspase-8 (55 kDa) by its cleavage to caspase-8 (18 kDa); this smaller protein together with caspase-3 med- iates the rapid dismantling of cellular organelles and architecture [28]. EGFR is usually activated in response to extracellular ligands (EGF) by its phosphorylation; ligand binding leads to homo- or heterodimerization with another ligand-bound ErbB receptor, and transmits extracellular Page 5 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Bcl2 MCF7 0 20 40 60 80 100 120 EPADHA CTR TREATMENT Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPADHA CTR TREATMENT A MCF7 Bcl2 ȕ-actin B MDA-MB-231 CTR EPA CTR DHA Bcl2 ȕ-actin CTR EPA CTR DHA Figure 2 Effects of n-3 PUFA on Bcl2 expression. Both cell lines were treated with DHA (200 μM) or EPA (230 μM) for 72 h. Control and treated cell lysates are separated on 10% SDS-PAGE and transferred to PVDF membrane. The expression of the anti-apoptotic protein Bcl2 was assessed by western blot and semi-quantitative analysis performed by plate scanning. b actin was used to normalize results of protein content. A: MCF7 cells, B MDA-MB-231 cells. CTR EPA CTR DHA Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT CTR EPA CTR DHA Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT B MDA-MB-231 -actin ProCASPASE 8 ProCASPASE 8 A MCF7 -actin Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment with DHA (200 μM) and EPA (230 μM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. B: MDA-MB-231 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. Semi-quantitative analysis performed by plate scanning. b-actin was used to normalize results of protein content. Total fatty acid profile after treatment with PUFA y p Treatment with AA, EPA or DHA alters the FA profile in MDA-MB-231 and MCF7 cells compared with con- trol cells (Table 1). Treatment of both cell lines with AA resulted in a sig- nificant increase of AA content in total cell lipids, from 14.40% to 46.85% in MDA-MB-231 and from 12.73% to Figure 4 reports the effects of EPA and DHA treat- ments on expression and activation of EGFR in presence Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 6 of 16 EGFR 0 50 100 CTR EPA EGF EGF/EPA EGFR 0 50 100 CTR DHA EGF EGF/DHA p-EGFR 0 50 100 150 CTR EPA EGF EGF/EPA * ** p-EGFR 0 50 100 150 CTR DHA EGF EGF/DHA * EGF CTR DHA DHA/EGF B pEGFR EGFR -actin CTR EGF EPA/EGF EPA pEGFR EGFR -actin A Figure 4 EGFR expression and EGF stimulation (p-EGFR) in MDA-MB-231 breast cancer cells after n-3 PUFAs treatment. A: MDA-MB-231 treated with 230 μM EPA for 72 h, lane 1 control, lane 2 230 μM EPA, lane 3 EGF, lane 4 EGF+EPA. B: MDA-MB-231 treated with 200 μM DHA for 72 h, lane 1 control, lane 2 200 μM DHA, lane 3 EGF, lane 4 EGF+DHA. The relative intensities of band signals, reported in graphics, are determined by digital scanning densitometry. b-actin was used to normalize results of protein content. * p < 0.05; ** p < 0.01 compared to control cells; n = 3. A B p-EGFR 0 50 100 150 CTR EPA EGF EGF/EPA * ** p-EGFR 0 50 100 150 CTR DHA EGF EGF/DHA * Figure 4 EGFR expression and EGF stimulation (p-EGFR) in MDA-MB-231 breast cancer cells after n-3 PUFAs treatment. A: MDA-MB-231 treated with 230 μM EPA for 72 h, lane 1 control, lane 2 230 μM EPA, lane 3 EGF, lane 4 EGF+EPA. B: MDA-MB-231 treated with 200 μM DHA for 72 h, lane 1 control, lane 2 200 μM DHA, lane 3 EGF, lane 4 EGF+DHA. The relative intensities of band signals, reported in graphics, are determined by digital scanning densitometry. b-actin was used to normalize results of protein content. * p < 0.05; p < 0.01 compared to control cells; n = 3. Total fatty acid profile after treatment with PUFA Table 1 Total fatty acid composition of PUFA-treated breast cancer cells MDA-MB-231 MCF-7 CTR AA EPA DHA CTR AA EPA DHA C:16:0 13.91 ± 2.19 13.48 ± 2.91 6.57 ± 0.91 8.66 ± 1.25 16.03 ± 4.06 15.52 ± 1.56 14.7 ± 1.76 11.14 ± 3.59 C 16:1 1.61 ± 0.85 1.19 ± 0.87 0.87 ± 0.58** 0.77 ± 0.42* 8.25 ± 3.27 2.97 ± 0.86 3.95 ± 0.54 2.94 ± 1.53** C 18:0 17.57 ± 1.97 10.46 ± 2.05* 5.15 ± 1.03 6.39 ± 1.30 14.83 ± 1.58 15.73 ± 1.76 10.41 ± 2.49 9.46 ± 0.89 C 18:1 26.14 ± 4.74 15.91 ± 4.21* 8.15 ± 0.95 9.65 ± 1.29 30.14 ± 2.91 12.12 ± 1.19 12.21 ± 1.94 9.77 ± 1.12 C 18:2 6.05 ± 2.38 3.07 ± 0.65 2.38 ± 0.17 2.46 ± 0.32 4.23 ± 0.73 2.48 ± 1.27 2.26 ± 0.42 1.66 ± 0.21 C 18:3 1.54 ± 4.10 0.68 ± 0.37 0.22 ± 0.16 0.32 ± 0.10 1.32 ± 0.87 0.96 ± 0.53* 0.59 ± 0.36* 0.63 ± 0.61* C 20:3 1.64 ± 0.21 1.60 ± 0.10 0.72 ± 0.37** 1.18 ± 0.70* 2.41 ± 0.67 1.28 ± 0.11 0.67 ± 0.09 0.78 ± 0.16** C 20:4 (AA) 14.40 ± 2.92 46.85 ± 10.48* 2.56 ± 0.87 3.91 ± 0.50 12.73 ± 2.90 44.26 ± 3.80 3.13 ± 0.57 3.20 ± 0.55 C 20:5 (EPA) 2.42 ± 0.70 0.70 ± 0.23 38.75 ± 3.52 1.98 ± 0.43 3.22 ± 0.98 0.74 ± 0.75 34.16 ± 3.89 4.90 ± 0.51 C 22:5 (DPA) 7.04 ± 1.10 3.61 ± 0.44 33.30 ± 1.83 2.94 ± 0.62 1.10 ± 0.71 1.29 ± 0.45 15.80 ± 3.32 0.96 ± 0.14 C 22:6 (DHA) 7.68 ± 1.55 2.45 ± 0.60 1.29 ± 0.35 61.76 ± 3.93 5.73 ± 2.36 2.65 ± 0.68 2.12 ± 0.31 54.55 ± 6.02 SFA 33.35 ± 5.50 23.94 ± 4.88* 11.76 ± 1.90 15.05 ± 2.52 30.86 ± 3.21 31.24 ± 2.89 25.11 ± 3.81 20.60 ± 4.18 MUFA 27.74 ± 4.69 17.10 ± 4.61* 9.01 ± 0.82 10.41 ± 1.06 38.39 ± 4.36 15.09 ± 1.62 16.16 ± 2.02 12.71 ± 2.27 n-3 PUFA 18.68 ± 3.93 7.44 ± 0.56 73.56 ± 3.16 67.00 ± 3.45 11.37 ± 3.71 5.63 ± 1.58 52.67 ± 6.24 61.05 ± 5.96 n-6 PUFA 22.09 ± 3.15 51.52 ± 9.81 5.67 ± 1.37 7.55 ± 0.70 19.38 ± 3.58 48.03 ± 4.00 6.06 ± 0.76 5.64 ± 0.59** Breast cancer cells were treated with solvent (ethanol) as control, AA (20:4, n-6), EPA (20:5, n-3), and DHA(22:6, n-3). Discussion After treatment with AA, the content of this n-6 PUFA was increased significantly in PE and PC in MDA-MB-231 (Figure 5, Table 2). The treatment induced a reduction of omega-3 PUFAs, particularly EPA, that was significantly decreased in all phospholi- pids but SM; while DHA content was decreased after AA treatment only in PE and PC, the other two omega- 3 fatty acids namely DPA and ALA (C18:3) were decreased in all phospholipids but SM. Breast cancer is the leading cause of the death among women in the world. The principal effective endocrine therapy for treatment on this type of cancer is anti- estrogens, but therapeutic choices are limited for estro- gen receptor (ER) negative tumor, which are more aggressive. Moreover the development of ER positive cancer cells that are resistant to chemotherapeutic agents is a major factor responsible to the successful treatment of breast cancer. This is a strong input to dis- cover new approaches in vitro. Incubation with EPA caused an increase of EPA con- tent in all PLs in MDA-MB-231 cells. In particular, the incorporation of EPA was different in relation to the PL moiety with highest levels of incorporation in PI and PC. There was a decrease of monounsaturated FA in PE and PC, and a significant increase of polyunsaturated FA in PI and PC. Furthermore, an increase of DPA con- tent was found in all PLs, especially PC. The content of AA was significantly decreased in all phospholipids but SM. Several epidemiologic and clinical studies have shown that n-3 PUFAs are able to provide beneficial effects in a wide variety of pathologies ranging from autoimmune and inflammatory diseases to neurological and psychia- tric disorders and, in particular, to several types of malignancy, including ovarian, pancreatic, prostate, renal, colorectal and breast cancer [31-33]. This study was prompted by the observation that MDA-MB-231 and MCF7 breast cancer cell lines showed a significant reduction in cell number following treat- ment with n-3 PUFAs. The same conclusion is not possi- ble for the AA incubation. We hypothesize that this reduction in cell number results from both proliferation reduction and induction of apoptosis. Apoptosis is a genetically controlled form of cell death that is conserved from worms to humans. Deregulation of apoptosis is a hallmark of all cancer cells and the agents that activate apoptosis in cancer cells could be considered as anti- cancer therapeutics [34]. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 7 of 16 Page 7 of 16 PC. The concentration of DHA was significantly decreased in PE and PI, whereas the AA content was significantly reduced in PE, and PI. 44.26% in MCF7. Furthermore, the data for MDA-MB- 231 show a significant decrease of EPA, docosapentae- noic acid (DPA) and DHA, whereas the data for MCF7 cells show a significant reduction of only EPA and DHA. The exposure of MCF7 cells to DHA determined a significant increase of DHA in all PLs, but not in SM, and of EPA content in PE, PI, and PC, whereas the con- tent of AA was significantly reduced only in PE, PI and PS. When both cell lines were treated with EPA, the con- tent of this FA in total cell lipids was increased signifi- cantly and we observed a significant reduction of AA. Unexpectedly, we found an increase of DPA content, indicating that EPA is incorporated into cells and is further metabolized by elongation. Also in these cells a significant decrease of monounsa- turated fatty acids is always present when the cells are treated with n-6 and n-3 PUFAs; while saturated fatty acids are in most cases constant. The treatment with DHA determines a significant increase of DHA content in both cell lines and an increase of EPA content in MCF7, probably due to a retro conversion; a significant reduction of AA content was also measured. Moreover also 18:2, 18:3 (n-3) and 20:3 (n-6) are sig- nificantly decrease after PUFA treatment. As far as phospholipids content concerns, it is worth noting the significant decrease of SM content (from 11.32% to 9.02%, data not shown) in MCF7 after treat- ment with DHA even if, sphingomyelin is the phospholi- pid less influenced in its fatty acid composition by PUFA treatment. The other treatments did not modify the distribution of PL in both cell lines. Effects of treatment with PUFAs on PL composition in breast cancer cells Table 2 and 3 give the fatty acid composition of specific PLs in MDA-MB-231 and MCF7 cells treated with n-3 or n-6 PUFAs; to simplify the tables SD, are reported as plain numbers above the bold mean value. Total fatty acid profile after treatment with PUFA Fatty acid composition was analyzed and expressed as percentage of total fatty acids (mean ± SD). Asterisks indicate the significant differences between treated- and control cells (n=10, P < 0.05; P < 0.01). Table 1 Total fatty acid composition of PUFA-treated breast cancer cells MDA-MB-231 Discussion In some mammalian cells, apoptosis can be triggered by members of the Fas/TNF receptor family. When activated by receptor aggregation, After treatment of MDA-MB-231 cells with DHA, the content of this fatty acid was significantly increased in all cell membrane PLs, but not in SM. We measured a significant decrease of the content of EPA in PE, PS, PI and PC. The concentration of AA was significantly reduced in PE and PI and SM as the result of treatment with DHA. In MCF7 cells (Figure 6, Table 3), the treatment with AA induced a significant increase of this fatty acid in all PLs, except in SM; a significant reduction of EPA and of DHA in PE, PI, PS and PC was also measured. After treatment with EPA, the EPA and DPA content was significantly increased, especially in PE, PI, PS and Table 2 Phospholipids fatty acid composition of PUFA-treated MDA-MB-231 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:3 C20:4 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/Omega-3 AA/EPA AA/DHA PE CTR 5.13 0.80 19.58 18.11 3.12 0.62 1.58 30.38 3.65 6.14 7.85 24.71 18.90 53.34 35.08 18.26 1.95 8.96 3.93 s.d. 1.87 0.59 2.40 2.59 0.97 0.19 0.37 4.39 0.90 1.01 1.06 3.16 2.80 4.03 3.42 2.01 0.30 2.99 0.79 PE AA 6.16 1.29 22.60 15.64* 1.52* 0.30* 1.15* 40.72* 0.66* 3.75* 3.09* 28.76* 16.93* 51.08* 43.39* 7.70* 5.69* 81.49* 13.38* s.d. 0.86 1.12 0.88 0.84 0.14 0.03 0.35 1.29 0.49 0.74 0.45 1.73 0.52 0.89 1.16 0.80 0.61 37.02 1.79 PE EPA 5.64 0.55 20.44 13.68* 1.66* 0.33* 0.84* 8.13* 22.52* 18.86* 1.62* 26.09 14.23* 53.96 10.63* 43.34* 0.25* 0.36* 5.10 s.d. 1.35 0.24 5.47 0.92 0.35 0.07 0.28 1.21 1.33 2.80 0.26 5.28 1.07 3.47 1.13 3.78 0.04 0.07 1.14 PE DHA 6.44 1.14 19.87 8.80* 1.34* 0.27* 0.84* 12.15* 2.10* 2.26* 42.31* 26.31 10.57* 56.55 13.46* 47.24* 0.26* 6.41 0.25* s.d. 1.86 0.64 3.99 0.62 0.46 0.09 0.45 2.73 0.71 0.45 3.04 5.38 1.95 7.81 3.21 2.96 0.01 3.60 0.02 PI CTR 4.32 1.31 34.32 14.48 2.50 0.50 2.88 25.54 1.44 4.65 3.98 40.05 14.35 41.67 30.34 11.33 3.23 22.84 8.98 s.d. 1.73 1.31 5.38 4.35 1.74 0.35 0.66 3.93 0.65 1.12 1.74 5.37 5.32 5.09 6.33 3.34 1.68 11.79 9.39 PI AA 8.23* 2.40 33.71 13.31 1.44* 0.29* 0.65* 30.03* 0.40* 2.16* 2.94 41.95 15.26 37.99 32.11 5.87* 5.51* 75.95* 10.23 s.d. Discussion 2.67 1.24 2.05 1.54 0.40 0.08 0.30 2.86 0.04 0.15 0.36 3.09 2.39 2.53 2.70 0.40 0.69 15.180 1.75 PI EPA 6.16* 1.12 24.38 14.81 2.34 0.47 1.15* 11.53* 14.30* 16.40* 1.32* 30.55* 15.93 48.87* 15.02* 33.85* 0.45* 0.84* 10.35 s.d. 1.23 0.50 2.72 6.18 1.19 0.24 0.48 3.47 5.28 4.92 0.74 2.27 6.31 6.89 2.60 5.10 0.07 0.15 6.91 PI DHA 10.09* 2.99 32.88 10.90 1.96 0.39 1.81* 10.22* 0.96* 2.55* 20.43* 42.96 13.89 39.71 16.48* 22.57* 0.81* 10.77* 0.53* s.d. 4.52 1.72 4.57 3.28 0.51 0.10 0.91 3.93 0.24 0.36 2.58 8.07 4.59 6.02 6.13 4.12 0.48 3.14 0.15 PS CTR 6.22 2.16 26.07 23.76 3.69 1.10 2.27 13.12 2.50 5.74 7.52 32.40 24.94 38.00 20.12 16.73 1.25 6.03 1.90 s.d. 1.88 1.96 9.75 6.53 1.62 1.07 0.99 7.67 1.20 1.69 3.71 9.19 7.87 11.64 10.49 4.72 0.68 3.59 1.64 PS AA 13.48* 4.69* 21.00 25.00 2.00* 0.48* 0.29* 14.87 0.41* 1.82* 6.07 34.38 29.80 26.37* 17.81 8.56* 2.12* 47.07* 2.86 s.d. 1.77 1.71 4.77 4.62 0.61 0.21 0.16 4.00 0.23 0.63 1.75 3.20 3.57 4.01 4.27 0.98 0.70 29.02 1.92 PS EPA 6.17 1.38 14.67* 18.78 1.76* 0.35* 1.34 6.96* 16.53* 23.17* 2.48* 27.92 19.97 48.44 10.06* 38.38* 0.29* 0.48* 2.18 s.d. 1.30 0.65 4.30 3.79 0.55 0.11 1.12 4.17 0.34 7.45 0.59 13.86 4.06 13.24 4.27 12.21 0.14 0.27 0.83 PS DHA 11.48* 2.44 28.32 10.43* 1.60* 0.32* 0.42* 9.99 1.23* 2.41* 30.84* 36.77 12.26* 45.48 10.63 34.49* 0.32* 9.65 0.33* s.d. 3.46 1.48 13.14 1.56 0.29 0.06 0.13 2.22 0.40 0.83 8.78 17.15 3.15 11.23 3.27 8.40 0.09 3.81 0.05 PC CTR 23.82 3.13 13.90 35.51 6.45 1.29 1.43 8.17 1.20 1.88 2.29 37.73 38.64 22.62 16.04 6.58 2.50 7.50 3.86 s.d. 3.82 1.19 2.11 3.70 0.84 0.17 0.37 2.43 0.44 0.64 0.72 3.25 3.55 2.82 2.26 1.15 0.47 3.56 1.43 PC AA 27.29* 3.84 11.20* 21.03* 2.66* 0.53* 1.02* 27.61* 0.10* 1.45* 1.33* 38.50 24.87* 34.80* 31.29* 3.51* 8.89* 277.85* 18.21* s.d. 1.51 0.42 0.69 1.98 0.18 0.04 0.13 3.19 0.01 0.23 0.28 1.43 2.03 3.27 3.13 0.15 0.56 34.12 2.17 PC EPA 25.10 1.63* 9.05* 20.73* 4.33* 0.87* 0.83* 3.62* 15.71* 14.26* 1.36 34.14 22.36* 40.96* 8.77* 32.17* 0.28* 0.24* 3.42 s.d. Discussion 3.84 0.25 1.94 1.79 0.66 0.13 0.42 0.53 2.09 5.43 0.93 4.74 1.92 6.92 0.36 6.75 0.05 0.04 1.29 PC DHA 23.54 2.89 13.68 16.85* 3.07* 0.61* 0.96* 7.16 1.78* 1.80 23.90* 37.41 20.77* 39.77* 11.80* 27.96* 0.43* 4.12* 0.33* s.d. 2.72 0.88 1.65 3.35 0.37 0.08 0.25 0.98 0.45 0.66 4.15 4.53 1.62 3.56 0.42 3.75 0.08 0.54 0.09 Table 2 Phospholipids fatty acid composition of PUFA-treated MDA-MB-231 Table 2 Phospholipids fatty acid composition of PUFA-treated MDA-MB-231 (Continued) SM CTR 20.03 2.74 14.34 21.96 3.21 0.80 1.11 8.09 1.17 3.16 6.05 33.78 22.98 24.11 12.93 11.18 1.34 11.07 1.73 s.d. 9.10 1.62 8.11 7.61 1.60 0.73 1.09 3.47 1.17 2.87 3.57 16.17 9.73 6.62 4.26 4.31 0.45 7.33 0.83 SM AA 18.75 2.94 10.40 20.80 2.24 0.45 0.53* 12.30* 0.75 0.63* 6.68 33.32 23.22 22.58 14.80 8.45* 1.76* 30.33* 1.86 s.d. 5.27 1.67 1.00 4.33 0.93 0.19 0.15 0.96 0.54 0.34 0.94 14.58 4.99 1.84 1.45 0.29 0.23 10.50 0.26 SM EPA 12.67* 1.99 6.43* 24.50 2.07* 0.41 0.38* 7.85 4.38* 6.28* 2.93* 19.11* 24.70 26.51 13.17 16.24 0.90 1.78* 3.47 s.d. 3.19 0.78 0.67 2.85 0.71 0.14 0.06 2.91 1.67 2.13 1.48 3.51 7.82 6.22 5.26 7.02 0.45 0.83 2.00 SM DHA 19.07 5.01* 14.14 13.62* 2.89 0.58 1.72 5.60* 1.20 1.24* 8.35 33.21 19.47 25.69 10.21* 15.48 0.79* 5.76* 0.70* s.d. 6.89 0.79 3.32 1.01 0.42 0.08 0.73 1.88 0.53 0.23 2.52 9.19 2.32 7.32 1.37 7.31 0.37 3.21 0.37 Values are expressed as percentage of total fatty acids (means bold numbers, SD plain numbers). CTR n = 15, Treated n=6; * P < 0.01 Corsetto et al. Lipids in Health and Diseas http://www.lipidworld.com/content/10/1/ 2 Phospholipids fatty acid composition of PUFA-treated MDA-MB-231 (Continued) Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Table 3 Phospholipids fatty acid composition of PUFA-treated MCF7 Table 3 Phospholipids fatty acid composition of PUFA-treated MCF7 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:3 C20:4 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/Omega-3 AA/EPA AA/DHA PE CTR 7.76 4.23 22.01 23.50 3.46 0.69 1.70 24.93 5.28 0.72 5.71 29.77 27.73 42.50 30.09 12.41 2.47 4.75 4.58 s.d. 1.18 1.91 1.92 3.14 0.69 0.14 0.28 3.13 0.69 0.56 1.37 1.86 3.83 3.88 2.66 1.96 0.37 0.50 1.04 PE AA 8.24 1.52* 33.44* 8.05* 1.38* 0.28* 0.49* 42.32* 0.70* 1.03 2.56* 41.68* 9.57* 48.75* 44.19* 4.57* 9.97* 76.64* 17.85* s.d. Discussion 1.62 0.95 1.22 1.04 0.20 0.04 0.25 2.15 0.47 0.39 0.81 1.44 1.57 2.19 1.94 0.86 1.80 30.68 5.10 PE EPA 9.52 2.19* 27.35* 8.58* 1.50* 0.30* 0.53* 7.37* 33.91* 7.68* 1.07* 36.87* 10.77* 52.36* 9.40* 42.96* 0.23* 0.22* 5.42 s.d. 2.55 1.22 2.29 2.80 0.52 0.10 0.26 1.10 4.88 2.26 0.50 1.58 4.00 5.13 1.76 6.77 0.08 0.06 0.45 PE DHA 9.26 1.36* 25.39 6.97* 2.08* 0.42* 1.03 6.62* 9.45* 0.52 36.92* 34.65* 8.33* 57.02* 9.73* 47.30* 0.21* 0.71* 0.19* s.d. 2.80 1.10 4.61 2.50 1.24 0.25 1.00 1.43 2.19 0.33 7.43 4.38 2.66 6.35 1.95 6.27 0.05 0.13 0.08 PI CTR 10.84 2.51 31.95 18.13 3.43 0.69 5.50 14.10 2.22 1.64 8.99 42.79 20.65 36.56 23.02 13.54 1.80 8.04 1.84 s.d. 5.40 2.32 5.42 6.55 1.29 0.26 1.53 3.89 1.65 1.00 3.07 5.35 7.91 7.50 4.66 3.77 0.47 4.32 1.20 PI AA 8.22 1.04* 35.22* 6.79* 1.45* 0.29* 1.01* 40.65* 0.96* 1.52 2.83* 43.44 7.83* 48.73* 43.12* 5.61* 8.34* 69.09* 23.36* s.d. 1.78 0.73 2.36 3.69 0.34 0.07 0.13 3.84 0.80 1.41 1.77 2.35 4.03 2.81 3.70 1.65 2.63 49.53 17.92 PI EPA 8.81 1.66 33.04 6.85* 2.18* 0.44* 1.48* 10.41* 24.41* 8.60* 2.13* 41.84 8.51* 49.65* 14.07* 35.57* 0.40* 0.44* 4.76 s.d. 1.58 1.04 1.55 2.31 0.79 0.16 0.61 0.92 4.29 1.69 2.18 1.73 2.04 2.96 1.61 3.59 0.08 0.10 3.02 PI DHA 9.06 1.23* 24.60* 13.54 4.52 0.91 3.11 7.63* 7.71* 0.87* 26.82* 33.66* 14.77* 51.57* 15.26* 36.31* 0.43* 1.16* 0.31* s.d. 3.81 0.91 5.96 5.74 2.54 0.51 3.09 1.55 3.67 0.76 6.04 6.91 5.63 7.46 5.64 5.29 0.19 0.48 0.17 PS CTR 10.79 3.53 24.95 27.92 4.30 0.86 4.21 11.93 3.18 2.19 6.14 35.75 31.45 32.81 20.43 12.37 1.83 5.17 2.49 s.d. 3.01 2.22 8.41 7.49 1.77 0.35 1.57 5.37 1.91 2.00 3.36 8.35 8.32 8.96 7.21 4.27 0.84 4.46 1.57 PS AA 11.97 1.92 29.81 12.97* 2.24* 0.45* 1.07* 33.50* 1.21* 1.69 3.18* 41.78* 14.89* 43.33* 36.80* 6.53* 6.49* 38.97* 12.22* s.d. 4.28 2.83 7.69 4.92 1.33 0.27 0.43 11.48 0.63 2.12 1.47 5.84 7.35 9.32 10.79 2.15 3.45 31.62 6.48 PS EPA 16.78* 1.07* 20.16 7.31* 1.27* 0.25* 1.36* 7.62 33.04* 8.11* 3.05 36.93 8.37* 54.70* 10.25* 44.45* 0.22* 0.22* 4.62 s.d. Discussion 3.53 0.69 11.14 3.65 0.53 0.11 0.66 3.42 12.94 3.14 3.04 11.06 3.52 14.23 3.84 10.79 0.06 0.06 4.12 PS DHA 9.83 1.78* 24.73 13.57* 3.40 0.68 1.75* 6.67* 5.00 1.55 31.06* 34.56 15.35* 50.10* 11.82* 38.28* 0.32* 1.85* 0.22* s.d. 3.89 1.47 6.84 3.83 1.70 0.34 1.30 3.26 2.94 0.59 2.17 6.22 3.88 4.33 5.70 4.53 0.18 1.50 0.12 PC CTR 28.42 13.13 6.51 36.22 4.19 0.84 1.52 5.18 1.18 0.59 2.23 34.93 49.35 15.73 10.89 4.83 2.61 5.70 3.91 s.d. 4.83 5.72 1.57 4.15 0.85 0.17 0.66 1.79 0.62 0.92 2.01 3.83 4.82 5.01 2.61 2.95 0.85 3.65 3.51 PC AA 28.28 4.97* 10.89* 16.67* 1.92* 0.38* 0.58* 34.61* 0.19* 0.29 1.21* 39.16* 21.65* 39.19* 37.11* 2.08* 18.12* 222.12* 30.38* s.d. 2.59 1.87 2.23 0.69 0.05 0.01 0.05 1.68 0.10 0.07 0.33 0.63 1.61 1.91 1.61 0.33 2.44 100.56 8.64 PC EPA 36.02 8.05 5.93 19.61* 2.64* 0.53* 0.56* 3.72 20.12* 1.75 1.07 41.95* 27.66* 35.69* 6.92* 23.48* 0.34* 0.21* 4.37 s.d. 4.52 3.18 0.62 4.19 0.29 0.06 0.34 1.29 8.27 1.39 0.63 0.91 6.34 0.53 1.68 9.30 0.17 0.10 2.54 PC DHA 29.09 3.68* 9.94* 14.94* 3.37* 0.67* 1.42 5.14 10.01* 0.39 21.34* 39.03* 18.62* 42.35* 9.93 32.42* 0.32* 0.51* 0.26* s.d. 5.46 2.60 1.71 3.86 0.46 0.09 0.39 0.99 1.36 0.20 5.63 4.11 5.19 6.44 1.47 6.01 0.07 0.06 0.08 Table 3 Phospholipids fatty acid composition of PUFA-treated MCF7 (Continued) SM CTR 29.95 3.73 15.64 18.03 3.22 0.64 1.86 13.35 4.32 3.66 5.59 45.60 21.76 32.64 18.43 14.22 1.60 4.45 6.27 s.d. 10.86 3.23 5.22 12.01 2.59 0.52 1.75 7.22 2.64 4.77 6.24 14.67 13.34 11.00 7.83 7.61 1.01 4.12 7.52 SM AA 17.58* 2.89 11.48* 20.36 2.56 0.51 2.16 17.74 4.16 6.86 5.97 29.06* 23.25 47.70* 22.46 15.86 1.50 6.56 3.41 s.d. 3.70 1.81 2.48 14.89 1.24 0.25 1.20 4.82 2.46 2.41 2.77 5.41 15.15 13.59 5.90 5.92 0.48 5.17 1.33 SM EPA 22.22* 13.52* 10.43* 5.06* 1.62* 0.32* 5.71 10.28 7.24 7.54* 16.06 32.65* 18.57 48.78* 17.62 31.16* 0.59* 1.49* 0.74* s.d. 2.85 2.24 1.59 0.20 0.65 0.13 3.16 3.56 2.52 1.84 8.51 3.98 2.43 5.54 4.86 6.18 0.22 0.52 0.37 SM DHA 27.05 5.19 11.33* 23.02 3.99 0.80 1.30 9.78 2.65* 4.38 10.53 38.38 28.21 33.41 15.06* 18.35 0.94 4.00 1.05* s.d. Discussion PE MDA 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * * PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * SM MDA 0 2 4 6 8 10 12 14 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * Figure 5 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 cells treated with LCPUFAs. MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Discussion 10.74 4.69 2.93 20.63 1.93 0.39 0.89 5.93 0.60 3.81 8.19 12.47 18.30 15.33 6.22 11.75 0.41 2.60 0.47 Values are expressed as percentage of total fatty acids (means bold numbers, SD plain numbers). CTR n=5, Treated n=6; * P < 0.01 Corsetto et al. Lipids in Health and Diseas http://www.lipidworld.com/content/10/1/ Table 3 Phospholipids fatty acid composition of PUFA-treated MCF7 (Continued) Page 11 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 12 of 16 PE MDA 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * * PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * SM MDA 0 2 4 6 8 10 12 14 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * Figure 5 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 LCPUFAs. MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an H PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Discussion PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * Figure 6 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated w cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fa determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are exogenous fatty acids. * p < 0.01; n = 3. Discussion PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * SM MDA 0 2 4 6 8 10 12 14 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * Figure 5 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 cells treated with LCPUFAs. MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Discussion Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 13 of 16 PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * Figure 6 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated with LCPUFAs. MCF7 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 14 of 16 Fas and TNFR1 induce the activation of a set of cysteine proteases called caspases. Studies designed to elucidate the mechanism(s) by which Fas and TNFR1 stimulation lead to caspase activation are underway. In the case of Fas, receptor aggregation by the Fas ligand induces the formation of a death-inducing signalling complex (DISC) of proteins comprising Fas itself, the adaptor protein FADD and the inactive zymogen form of caspase-8. Shortly after formation of the DISC, procaspase-8 is cleaved and the active protease is released. Once acti- vated, caspase-8 is thought to activate other downstream caspases by proteolytic cleavage of their zymogen forms, thus amplifying the caspase signal [35]. Our results demonstrate the activation of caspase-8 in response to incubation with n-3 PUFAs by a reduction of the levels of its zymogen form in both cell lines. In many cells, over-expression of either Bcl2 or Bcl-xl inhibits apopto- sis, affecting the release of cyt-c and apoptosis-inducing factor (AIF) from the mitochondrial intramembrane space to the cytosol. Once released, AIF is translocated to the nucleus where it is capable of inducing nuclear chromatin condensation and large-scale DNA fragmenta- tion that mediates a caspase-independent mitochondrial apoptotic pathway [36]. Cyt-c, together with dATP, binds to apoptotic proteinase activating factor-1 (Apaf-1) and this complex promotes procaspase-9 autoactivation. The active forms of caspase-8 and caspase-9 might activate the downstream effectors caspase-3, -6 and -7, resulting in the cleavage of crucial cellular proteins and apoptosis. We have observed a significant difference in the amount of Bcl2 present in the DHA-treated MDA-MB-231 cells and EPA-treated MCF7 cells compared to the control group. The absence of Bcl2 when compared to the con- trol is suggestive that the cell might be more likely to proceed to apoptosis. The entire mechanism by which n-3 PUFAs exert their beneficial effects is not fully understood. We have hypothesized that the induction of apoptosis, the reduc- tion of cell proliferation and the inhibition of EGFR activity by these fatty acids might be the consequences of cell membrane alterations induced by FA. Our data indicate that EPA and DHA are incorporated in breast cancer membrane. In particular the EPA treatment determines an increase of EPA and DPA content, and a reduction of SFA, MUFA and n-6 PUFA concentration in both cell lines. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 This suggests an incorporation of EPA which is further metabolised. In fact, EPA is converted to 22:5 n-3 (DPA) by elongase (Elovl)-5 and then by Elovl-2 to 24:5, n-3. The next step requires desaturation of 24:5 by Δ6 desaturase to produce 24:6, n-3. This pro- duct is translocated from the endoplasmic reticulum to the peroxisome, where the b oxidation pathway involves acyl chain shortening of C2 to produce DHA [40]. y g p Also DHA incubation determines an increase of EPA, of DHA, and in general of the unsaturation degree in both cell lines. We have also observed that PUFAs are incorpo- rated into the breast cancer membrane with different spe- cificity for each PL moiety. The enrichment is significant, especially in PE, PI and PC. The transbilayer distribution of lipids across biological membranes is asymmetric. The choline-containing lipids PC and SM are enriched primar- ily on the external leaflet of the plasma membrane or the topologically equivalent luminal leaflet of internal organelles. In contrast, the amine-containing glyceropho- spholipids PE and PS are located preferentially on the cytoplasmic leaflet. Other minor PLs, such as phosphatidic acid (PA), PI and phosphatidylinositol-4,5-bisphosphate (PIP2) are also enriched on the cytofacial side of the mem- brane. Specific alterations of the molecular composition of the plasma membrane occur during apoptosis. Hence, cells undergoing apoptosis express signals, including lipids, proteins and modified sugar moieties that facilitate recog- nition and ingestion by macrophages. Loss of transmem- brane PL asymmetry, with consequent exposure of PS in the external monolayer, occurs in both normal and patho- logic conditions. PS externalization is induced early in the process of apoptosis. On the basis of our findings, the data suggest that the incorporation of n-3 PUFAs is mainly into cytofacial leaflet PLs, altering the membrane environ- ment to impact on the activation of cell signalling. More- over, a significant decrease of SM was evident in cells treated with DHA. EGFR is an interesting target for tumour therapy, because it is over-expressed in many human tumours such as lung and breast cancers [37]. MDA-MB-231 cells express high levels of EGFR and are a good model to study EGFR modulation by n-3 PUFAs. This receptor is a member of the ErbB receptor tyrosine kinase family, which consists of EGFR (or HER1 or ErbB1), HER2/ ErbB2, HER3/ErbB3 and HER4/ErbB. Discussion PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * Figure 6 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated with LCPUFAs. MCF7 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 References 1. Bartsch H, Nair J, Owen RW: Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20:2209-18. 1. Bartsch H, Nair J, Owen RW: Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20:2209-18. Moreover PUFA incorporation induces an alteration of SFA, MUFA and PUFA content in membrane phos- pholipids; these data suggest a metabolic rearrangement in cells in order to try to balance the ratio between satu- rated and unsaturated fatty acids. 2. Rose DP, Connolly JM: Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther 1999, 83:217-44. 3. Hardman WE: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132:3508S-12S. 4. Lee CY, Sit WH, Fan ST, Man K, Jor IW, Wong LL, Wan ML, Tan-Un KC, Wan JM: The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 2010, 36(4):991-8. 4. Lee CY, Sit WH, Fan ST, Man K, Jor IW, Wong LL, Wan ML, Tan-Un KC, Wan JM: The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 2010, 36(4):991-8. In addition membranes constitute a meeting point for lipids and proteins. Thousands of cellular proteins inter- act with membranes in different ways, for example inte- gral (transmembrane, as EGFR) proteins are embedded in the lipid bilayer and their activity is sensitive to changes in the lipid environment. Recently, multiple stu- dies demonstrated very rapid ERa actions at level of the plasma membrane [45]. O’Malley and collaborators have demonstrated that ERa on the membrane initially acti- vates cytoplasmic kinases, which in turn phosphorylate and activate coactivators proteins in the cytoplasm. These coactivators then travel to the nucleus and modu- late ERa-mediated transcriptional events [46]. 5. Habermann N, Schön A, Lund EK, Glei M: Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo. Apoptosis 2010, 15:621-630. 5. Habermann N, Schön A, Lund EK, Glei M: Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo. Apoptosis 2010, 15:621-630. 6. Larsson SC, Kumlin M, Ingelman-Sundberg M, Wolk A: Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Authors’ contributions PAC carried out cell treatments, MTT tests, WB assays and drafted the manuscript, GM performed lipid analysis, SZ was responsible for cell cultures, IEJ performed lipid analysis, AC performed lipid analysis, BB Coordinated the study, AMR conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. PAC carried out cell treatments, MTT tests, WB assays and drafted the manuscript, GM performed lipid analysis, SZ was responsible for cell cultures, IEJ performed lipid analysis, AC performed lipid analysis, BB Coordinated the study, AMR conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. All authors have read and approved the final manuscript. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 6 April 2011 Accepted: 12 May 2011 Published: 12 May 2011 Received: 6 April 2011 Accepted: 12 May 2011 Published: 12 May 2011 References Am J Clin Nutr 2004, 79:935-945. 7. Serini S, Piccioni E, Merendino N, Calviello G: Dietary polyunsaturated fatty acids as inducers of apoptosis: implications for cancer. Apoptosis 2009, 14(2):135-52. 8. Field CJ, Schley PD: Evidence for potential mechanisms for the effect of conjugated linoleic acid on tumor metabolism and immune function: lessons from n-3 fatty acids. Am J Clin Nutr 2004, 79:1190S-8S. 8. Field CJ, Schley PD: Evidence for potential mechanisms for the effect of conjugated linoleic acid on tumor metabolism and immune function: lessons from n-3 fatty acids. Am J Clin Nutr 2004, 79:1190S-8S. 9 D UN A di l h M d S i M i 2002 8 RA79 92 lessons from n-3 fatty acids. Am J Clin Nutr 2004, 79:1190S-8S. 9. Das UN: A radical approach to cancer. Med Sci Monit 2002, 8:RA79-92. K hh SK D PP S RH S SS Gh h SN Eff f y 9. Das UN: A radical approach to cancer. Med Sci Monit 2002, 8:RA79-92. 10. Kachhap SK, Dange PP, Santani RH, Sawant SS, Ghosh SN: Effect of n-3 fatty acid (docosahexaenoic acid) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem 2001, 16:257-63. 10. Kachhap SK, Dange PP, Santani RH, Sawant SS, Ghosh SN: Effect of n-3 fatty acid (docosahexaenoic acid) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem 2001, 16:257-63. Then n-3 PUFAs, modifying the unsaturated degree, the permeability, the flip-flop process and the fluidity of the plasma membrane, might alter the activity of these proteins. This hypothesis will be investigated in our laboratory. 11. Albino AP, Juan G, Traganos F, Reinhart L, Connolly J, Rose DP, Darzynkiewicz Z: Cell cycle arrest apoptosis of melanoma cells by docohexaenoic acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60:4139-45. 11. Albino AP, Juan G, Traganos F, Reinhart L, Connolly J, Rose DP, Darzynkiewicz Z: Cell cycle arrest apoptosis of melanoma cells by docohexaenoic acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60:4139-45. 12. Chiu LCM, Ooi VEC, Wan JMF: Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 2001, 19:845-9. 12. Chiu LCM, Ooi VEC, Wan JMF: Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 2001, 19:845-9. Acknowledgements This paper is dedicated to the memory of Prof. Bruno Berra, who dedicated his life to the biochemistry of lipids. This paper is dedicated to the memory of Prof. Bruno Berra, who dedicated his life to the biochemistry of lipids. y p Financial support to Dr Angela M. Rizzo came from VI PQ: Nutra Snack Project and from the Italian Space Agency (ASI) Financial support to Dr Angela M. Rizzo came from VI PQ: Nutra Snack Project and from the Italian Space Agency (ASI). Abbreviations PL: (phospholipid); FA: (fatty acid); PUFA: (polyunsaturated fatty acid); MUFA: (monounsaturated fatty acid); SFA: (saturated fatty acid); DHA: (docosahexaenoic acid); EPA: (eicosapentaenoic acid); AA: (Arachidonic acid); PE: (phosphatidylethanolamine); PI: (phosphatidylinositol); PC: (phosphatidylcholine); PS: (phosphatidylserine); SM: (sphingomyelin). Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 15 of 16 Page 15 of 16 described that are responsible for the ATP-dependent transport of lipids. The best characterized activity is flip- pase, which transports PS from the outer monolayer to the cytoplasmic surface of the plasma membrane and requires ATP and Mg2+ but its activity is inhibited by Ca2+. A second ATP-dependent activity, catalysed by flip- pases, transports lipids in the opposite direction. The third class of lipid transporter consists of the Ca2+-acti- vated scramblases that catalyse the PS externalization [41,42]. Growing evidence indicates that excessive con- centrations of FA affect cell functions by altering the activity of various ion transporters and channels, includ- ing Ca2+. Zhang et al. have found that PUFAs, but not monounsaturated or saturated FAs, cause [Ca2+]i mobili- zation in NT2 human tetracarcinoma cells by causing release of this proton from mitochondria [43]. Further- more, Djemli-Shipkolye et al. showed that FA modifica- tions in membranes could be correlated with the variations observed in the activity of ATPase, for instance of Mg-ATPase [44]. This effect could influence the flip- pase and scramblase activities, and thus the transbilayer lipid asymmetry. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Ligand binding to EGFR induces its dimerization with another EGFR or with other members of the ErbB family, and activates tyrosine kinase residues on the intracellular domains of the protein through autophosphorylation. EGFR recruits downstream signalling proteins, triggering signal cas- cades along a number of pathways that eventually lead to cell growth, migration and apoptosis resistance [38,39]. We found that the phosphorylated EGFR levels are reduced after treatment with n-3 PUFAs (EPA or DHA) in MDA-MB-231 cells, whereas the EGFR level was decreased only after incubation with DHA. Once lipid asymmetry has been established, it is main- tained by a combination of slow transbilayer diffusion, protein-lipid interactions and protein-mediated trans- port. The most significant contributors to the mainte- nance and dissipation of transbilayer lipid asymmetry are proteins that catalyse the movement of lipids across the membrane. Two classes of transport activities have been Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Conclusions 13. Lai PB, Ross JA, Fearon KC, Anderson JD, Carter DC: Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed eicosapentaenoic acid in vitro. Br J Cancer 1996, 74:1375-83. 13. Lai PB, Ross JA, Fearon KC, Anderson JD, Carter DC: Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed eicosapentaenoic acid in vitro. Br J Cancer 1996, 74:1375-83. We suggest that n-3 PUFAs induce modifications of membrane structure and function of breast cancer cells, thereby increasing the degree of unsaturation. 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https://openalex.org/W1986980976	http://www.bioinformation.net/004/009800042010.pdf	English	null	Classification of DNA sequences based on thermal melting profiles	Bioinformation	2,010	cc-by	3,832	Methodology: The first exon of the β -globin gene is often used as a standard example in many DNA-based graphing methods [32] is used as the first example. The gene family of β -globin varies between 86-105 bases and has a significant biological role in oxygen transport. β - globin gene across the 11 species is listed in Table 1 (see supplementary material). The class II MHC α-chain sequences were obtained from the original reference of Takahashi and co- workers [33] (see supplementary material for data). The average length of the class II MHC α-chain is 612 and a total of 31 different species were used for the analysis. The melting profile was separately calculated for each individual sequence using the MELTSIM program [34, 35]. The melting profiles were calculated from 60°C to 120°C in steps of 0.1°C using the default setting (75mM of NaCl). Classification of the calculated profiles were performed using the Euclidian distance measure, unweighted arithmetic average for clustering with a 10 fold bootstrapping for revalidation. Clustering and validation of the profiles were performed using a combination of codes written in Matlab [36] and R [37, 38 ]. For the same DNAs, sequence-based evolutionary trees were constructed using MEGA [39]. All phylogenetic output files were generated in newick tree format (http ://evolution.genetics.washington.edu/phylip/newicktree.html), and visualized using Treedyn [40]. Melting curve analysis has been used in many applications such as the detection of single nucleotide polymorphisms (SNPs) [5-7] and has recently been proposed as an approach to DNA sequencing [8]. DNA melting profile analysis has also been used in many clinical research applications [9]; these include genotyping [10-13], mutation scanning [14, 15] and simultaneous genotyping and mutation scanning [16-18]. Experiments based on melting profiles have also been used as a rapid, economical means of screening close relatives for transplant compatibility [19]. The melting behavior and melting temperature of oligonucleotides can be predicted by a wide range of thermodynamic models which assumes that the stability of a DNA duplex depends on the identity and orientation of the neighboring base pairs [5-7, 20-25]. The idea of using the thermal stability, in particular the melting temperature to differentiate between DNA sequences was originally suggested in the pioneering work by King and Wilson [26] and later followed by others [26- 29]. King and Wilson used the nucleic acid hybridization melting temperature to quantify the resemblance between human and chimpanzee genes [26]. Bioinformation Bioinformation Bioinformation www.bioinformation.net open access Hypothesis Background: demonstrated the use of DNA hybridization as a guide to phylogeny using a model system of heteroduplexes formed between human β globin CDNA and four β -like globin genes isolated from a different species (chimpanzee). g The DNA double helix has more information built into its structure, that are both local such as variation in base pairing interactions and stacking interactions, as well as long range such as dynamic superhelical stress [1-3]. These interactions are responsible for the physical chemistry of the sequences and they are reflected in the thermodynamic properties such as the melting temperature. Therefore, sequences that have a high homology are expected to have similar thermodynamic parameters such as the melting temperature (Tm). Melting of a DNA molecule involves the denaturation the double-stranded DNA molecule into two single strands and it is the reverse process of hybridization. The denaturation process can be affected by many means such as an increase in temperature or denaturant concentrations [4]. The melting curve represents the denaturation process as a function of increasing sample temperature. Experimentally, the melting profile can be monitored by optical techniques such as absorption and fluorescence microscopy as the interactions among stacked bases cause a decrease in UV absorption. Melting of double-stranded DNA at elevated temperatures involves breaking the hydrogen bonds of the base pairs and a decrease of base stacking. This results in an increase in UV absorption, a hyperchromicity, which can be measured with a spectrophotometer [4]. In this study, we present a simple method for classifying the nucleotide sequences using simulated melting profiles. We demonstrate the utility of this method in β -globin and gene clusters of MHC class II α-chain proteins across multiple mammalian species. Comparison of the melting profile generated phylogenetics with that of the conventional sequence based approach reproduces many of the major features, but do not show a perfect match. The major advantage of this method is that it provides a way to verify phylogenetics constructed only from the DNA sequence and the molecular evolutionary process experimentally. Methodology: The difference in melting temperature between the reannealed human DNA and human-chimpanzee hybrid DNA is about 1.1° C, and that to the sibling species of Drosophila, congeneric species of mice and congeneric species of Drisophila are 3° C, 5° C and 19° C, respectively. Higher the difference in the Tm, larger the evolutionary distance between the species. As longer DNA sequences tend to have several localized melting events [30, 31], the melting profile of the DNA sequence has additional information [9]. In one of the early works, Schmid and Marks [28] Key words:DNA, hybridization, melting profiles, classification. Key words:DNA, hybridization, melting profiles, classification. ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) Abstract: A new classification scheme based on the melting profile of DNA sequences simulated thermal melting profiles presented. This method was applied in the classification of (a) several species of mammalian - β globin and (b) α-chain class II MHC genes. Comparison of the thermal melting profile with the molecular phylogenetic trees constructed using the sequences shows that the melting temperature based approach is able to reproduce most of the major features of the sequence based evolutionary tree. Melting profile method takes into account the inherent structure and dynamics of the DNA molecule, does not require sequence alignment prior to tree construction, and provides a means to verify the results experimentally. Therefore our results show that melting profile based classification of DNA sequences could be a useful tool for sequence analysis. Classification of DNA sequences based on thermal melting profiles Edward Reese1, Vishwanathan V. Krishnan1,2,3,* 1Department of Chemistry, California State University Fresno, Fresno CA 93740; 2Department of Pathology & Laboratory Medicine, University of California Davis Medical Center, Sacramento, CA 95837 ; 3Department of Applied Science, University of California, Davis, CA, 95616. V. V. Krishnan -Email id : vvkrishnan@ucdavis.edu. Tel. +530 746-8220. Fax. +559 278-4402 Received December 03, 2009; accepted December 13, 2009; published April 30, 2010 www.bioinformation.net The first derivative of the melting profile is more illustrative of the process of denaturation, as the melting temperature is represented as a peak (dθ/dT). Each derivative profile shows peak value that corresponds to the Tm and additional features such the width, shape and other low intensity peaks are manifestations of the sequence composition [41, 42]. As β-globin is represented by a relatively small number of bases such distinct features are not pronounced, except for the opossum (highlighted with dark curve in Figure 1). The DNA sequence of opossum has the lowest of the GC content of the sequences and consequently has the lowest Tm [43]. Melting profile based phylogenetics of β -globin is shown in Figure 2a, while the similar profile using the sequence is shown in Figure 2b. Most of the tree structures are reproduced in the melting profile based phylogenetics, with few notable differences. In both approaches the sequence of Opossum is clearly differentiated and the relative distances between Rat, Lemur and Gallus are also reproduced. Sequence based approach shows the cluster of Human, Chimpanzee and Gorilla, while the melting profile based approach keeps only the cluster of Chimpanzee and Gorilla together. origins and divergence times of mammalian class II MHC genes have been studied in detail by Takahashi et al [33] and the data presented here is one of the subgroups of gene clusters studied elsewhere ([33]). The sequence lengths of class II MHC α-chain genes are much larger than that of β -globin. Total of 31 different sequences and sequence length close to 612 for most sequences (see supporting material for the list genetic sequences) were used. As expected the melting profiles of these genes are complex suggesting the presence of additional features that could be useful to differentiate one sequence from the other. Original sequence based analysis of class II MHC α-chain genes showed four sequences (Zebra fish A1, A2 and A3 and shark) belong to an out-group and the melting temperature analysis (Fig. 4) also reproduces the same result. Mammalian class II MHC genes are clustered into four major groups, DRA, DPA, DQA and DNA [33]. Melting temperature based phylogeny is able to reproduce three of the four clusters only with overlaps between the closer clusters DQA and DPA. www.bioinformation.net Figure 2: Evolutionary trees of β-globin genes: Evolutionary trees constructed using the thermal melting profiles (left) and that from the respective gene sequence after sequence alignment. Figure 1: Melting profiles of beta-globin genes: (a) and their respective first derivatives (b). Some of the sequences are labeled in (b) and the melting profile of Opossum is shown in dark lines. Figure 1: Melting profiles of beta-globin genes: (a) and their respective first derivatives (b). Some of the sequences are labeled in (b) and the melting profile of Opossum is shown in dark lines. The shape of the melting curve and the melting temperature (Tm) are sensitive to the salt concentration in the sample [44]. All the calculations in figure 1 are performed with the salt concentration of 75mM. To highlight the effect of salt concentration on the melting profile, the β-globin sequence of opossum that shows additional features in the melting profile is simulated as a function of salt concentration. Figure 3a shows the plot of the profiles and the change in the Tm is shown in Figure 3b. Increase in salt concentration increases Tm (70°C at 0.01M to 98°C at 0.5M) and follows the empirical relationship between Tm vs. log(Concentration) [44]. Increasing the salt concentration also drastically changes the melting profile such as drastic loss of fine features that represent local melting events. Both melting profile and the Tm are important to differentiate the sequences in determining the phylogenetics and therefore optimal concentration of the salt is expected to be critical. Figure 2: Evolutionary trees of β-globin genes: Evolutionary trees constructed using the thermal melting profiles (left) and that from the respective gene sequence after sequence alignment. Bioinformation open access Hypothesis ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) Results: The gene family of β-globin across the 11 species are listed in Table 1 along with the percentage of the various bases, GC content as well the estimated Tm, for each sequence. Figure 1 shows the simulated melting profile using the program MELTSIM of the denaturation process (θ vs. temperature) (in Figure.1a) for all the 11 sequences and its first derivative dθ/dT (in Figure. 1b). The population value 0.5 and -0.5 represents the double-helical and denatured (single strand) DNAs, respectively and the melting temperature (Tm) is defined to be where these populations are equal. 463 ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) © 2010 Biomedical Informatics References [37] RC Gentleman J Comp Graphics (1996) 5: 299 314 [1] CJ Benham, SP Mielke, Annu Rev Biomed Eng (2005) 7:21. [3] NR Cozzarelli, Science (1980) 207:953. [4] VA Bloomfield et al. (2000) Sausalito, California, University Science Books, 794. [5] CD Bennett et al. Biotechniques (2003) 34:128 ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) 4 Figure 4: Application to Class II alpha-chains of MHC sequences: (a) Melting profiles of the 31 class II alpha-chains of MHC proteins simulated using MELTSIM. (b) Melting profile derived molecular evolutionary tree of class II alpha-chains of MHC proteins. DPA, DQA, DNA and DRA refer to the gene clusters originally [33]. [6] E Lyon, Expert Rev Mol Diagn (2001) 1:92. [7] NM von Ahsen et al. Clin Chem (1999) 45:2094. [8] YJ Chen, X Huang, Anal Biochem (2009) 384:170. [9] RPalais, CT Wittwer, Methods Enzymol (2009) 454:323. [10] R.Graham et al. Clin Chem (2005) 51:1295. [11] MR Liew et al. Clin Chem (2004) 50:1156. [12] RA Palais et al. Anal Biochem (2005) 346:167. [13] CT Wittwer et al. Clin Chem (2003) 49:853 [14] CN Gundry et al. Clin Chem (2003) 49:396. [15] C Willmore et al. Am J Clin Path (2004) 122:206. [16] SF Dobrowolski et al. Hum Mutat (2005) 25:306. [17] J Montgomery et al. Nat Protoc (2007) 2:59. [18] L Zhou et al. Clin Chem (2005) 51:1770. [19] L Zhou et al. Tissue Antigens (2004) 64:156. ( ) [20] PN Borer et al. J Mol Biol (1974) 86:843. ( ) [21] KJ Breslauer et al. Proc Natl Acad Sci USA (1986) 83:3746. [22] RA Dimitrov, M Zuker, Biophys J (2004) 87:215. ( ) [23] J SantaLucia Jr, Proc Natl Acad Sci U S A, (1998) 95 [24] H SantaLuciaJr et al. Biochemistry (1996) 35:3555. [25] N SugimotoS et al. Nucleic Acids Res (1996) 24:4501. [26] CM King, AC Wilson, Science (1975) 188:107. [27] FM Catzeflis et al. Mol Biol Evol (1987) 4:242 [28] CW Schmid, J Marks, J Mol Evo (1990) 30:237. [29] CG Sibley, JE Ahlquist, J Mol Evol (1987) 26:99. [30] SP H.Tanaka et al. J Phys Chem B (2008) 112:16788. [31] H Gille, W Messer EMBO J (1991) 10: 1579. [32] PA He, J Wang, J Chem Inf Comput Sci, (2002) 42:1080. [33] K Takahashi et al. J Hered (2000) 91:198. [34] RD Blake et al. Bioinformatics (1999) 15:370. [35] RD Volker et al. Biopolymers (1999) 50:303. Discussion: Here, we are suggesting an approach to classify DNA sequences has potential implication for sequence analysis; DNA sequences could be classified purely from the experimental melting profiles and sequence information is not mandatory as it depends. This method is expected to find wider applications once the sensitivity of the results is established by experiments. Salt concentration is expected to play a significant role on the applicability of DNA melting based differentiation between the sequences (Figure 2). The hypochromicity of DNA, responsible for the denaturing of the double helix is explained in terms of the interaction of the bases when they are stacked in the double helical array as delineated by Watson - Crick Model [49-51]. Semi- quantitative models developed in the 1960s continue to provide significant insight to the melting profiles of DNAs [44, 52]. Another major variable generating the melting profiles of the DNA sequences is the choice of the simulation program. In this work we have used the one of the widely used method, MELTSIM (materials and methods). For short oligonucleotides (16-32 bases), Panjkovich and Melo [53] performed an extensive comparison of the various methods. In their study it was noted that large and significant differences in the estimations of Tm were obtained while using different methods and no conclusive recommendations were provided on the choice of simulation methods to determine the melting profiles or its accuracy. Here, we are suggesting an approach to classify DNA sequences has potential implication for sequence analysis; DNA sequences could be classified purely from the experimental melting profiles and sequence information is not mandatory as it depends. This method is expected to find wider applications once the sensitivity of the results is established by experiments. Acknowledgements: g The authors acknowledge Dr. K.W. P. Miller for critical reading of the manuscript. V.V.K. is supported by an NIH grant: Research Infrastructure for Minority Institutions P20MD002732. The authors acknowledge Dr. K.W. P. Miller for critical reading of the manuscript. V.V.K. is supported by an NIH grant: Research Infrastructure for Minority Institutions P20MD002732. Discussion: With the advent of new molecular biology techniques including sophisticated cloning, sequencing and monitoring genomes allows the characterization of the single species without need for cross- hybridization techniques. For example Liu et al. [45] have applied a melting map calculation to the complete human genome. (http://meltmap.uio.no). DNA melting curves analysis is a valuable technique in sequencing, differentiate between coding and non- coding regions [46-48], genotyping [46-48] in the design of oligonucleotide probes in microarray experiments [46-48] and clinical applications [19] Classification of the profiles presented in this paper adds to an additional dimension to the power of melting profile analysis. Figure 4 show the melting profiles and molecular phylogenetics, respectively for the genes of class II MHC α-chain genes [33]. The 464 ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) © 2010 Biomedical Informatics Bioinformation open access Hypothesis www.bioinformation.net Figure 3: Temperature effect of Melting profiles: Plots of the melting profiles of the Opossum sequence of the β-globin gene family as a function of salt concentration (a). The plot of the melting temperature (Tm) as a function of the log (concentration) follows a liner relationship (b). Salt concentration is expected to play a significant role on the applicability of DNA melting based differentiation between the sequences (Figure 2). The hypochromicity of DNA, responsible for the denaturing of the double helix is explained in terms of the interaction of the bases when they are stacked in the double helical array as delineated by Watson - Crick Model [49-51]. Semi- quantitative models developed in the 1960s continue to provide significant insight to the melting profiles of DNAs [44, 52]. Another major variable generating the melting profiles of the DNA sequences is the choice of the simulation program. In this work we have used the one of the widely used method, MELTSIM (materials and methods). For short oligonucleotides (16-32 bases), Panjkovich and Melo [53] performed an extensive comparison of the various methods. In their study it was noted that large and significant differences in the estimations of Tm were obtained while using different methods and no conclusive recommendations were provided on the choice of simulation methods to determine the melting profiles or its accuracy. References Figure 3: Temperature effect of Melting profiles: Plots of the melting profiles of the Opossum sequence of the β-globin gene family as a function of salt concentration (a). The plot of the melting temperature (Tm) as a function of the log (concentration) follows a liner relationship (b). References [1] CJ Benham, SP Mielke, Annu Rev Biomed Eng (2005) [2] JF Marko, ED Siggia, Science (1994) 265:506. [3] NR Cozzarelli, Science (1980) 207:953. [4] VA Bloomfield et al. (2000) Sausalito, California, U Science Books, 794. [5] CD Bennett et al. Biotechniques (2003) 34:1288. [6] E Lyon, Expert Rev Mol Diagn (2001) 1:92. [7] NM von Ahsen et al. Clin Chem (1999) 45:2094. [8] YJ Chen, X Huang, Anal Biochem (2009) 384:170. [9] RPalais, CT Wittwer, Methods Enzymol (2009) 454:32 [10] R.Graham et al. Clin Chem (2005) 51:1295. [11] MR Liew et al. Clin Chem (2004) 50:1156. [12] RA Palais et al. Anal Biochem (2005) 346:167. [13] CT Wittwer et al. Clin Chem (2003) 49:853 [14] CN Gundry et al. Clin Chem (2003) 49:396. [15] C Willmore et al. Am J Clin Path (2004) 122:206. [16] SF Dobrowolski et al. Hum Mutat (2005) 25:306. [17] J Montgomery et al. Nat Protoc (2007) 2:59. [18] L Zhou et al. Clin Chem (2005) 51:1770. [19] L Zhou et al. Tissue Antigens (2004) 64:156. [20] PN Borer et al. J Mol Biol (1974) 86:843. [21] KJ Breslauer et al. Proc Natl Acad Sci USA (1986) 83 [22] RA Dimitrov, M Zuker, Biophys J (2004) 87:215. [23] J SantaLucia Jr, Proc Natl Acad Sci U S A, (1998) 95 [24] H SantaLuciaJr et al. Biochemistry (1996) 35:3555. [25] N SugimotoS et al. Nucleic Acids Res (1996) 24:4501 [26] CM King, AC Wilson, Science (1975) 188:107. [27] FM Catzeflis et al. Mol Biol Evol (1987) 4:242. [28] CW Schmid, J Marks, J Mol Evo (1990) 30:237. [29] CG Sibley, JE Ahlquist, J Mol Evol (1987) 26:99. [30] SP H.Tanaka et al. J Phys Chem B (2008) 112:16788. [31] H Gille, W Messer EMBO J (1991) 10: 1579. [32] PA He, J Wang, J Chem Inf Comput Sci, (2002) 42:10 [33] K Takahashi et al. J Hered (2000) 91:198. [34] RD Blake et al. Bioinformatics (1999) 15:370. [35] RD Volker et al. Biopolymers (1999) 50:303. [36] Mathworks Inc, Matlab, (2005), Mathworks Inc., Na 01760, USA. References [36] Mathworks Inc, Matlab, (2005), Mathworks Inc., Natick, MA 01760, USA. Figure 4: Application to Class II alpha-chains of MHC sequences: (a) Melting profiles of the 31 class II alpha-chains of MHC proteins simulated using MELTSIM. (b) Melting profile derived molecular evolutionary tree of class II alpha-chains of MHC proteins. DPA, DQA, DNA and DRA refer to the gene clusters originally [33]. [37] RC Gentleman, J. Comp Graphics, (1996) 5: 299-314. [38] RC Gentleman et al. Genome Biol (2004) 5: R80. [39] KTamura et al. Mol Biol Evol (2007) 24: 1596. [40] F Chevenet et al. BMC Bioinformatics (2006) 7: 439. [41] YZ Chen, EW Prohofsky, Eur Biophys J (1996) 24: 203. ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) 465 © 2010 Biomedical Informatics open access Hypothesis ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) © 2010 Biomedical Informatics access othesis © 2010 Sequence GCAAGGTGAAAGTGGATGA TGTGGGGCAAGGTGAACGT CTCTGGGGCAAGGTCAATGT GGCAAGGTGAAAGTGGATGA TGTGGGGCAAGGTGAACGT TGTGGGGCAAGGTGAACGT TGTGGGGCAAGGTGGATGTA CTGTGGGCAAAGGTGAACCC GGTCTAAGGTGCAGGTTGACC TGTGGGGCAAGGTGAATGT TGTGGGGAAAGGTGAACCC .mhctab2seq; >DogDRA mhctab2seq; >HumanDQ 2seq; >PigDQA_M2993 ctab2seq; 10.mhctab2seq ;>ZfishA_ 60.5 60.2 58.7 54.3 57.5 53.2 58.9 55.5 of MHC 9.mhctab hctab2seq hctab2se 7.mhctab .mhctab2 eq ;>Sha open access Hypothesis [42] O Gotoh, et al. Biopolymers (1976) 15: 655. [43] M Mandel, J Marmur (1968) 12: 198-206. [44] C Schildkraut, Biopolymers (1965) 3: 195. [45] F Liu et al. PLoS Comput Biol (2007) 3: e93. [46] R. Koehler, T N Peyret, Comput Biol Chem, (2005) 29: 393. [47] DD Long et al. Biophys Chem (2004) 110: 25. [48] S Payungporn et al. J Virol Methods (2004) 120: 131. [49] H Devoe, I Tinoco Jr., J Mol Biol, (1962) 4: 500. [50] H Devoe, I. Tinoco Jr., J Mol Biol, (1962) 4: 518. [51] I Tinoco, Jr J Am Chem Soc (1960) 8: 4785. [52] L Kotin, J Mol Biol (1963) 7:309. [53] A Panjkovichand F Melo, Bioinformatics, (2005) 21. 711. [42] O Gotoh, et al. Biopolymers (1976) 15: 655. [43] M Mandel, J Marmur (1968) 12: 198-206. [44] C Schildkraut, Biopolymers (1965) 3: 195. [45] F Liu et al. PLoS Comput Biol (2007) 3: e93. [46] R. Koehler, T N Peyret, Comput Biol Chem, (2005) 29: 393. [47] DD Long et al. Biophys Chem (2004) 110: 25. [48] S Payungporn et al. J Virol Methods (2004) 120: 131. Edited by P. Kangueane Citation: Reese et al. Bioinformation 4(10): 468-467 (2010) License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium, for non- commercial purposes, provided the original author and source are credited. 466 ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 4(10): 463-467 (2010) © 2010 Biomedical Informatics © 2010 Biomedical Informatics cess esis © 2010 Biomedical Informatics Sequence AGGTGAAAGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAG GGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAA GGGGCAAGGTCAATGTGGCCGAATGTGGGGCCGAAGCCCTGGCCAG AGGTGAAAGTGGATGAAGTTGGTGCTGAGGCCCTGGGCAG GGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGG GGGGCAAGGTGAACGTGGATTAAGTTGGTGGTGAGGCCCTGGGCAG GGGCAAGGTGGATGTAGAGAAAGTTGGTGGCGAGGCCTTGGGCAG GGGCAAAGGTGAACCCCGATGAAGTTGGTGGTGAGGCCCTGGGCAGG TAAGGTGCAGGTTGACCAGACTGGTGGTGAGGCCCTTGGCAG GGGGCAAGGTGAATGTGGAAGAAGTTGGTGGTGAGGCCCTGGGC GGGGAAAGGTGAACCCTGATAATGTTGGCGCTGAGGCCCTGGGCAG ctab2seq; >DogDRA_L37332.mhctab2seq; >RatEA_Y00480.mhctab2seq tab2seq; >HumanDQA_M26041.mhctab2seq; >CowDQA_D50045.mhct ; >PigDQA_M29938.mhctab2seq; >HorseDQA_L33909.mhctab2seq; 2seq; hctab2seq ;>ZfishA_L19445.mhctab2seq ;>ZfishA_L19446.mhctab2seq nline) 0 0): 463- GGTATCA Biomedical Informatics AGTTGGTGGTGAGGCCCTGGGCAG GGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATC GGCCGAATGTGGGGCCGAAGCCCTGGCCAG AGTTGGTGCTGAGGCCCTGGGCAG GGATGAAGTTGGTGGTGAGGCCCTGGGCAGG GGATTAAGTTGGTGGTGAGGCCCTGGGCAG AGAGAAAGTTGGTGGCGAGGCCTTGGGCAG CGATGAAGTTGGTGGTGAGGCCCTGGGCAGG CAGACTGGTGGTGAGGCCCTTGGCAG GGAAGAAGTTGGTGGTGAGGCCCTGGGC TGATAATGTTGGCGCTGAGGCCCTGGGCAG _L37332.mhctab2seq; >RatEA_Y00480.mhctab2 QA_M26041.mhctab2seq; >CowDQA_D50045.m 8.mhctab2seq; >HorseDQA_L33909.mhctab2seq _L19445.mhctab2seq ;>ZfishA_L19446.mhctab2s nline) 0 0): 463
https://openalex.org/W4287162586	https://zenodo.org/record/6786171/files/CYRENE_D2.1_Supply%20Chain%20Analysis%20and%20Requirements_v1.0.pdf	English	null	D2.1: Supply Chain Analysis and Requirements	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	63,969	This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952690. D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 4 31/05/2021 Table of Contents List of Tables ............................................................................................................................. 6 List of Figures ........................................................................................................................... 8 List of Acronyms ....................................................................................................................... 9 Executive Summary .................................................................................................................. 10 1. Introduction ........................................................................................................................ 11 1.1 Scope ......................................................................................................................... 11 1.2 Relation with other work packages and tasks .............................................................. 12 1.3 Document Structure .................................................................................................... 13 2. Security of Supply Chains .................................................................................................. 14 2.1 Supply Chain Classifications ....................................................................................... 14 2.1.1 Overall business view ............................................................................................16 2.1.2 Technical View - Asset Interdependent SC ............................................................16 2.1.3 Sector Specific view - SC snapshot .......................................................................16 2.2 Security Aspects of the SCSs ..................................................................................... 17 2.2.1 SCS threat landscape ............................................................................................17 2.2.2 Legal framework ....................................................................................................19 2.2.3 SC security standards (families of ISO2800x) ........................................................27 2.2.4 Risk Management standards (families of ISO2700x) .............................................29 2.2.5 SC risk assessment methodologies & Tools ..........................................................29 3. EU Certification .................................................................................................................. 34 3.1 Security Certification - EU requirements ..................................................................... 34 3.1.1 Cybersecurity Act - The European certification legal instrument ............................34 3.1.2 Security Certification standards addressing EU requirements ................................35 3.1.3 The European Cyber Security Certification Schema (EUCC) .................................37 3.1.4 R&D certification projects and EU initiatives ..........................................................38 3.1.5 Examples of Certificates ........................................................................................43 3.2 Conformity Assessment (CA) ...................................................................................... 44 3.2.1 Basic Concepts and Requirements ........................................................................44 3.2.2 Target of Evaluations (ToE) - Security & Assurance Requirements .......................45 3.2.3 Methodologies for CAs ...........................................................................................48 project has received funding from the European Union’s Horizon 2020 research ation programme under grant agreement No 952690 Horizon 2020 Program ICT-02-2020 Building blocks for resilience in evolving ICT systems Certifying the Security and Resilience of Supply Chain Services D2.1: Supply Chain Analysis and Requirements Contractual Date of Delivery 31/5/2021 Actual Date of Delivery 31/5/2021 Deliverable Security Class Public Type Report Editor Jlenia Puma (CRF) Contributors Sofoklis Efremidis, Eleni-Maria Kalogeraki, Fabio De Donato, Cristiano Casadei (MAG) Jlenia Puma, Julien Mascolo (CRF) Danijela Boberic Krsticev (UNSPMF) Alexandra Michota, Nineta Polemi (FP) Nikos Argyropoulos (CLS) Manolis Chatzimpyrros (STS) Haralambos Mouratidis (SU) Building blocks for resilience in evolving ICT systems Certifying the Security and Resilience of Supply Chain Services D2.1: Supply Chain Analysis and Requirements Contractual Date of Delivery 31/5/2021 Actual Date of Delivery 31/5/2021 Deliverable Security Class Public Type Report Editor Jlenia Puma (CRF) Contributors Sofoklis Efremidis, Eleni-Maria Kalogeraki, Fabio De Donato, Cristiano Casadei (MAG) Jlenia Puma, Julien Mascolo (CRF) Danijela Boberic Krsticev (UNSPMF) Alexandra Michota, Nineta Polemi (FP) Nikos Argyropoulos (CLS) Manolis Chatzimpyrros (STS) Haralambos Mouratidis (SU) D2.1: Supply Chain Analysis and Requirements This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952690. D2.1 – Supply Chain Analysis and Requirements Gregory Chrysos (TSI) Farhan Sahito (PN) Pablo Giménez Salazar (VPF) Norma Zanetti (HYPER) Sophia Karagiorgou (UBI) Quality Assurance Nikos Argyropoulos (CLS) Reviewers George Spyridakis (ITML) Dora Kallipolitou (ZELUS) D2.1 – Supply Chain Analysis and Requirements Gregory Chrysos (TSI) Farhan Sahito (PN) Pablo Giménez Salazar (VPF) Norma Zanetti (HYPER) Sophia Karagiorgou (UBI) Quality Assurance Nikos Argyropoulos (CLS) Reviewers George Spyridakis (ITML) Dora Kallipolitou (ZELUS) 952690 CYRENE PROJECT PARTNERS 31/05/2021 2 2 D2.1 – Supply Chain Analysis and Requirements Revision History Version Date By Overview 0.11 18/1/2021 All WP2 partners Tentative ToC, unifying previous proposals 0.2 25/01/2021 CRF Final ToC 0.2 26/02/2021 MAG, CRF, UNSPMF, FP, CLS, STS, SU, TSI, PN First contributions provided 0.3 01/03/2021 CRF Integration of contributions from the partners 0.3.1 09/03/2021 CRF Minor modifications 0.3.1 19/03/2021 MAG, CRF, UNSPMF, FP, CLS, SU, VPF, HYPER Update and integration to first contribution 0.4 25/03/2021 CRF Integration of contributions from the partners 0.4.1 26/03/2021 CRF Minor modifications 0.4.2 09/04/2021 MAG, CRF Minor modifications 0.5 10/05/2021 CRF Integration of contributions 0.5.1 13/05/2021 CRF, MAG, UBI, UNSPMF Last contributions and modifications added before internal review 0.6 28/05/2021 CRF Updated version incorporating input and comments from internal reviewers 1.0 31/05/2021 CRF Final version 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 3 D2.1 – Supply Chain Analysis and Requirements Table of Contents 4 D2.1 – Supply Chain Analysis and Requirements List of Tables List of Tables Table 1 - Impact on assets after a successful attack .................................................................49 Table 2 - Mapping of motivation and capability of threat agents. ...............................................50 Table 3 - Conformity standards. ................................................................................................52 Table 4 - Business importance of SCS. .....................................................................................60 Table 5 - Security standards and guidelines adopted. ...............................................................60 Table 6 - SC process description template. ...............................................................................75 Table 7 - SCS Business Partners description template. ............................................................75 Table 8 - ToE’s infrastructure description template. ..................................................................76 Table 9 - “Vehicle Order Dispatch” process description. ............................................................79 Table 10 - “Contract Agreement on the Vehicle Purchase” process description. .......................79 Table 11 - “Chartering Agreement Preparation and Negotiation” process description. ..............79 Table 12 - “Ship Formalities Arrangements” process description. .............................................79 Table 13 - “Shipping Arrangements” process description. .........................................................80 Table 14 - “Port Call Request” process description. ..................................................................80 Table 15 - “Standard Cargo Manifest” process description. .......................................................80 Table 16 - “Entry Summary Declaration (ENS)” process description. ........................................80 Table 17 - “Loading and Discharge List” process description. ...................................................81 Table 18 - “Discharge Vehicles” process description. ................................................................81 Table 19 - “Customs Declarations” process description. ...........................................................81 Table 20 - “Transportation Order” process description. .............................................................81 Table 21 - Business partners involved in the “Vehicles Order Dispatch” process. .....................82 Table 22 - Business partners involved in the “Contract Agreement on the Vehicle Purchase” process. ....................................................................................................................................84 Table 23 - Business partners involved in the “Chartering Agreement Preparation & Negotiation” process. ....................................................................................................................................86 Table 24 - Business partners involved in the “Ship Formalities Arrangements” processs. .........88 Table 25 - Business partners involved in the “Shipping Arrangements” process. ......................89 Table 26 - Business partners involved in the “Port Call Request” process. ................................90 Table 27 - Business partners involved in the “Standard Cargo Manifest” process. ....................91 Table 28 - Business partners involved in the “Entry Summary Declaration (ENS)” process.......92 Table 29 - Business partners involved in the “Loading and Discharge List” process. ................93 Table 30 - Business partners involved in the “Discharge Vehicles” process. .............................95 Table 31 - Business partners involved in the “Customs Declarations” process. .........................96 Table 32 - Business partners involved in the “Transportation Order” process. ..........................97 Table 33 - Identified infrastructures of the “Port Call Request” process. .................................. 100 Table 34 - Identified infrastructures of the “Standard Cargo Manifest” process. ...................... 102 Table 35 - Identified infrastructures of the “Entry Summary Declaration (ENS)” process. ........ 103 Table 36 - Identified infrastructures of the “Loading and Discharge List” process. ................... D2.1 – Supply Chain Analysis and Requirements 3.2.4 Standards for CAs .................................................................................................51 3.2.5 Conformance monitoring ........................................................................................53 4. Methodology ...................................................................................................................... 54 4.1 Methodology for requirements elicitation ..................................................................... 54 4.2 Methodology for requirements validation ..................................................................... 56 4.3 CYRENE Questionnaire for enriching the requirements with feedback collected by external stakeholders ........................................................................................................... 57 4.3.1 Feedback from the questionnaire ...........................................................................57 5. CYRENE Conformity Assessment...................................................................................... 73 5.1 Targets of Evaluations (ToE) ...................................................................................... 74 5.1.1 ToE I: Business view of the VTS-SCS ...................................................................77 5.1.2 ToE II: Technical view of the VTS-SCS ..................................................................98 5.1.3 ToE III: Sectorial view of the VTS-SCS ................................................................ 109 5.2 Requirements for self-conformity assessing CYRENE ToEs ..................................... 119 5.2.1 Requirements for CYRENE ToE I ........................................................................ 119 5.2.2 Requirements for CYRENE ToE II ....................................................................... 121 5.2.3 Requirements for CYRENE ToE III ...................................................................... 123 6. Conclusion ....................................................................................................................... 126 7. References ...................................................................................................................... 127 Appendix A – Glossary and Examples .................................................................................... 132 Appendix B – Validation of CYRENE ToEs ............................................................................. 141 B.1 CYRENE Advisory Boards ........................................................................................... 141 B.2 First Project Workshop ................................................................................................. 142 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 5 List of Tables 105 Table 37 - Identified infrastructures of the “Discharge Vehicles” process. ............................... 105 Table 38 - Identified infrastructures of the “Customs Declarations” processs. ......................... 107 Table 39 - Identified infrastructures of the “Transportation Order” process. ............................. 108 Table 40 - Supply of partial-assembled components process description. ............................... 109 Table 41 - “Supply of finished components” process description. ............................................ 110 Table 42 - “Monitoring of components” process description. ................................................... 110 Table 43 - “Vehicle Assembly” process description. ................................................................ 110 952690 CYRENE PROJECT PARTNERS 6 List of Figures List of Figures Figure 1 - Relation of WP2 with the other project WPs ..............................................................13 Figure 2 - Ageron et al. Classification model .............................................................................14 Figure 3 - CYRENE circles of consideration for the CA process ................................................15 Figure 4 - ISO28000 .................................................................................................................27 Figure 5 - Overview of requirements elicitation and validation process .....................................54 Figure 6 - Overview of the methodology for requirements elicitation .........................................55 Figure 7 - Cybersecurity activities frequency .............................................................................59 Figure 8 - Awareness of EU Cybersecurity Certification Framework for ICT products and services .................................................................................................................................................60 Figure 9 - Measures to address cybersecurity issues ................................................................61 Figure 10 - Effective cybersecurity management plan applied to SCS ......................................62 Figure 11 - Awareness of security standards and best practices adopted .................................62 Figure 12 - Security procedure - Number of respondents ..........................................................62 Figure 13 - Compliance with legal and regulatory principles and EU directives .........................63 Figure 14 - Experience of cybersecurity issues in last years .....................................................63 Figure 15 - Probability of threats in the future ............................................................................64 Figure 16 - Addressee of risks and security threats ...................................................................65 Figure 17 - Relevant tools for secure service delivery ...............................................................66 Figure 18 - ICT Systems security measures applied to daily operations ....................................69 Figure 19 - Perform of periodic audits .......................................................................................70 Figure 20 - Analysis and evaluation of infrastructure security performed by external certified analyst ......................................................................................................................................70 Figure 21 - “Standard Cargo Manifest”: a business process model example .............................76 Figure 22 - Business process model for the “Vehicle Order Dispatch” process .........................83 Figure 23 - Business process model for the “Contact Agreement on the Vehicle Purchase” process .....................................................................................................................................84 Figure 24 - Business process model for the “Chartering Agreement Preparation & Negotiation” process .....................................................................................................................................86 Figure 25 - Business process model for the “Ship Formalities Arrangements” process .............88 Figure 26 - Business process model for the “Shipping Arrangements” process .........................89 Figure 27 - Business process model for the “Port Call Request” process ..................................90 Figure 28 - Business model for the “Standard Cargo Manifest” process ....................................92 Figure 29 - Business process model for the “Entry Summary Declaration (ENS)” process ........93 Figure 30 - Business process model for the “Loading and Discharge List” process ...................94 Figure 31 - Business process model for the “Discharge Vehicles” process ...............................95 Figure 32 - Business process model for the “Customs Declarations” process ...........................96 Figure 33 - Business process model for the “Transportation Order” process .............................97 Figure 34 - Business process model for the”Supply of partial-assembled components” process ............................................................................................................................................... D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements Table 44 - Business partners identification and analysis of the “Supply of partial-assembled components” process. ............................................................................................................. 111 Table 45 - Business partners identification and analysis of the “Supply of finished components” process. .................................................................................................................................. 111 Table 46 - Business partners identification and analysis of the “Monitoring of components during transportation” process. .......................................................................................................... 112 Table 47 - Business partners identification and analysis of the “Vehicle Assembly” process. .. 112 Table 48 - Identified infrastructures of the “Supply of partial-assembled components” process. ............................................................................................................................................... 113 Table 49 - Identified infrastructures of the "Supply of finished components" process. ............. 115 Table 50 - Identified infrastructures of the "Monitoring of components" process. ..................... 117 Table 51 - Identified infrastructures of the "Vehicle Assembly" process. .................................. 118 Table 52 - Requirements for CYRENE ToE I. ......................................................................... 121 Table 53 - Requirements for CYRENE ToE II.......................................................................... 123 Table 54 - Requirements for CYRENE ToE III......................................................................... 125 Table 55 - Extract from Security and Certification concept glossary. ....................................... 133 Table 56 - Extract from Supply Chain and Business concept glossary. ................................... 137 Table 57 - Extract from Maritime Transport concept glossary. ................................................. 140 Table 58 - Workshop agenda. ................................................................................................. 143 Table 59 - Suggestions from the Advisory Boards. .................................................................. 144 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 7 7 List of Figures 114 Figure 35 - Business process model for the "Supply of finished components" process ........... 116 Figure 36 - Business process model for the "Monitoring of components" process ................... 118 Figure 37 - Business process model for the “Vehicle Assembly” process ................................ 119 952690 CYRENE PROJECT PARTNERS 8 31/05/2021 8 D2.1 – Supply Chain Analysis and Requirements List of Acronyms List of Acronyms Acronym Description AIS Automatic identification system BP Business Partner CA Conformity Assessment CC Common Criteria CII Critical Information Infrastructure CSA Cybersecurity Act ENS Entry Summary Declaration LNG Liquefied Natural Gas MRA Mutual Recognition Agreement PCS Port Community System PDCA Plan-Do-Check-Act RA Risk Assessment SC Supply Chain SCADA Supervisory Control And Data Acquisition SCRM Supply Chain Risk Management SCM Standard Cargo Manifest SCS Supply Chain Service SOG-IS Senior Officials Group Information Systems Security ToE Target of Evaluation TVRA Threat Vulnerability Risk Analysis VTS Vehicle Transport Service 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 9 9 D2.1 – Supply Chain Analysis and Requirements 1.1 Scope This document is a record of the requirements that have been collected from several stakeholders, based on which the specifications of certification scheme, the definition of the conformity assessment processes, as well as the development and integration of tools within the CYRENE project will be developed. The described outputs are the results of the activities performed during the followin  T2.1: Conformity and certification assessment scheme state of the art revisio  T2.2: Large-scale European Supply Chain requirement gathering, analysis and tracking;  T2.3: Legal and ethics requirements;  T2.4: Classification of Supply Chains. During the first task, the activities of CYRENE’s phase 1 were initiated, focusing on the definition of a solid basis for setting up the CYRENE Conformity Assessment scheme. An updated state of the art analysis was carried out, consulting scientific papers, related projects, and relevant reference conformity assessment and certification schemes for cyber-security in related domains. In particular, during the task, the CYRENE consortium consulted and built upon the baseline security requirements recommended by ENISA [1]. The existing relevant schemes are mapped to the four CYRENE circles of consideration (as described in Figure 3), as well as to the three main aspects of CYRENE certification: business, infrastructure, and individual devices. Based on the described analysis, the task creates a basis for the conformity assessment scheme which grounds the multi-level evidence-driven supply chain risk assessment process. During Task 2.2, in parallel to Task 2.1, the CYRENE consortium identified Supply Chains’ Conformity Assessment requirements, along with legal/forensic, security and privacy requirements and covered the following aspects: (i) identification of requirements and specific needs of the participating Supply Chain Services, which represent different industry sub-sectors and with different needs with regard to IT security; (ii) substantial engagement of the participating SCs operators (representing different industrial sectors) so as to gain feedback regarding their needs and priorities in the frame of the project; (iii) specification of criteria associated to the nature of their IT system and infrastructure of SCs (such as size, interdependencies with other IT systems, services offered, etc.); (iv) analysis and documentation of the requirements of the various stakeholders, i.e. port authorities and operators, security systems integrators, policy makers etc.) in terms of the handling of multi-order dependencies and cascading effects; (v) identification and classification of dependencies between infrastructures and between SC operators, as well the dependencies among the business inter-organizational, infrastructure, and individual assets/devices. D2.1 – Supply Chain Analysis and Requirements Executive Summary The objective of this deliverable is to report the results of the activities performed in the first phase of CYRENE’s Work Package 2. The main output is related to the requirements that have been collected from relevant standards and literature review, project pilot partners, as well as external stakeholders. The document can be divided in four main parts. In the first one, an overview of the Supply Chains is given, describing both their classification, including three different views (business, technical and sectorial) of the SCs, and their security aspects, consisting of the threat landscape, legal framework, SC security and Risk Management standards and SC risk assessment methodology and tools. In the second part, an overview of the EU Certification schemes is provided, encompassing the general definition and requirements (policy, legal, standards, methodologies, technical) regarding the security certification. Moreover, in the third part, the document reports on the methodology used for collecting, analyzing and validating the requirements through the project’s Advisory Boards. The feedback obtained from the proposed questionnaire for requirements validation are presented in this part and conclusions are drawn afterwards. Finally, the fourth part of the deliverable deals with the three Targets of Evaluation (ToEs), namely, the Business, Technical, and Sectorial. Their descriptions and the respective validated requirements are provided in this section. Two appendixes are included in the document. The first one gives information on a glossary that forms the basis of the concepts used in the project, while the second one gives details regarding the first workshop organized with the Advisory Boards at the end of the first six months of the project. 952690 CYRENE PROJECT PARTNERS 31/05/2021 10 D2.1 – Supply Chain Analysis and Requirements services offered, number of administrators and IT security awareness level, etc.) based on which the categorization of the enterprise target group will occur. services offered, number of administrators and IT security awareness level, etc.) based on which the categorization of the enterprise target group will occur. 1.1 Scope The output of Task 2.3 is the identification, analysis and report of relevant legal and ethics requirements for CYRENE. The regulatory framework applicable to the project is analyzed to define requirements that are not dealt within the previous tasks. Finally, Task 2.4 activities are focused on the specification of the criteria associated to the nature of IT systems and infrastructure of SCs (such as size, interdependencies with other IT systems, 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 11 1.2 Relation with other work packages and tasks This document describes in detail the results of the Phase 1 activities of the projec During Phase 2 of the project, whose main task is T2.5, the Conformity/Certification Assessment scheme set-up will be described. In fact, based on the results described in this document, the activities of this task will set up the proposed Conformity Assessment scheme for ensuring the security and resilience of Supply Chain Services. The proposed scheme will be the basis for the Conformity Assessment processes implemented in WP3 and WP4. Moreover, Task 2.6 will propose a refined specification for CYRENE architecture based on a thorough understanding of the challenges, technologies, requirements and state-of-the-art introduced in all previous tasks of Work Package 2. The result of this task will serve as a reference for the implementation phase of the CYRENE approach, defined in WP3 and WP4. In addition, it will serve as a reference and a starting point for the integration activities described in WP5. The interdependencies between WP2 and the rest of the Work Packages is depicted in Figure 1. 952690 CYRENE PROJECT PARTNERS 31/05/2021 12 D2.1 – Supply Chain Analysis and Requirements Figure 1 - Relation of WP2 with the other project WPs. Figure 1 - Relation of WP2 with the other project WPs. D2.1 – Supply Chain Analysis and Requirements 1.3 Document Structure The rest of the document is structured as follows:  Chapter 2 presents an overview of the Security of Supply Chains, describing how Supply Chains are classified and the security aspects of the involved Supply Chain Services.  Chapter 2 presents an overview of the Security of Supply Chains, describing how Supply Chains are classified and the security aspects of the involved Supply Chain Services.  Chapter 3 gives an overview of the EU Certification schemes, presenting definitions and requirements that address the Security Certification process.  Chapter 3 gives an overview of the EU Certification schemes, presenting definitions and requirements that address the Security Certification process.  Chapter 4 presents the Methodology used for requirements elicitation and their validation strategy.  Chapter 4 presents the Methodology used for requirements elicitation and their validation strategy.  Chapter 5 presents the CYRENE Conformity Assessment process, including the description of the Targets of Evaluation and their requirements.  Finally, Chapter 6 concludes the document. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 13 2.1 Supply Chain Classifications 2.1 Supply Chain Classifications According to Mentzer [2], a Supply Chain (SC) is defined as a globally distributed, interconnected set of entities (i.e., organizations, individuals or/and CIs), processes and services that relies upon an interconnected web of ICT infrastructures and cyber networks to leverage the flows of products, services and information from a source to a customer. The literature is rich with efforts [3] to classify SCs. Ageron et al [4] developed a classification model that is focused on the practices related to Supply Chains (see Figure 2). The model, which is depicted as a pyramid, puts at the top the Managerial Process, on the second level the Information Sharing and Information Technologies, and on the bottom level the Operational process. Figure 2 - Ageron et al. Classification model. Figure 2 - Ageron et al. Classification model. Jabbour et al. [5] mapped supply chain practices into four constructs of supply chain management: (i) Supply chain integration for production planning and control support; (ii) Information sharing about products and targeting strategies; (iii) Strategic relationship with customer and supplier; (iv) Support customer order. Although these works are important and provide clear answers for the purposes of the related research, they failed to consider a conceptual representation of supply chains, which is practical for conformity and certification purposes for Supply Chain Services (SCSs). To address this challenge, CYRENE has developed a conceptual representation of SCSs based on the four circles of consideration. (Figure 3). 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 14 D2.1 – Supply Chain Analysis and Requirements represent the main block for the CYRENE Conformity Assessment. It also helps us to investigate a Supply Chain from a different perspective, in order to analyze the effects of business, technical and sector specific aspects on Supply Chain Services. In the next sub-sections, we provide an outline of the three views. represent the main block for the CYRENE Conformity Assessment. It also helps us to investigate a Supply Chain from a different perspective, in order to analyze the effects of business, technical and sector specific aspects on Supply Chain Services. In the next sub-sections, we provide an outline of the three views. 2.1.1 Overall business view In the context of CYRENE, the business view focuses on the identification, analysis and assessment of any Supply Chain elements that have direct input on the business perspective of an organization. As such, in this view, details of organizational processes, business partners that contribute to such processes (e.g. suppliers), stakeholders, facilities, related business logic (e.g. data and information flows, decision making), and any legal/regulatory restrictions are considered. As a result, all business-driven elements in a Supply Chain which have impact on the Supply Chain Services are taken into consideration. 2.1.2 Technical View - Asset Interdependent SC The technical view of the SCs includes an asset-based interdependent view of the Supply Chain, which is focused on the ICT assets within the SC used to carry out the activities related to the provision of the SCS. In such view, the SCS elements are all ICT assets (e.g. networks, IoT sensors, communication devices, local servers) hosted by the business partners’ infrastructures (and/or their subcontractors) and are utilized for the provision of the SCS. D2.1 – Supply Chain Analysis and Requirements Figure 3 - CYRENE circles of consideration for the CA process. Figure 3 - CYRENE circles of consideration for the CA process. In such representation, the established SCS interconnections reflect the relationships that exist between the involved entities representing how one process, activity or resource relies upon another. For example, an entity could be dependent on receiving information from another entity or organization as an input to one of its critical business processes. In such representation, the established SCS interconnections reflect the relationships that exist between the involved entities representing how one process, activity or resource relies upon another. For example, an entity could be dependent on receiving information from another entity or organization as an input to one of its critical business processes. The first inner circle, our starting point, includes all the ICT systems, equipment and devices (e.g. IoT systems, cyber-physical systems, SCADA systems, communication network assets, local hubs, switches, gateways, servers) used by the business partners, which support the operation of the SCSs. The second circle encapsulates the previous one and incorporates the individual Critical Information Infrastructures (CIIs) operated by the individual Business Partners involved in the SCSs. The third circle encloses the two previous ones and represents the Interdependent CIIs composing the ICT-empowered SCSs. Finally, the fourth circle contains all the already mentioned circles, complemented with the Business Perspective of the SCSs such as processes information exchange, business logic, ICT assets, Business Partners, etc. These four circles have been identified and distinguished based on the homogeneity of characteristics (safety, technical requirements, architectures etc.) identified in each one of them. As mentioned earlier, Supply Chain encompasses entities, assets, individuals, organizations and processes to leverage the flows of products and services to end users. In other words, supply chain provides the required infrastructure and resources for delivering a supply chain service or building a product. We have mapped the four circles of consideration to three different but interdependent views of SCSs, the Business view, the Technical view and the Sector specific view. This is important since it allows us to define views that can be directly translated to Targets of Evaluation (ToEs), which 952690 CYRENE PROJECT PARTNERS 31/05/2021 15 1 Port cybersecurity : good practices for cybersecurity in the maritime sector. ENISA, 2019, [Online]. Available: https://www.enisa.europa.eu/publications/port-cybersecurity-good-practices-for-cybersecurity- in-the-maritime-sector. Accessed: March 15, 2021. D2.1 – Supply Chain Analysis and Requirements connections/relations, elements. Last, each sub-sector can be categorized based on its functional aspect, where the main categorization criterion is the internal status of each sub-sector. connections/relations, elements. Last, each sub-sector can be categorized based on its functional aspect, where the main categorization criterion is the internal status of each sub-sector. Another sector-specific view of the Supply Chains, according to Mattsson [8], is that companies and Supply Chains can be regarded as open systems, where their components can be linked not only to each other but also to the surroundings, i.e. suppliers, customers, competitors, and the authorities. Such view leads to a more generic description, where the sub-systems in a supply chain can be considered as the functions within the Supply Chain that add value to the final product. Based on that, the sub-systems in a Supply Chain system are the individual companies. Finally, a really important Sector Specific view is based on the internal and external Supply Chain linkages [9]. Specifically, Mark Barrat and Ruth Barrat proved that internal linkages between sub- sectors are really important as the information flow improves operational performance of the whole Supply Chain. Additionally, this work concluded that organizations need to recognize the combining role of internal and external information-based linkages in order to extend the visibility across the entire Supply Chain. This section has analyzed the different Supply Chain classifications based on sub-sector view in order to investigate the effects of each sub-sector on Supply Chain services. Also, it has shown the importance of linkages among the sub-sectors as far as the entire Supply Chain visibility. 2.2 Security Aspects of the SCSs General aspects and requirements (legal, policy, standards, technical) related to the security of the SCs will be addressed in this section. General aspects and requirements (legal, policy, standards, technical) related to the security of the SCs will be addressed in this section. 2.1.3 Sector Specific view - SC snapshot Business Partners within a SC can belong to different sectors. For example, for the Vehicle Transport Service SC provision, the business partners belong to different industries and sectors (e.g. automotive industry, transport, government). The sector-specific view of a SC is the view that an individual business partner adopts to analyze the SCS. The sector-specific view of the SC consists of the specific processes that this business partner is participating and his ICT assets that are involved in the provision of the SCS. The Supply Chain framework is based on components, which are complex designs, consisting of various industry sub-sectors that focus on different IT security levels depending on their needs [6]. Based on that, the SCS can be analyzed according to their sector-specific schemes, which ensure vertical and horizontal security and resilience of Supply Chain Services. This section describes generic Supply Chains classifications that are based on sub-sector specific views. First, the sub-sector specific classification is based on the three different specific view aspects for the Supply Chains and their components: hierarchical, structural and functional [7]. According to the hierarchical aspect, the parts of a Supply Chain system can be considered as submodules and the system itself can be part of a more comprehensive system, i.e., a supersystem. Second, each Supply Chain sector can be classified based on the set of the sub-sectors’ elements and their interconnections. Such classification is based on the sub-sectors’ structural architecture, i.e. 31/05/2021 31/05/2021 952690 CYRENE PROJECT PARTNERS 16 D2.1 – Supply Chain Analysis and Requirements help mitigate the risk. For the purpose of this work three threat scenarios of SCS are presented, as well as the most common individual threats1 that can lead to such a situation. help mitigate the risk. For the purpose of this work three threat scenarios of SCS are presented, as well as the most common individual threats1 that can lead to such a situation. Threat scenario in the Container Management SCS A terrorist group wants to commit an attack at a port by placing a bomb in a container, which will be detonated before the inspection process of the security authorities, aiming for the greatest number of injuries and deaths. Knowing that the sealed container leaves the company’s factory and gets packed at the warehouse of a third-party company before being delivered to the port, they gain access to the third party’s IT systems, specifically to the container management system. In this way, they replace the information of a particular shipping container with another one that carries out a bomb, which they make sure it has been placed in the warehouse. After the moving of the malevolent container to the port, they gain access to the container shipping system, which keeps the routing or scheduling of the containers, so that they can change the containers’ details of location and maximize the number of victims. Considering that the containerized freight is almost one third of total trade exchanges measured in monetary value, according to Eurostat 184/2016 statistics, and when kilometres or tonne-kilo- metres are measured, the percentage of maritime transport in relation to total transported is even higher, it turns out that the impact of realizing such a possibility would be significant. 2.2.1.1 Threat scenarios in the Supply Chain Services (SCS) In the following threat scenarios [10][11] it is depicted how a security breach in a specific node of the SCS can cause damage to a larger scale, even to a physical level. 2.2.1 SCS threat landscape As the SCSs become more and more complex and technology-dependent, the SC threat landscape is getting broader. Their complexity is directly related to the interdependence among the abundance of sectors, ICT assets and services. Hence, the SCS stakeholders must encounter a variety of cyber/physical threats, either internal, external or diffused, coming from the whole SC. The combination of these threats can create numerous threat scenarios, which may affect the confidentiality, integrity, and / or availability of the SCS’s information and ICT systems. Among others, the result of such scenarios may include reduced or unwanted functionality, tampering, theft, counterfeiting or malicious content and may aim at gaining financial, political, military, or even ideological benefits. ENISA has proposed good practices for cybersecurity in the maritime sector in a report1 which describes multiple key cyberattack scenarios, targeted at the port ecosystem, concerning both IT and OT infrastructures and able to result in cyber-physical consequences. Each of these real-life scenarios is associated with the impacts that could cause, the assets affected, the stakeholders involved, the details of the attack, as well as the main security measures that could be taken to 952690 CYRENE PROJECT PARTNERS 31/05/2021 17 Threat scenario in the Vehicle Transport SCS A team of criminals launches a series of cyber-attacks, including phishing information from the port authorities and key officials as well as remote exploitation of software-related vulnerabilities using malware, to steal vehicles from the vehicle terminal of a port. Eventually they manage to compromise a few computers and systems, get access to the vehicles’ vast network of intercon- nected On-Board Units (OBUs) and spoof their geolocation. Having access to the in-port vehicle scheduling processes, they can continue changing the vehicles’ route and location to the values of their interest, without the port system administrator being able to detect it. They can also pen- etrate into the port’s surveillance system that controls the CCTV video cameras, so that they gain access and delete video streams that could disclose their malicious activities. The Vehicle Transport Service is supported by many stakeholders, for example port authorities and shippers, and involves the transfer of a plethora of vehicles and equipment, such as trucks, cranes, container terminals and providers of Dockers. This fact, together with the involvement of actors such domestic and international transportation, IT, warehouse management, order and inventory control, materials handling, and import/export facilitation, make the likelihood of a sce- nario like this high and its scope wide and complex. D2.1 – Supply Chain Analysis and Requirements a result, the terrorists can exploit vulnerabilities to delve deeper into the network of the oil com- pany’s monitoring software, which they can use to break into the system by performing remote tank monitoring, asset tracking, and data reporting services, and empty the oil tank without being detected. a result, the terrorists can exploit vulnerabilities to delve deeper into the network of the oil com- pany’s monitoring software, which they can use to break into the system by performing remote tank monitoring, asset tracking, and data reporting services, and empty the oil tank without being detected. As liquefied natural gas (LNG) is mainly methane, which is odorless, colorless, non-toxic and non- corrosive, in a liquid form for easier storage and transport, its vaporization could cause inflamma- tion, freezing or asphyxia. In addition, a potential explosion of an LNG tanker can be compared to a nuclear bomb’s, since it contains more than 100,000 m3 of LNG. 2.2.1.2 Individual threats in the SCS assets As can be seen from the above-mentioned scenarios, combining individual threats can lead to a variety of impacts, such as cargo and goods stealing, sensitive and critical data theft, illegal traf- ficking, systems damage or destruction, environmental disaster, or even human injuries and death. In addition, an organization’s paralysis, financial loss and costs, kidnapping, fraud and money theft are also included in this long list, and all of the above are usually accompanied by tarnished reputation, and /or loss of competitiveness. p p The assets (physical, digital, people, processes, smart objects) used to provide a SCS, hosted and operated by the various SCS business partners involved are called SCS assets. The assets (physical, digital, people, processes, smart objects) used to provide a SCS, hosted and operated by the various SCS business partners involved are called SCS assets. Examples of potential threats to the digital SCS assets are eavesdropping, interception of emis- e assets (physical, digital, people, processes, smart objects) used to provide a S d operated by the various SCS business partners involved are called SCS assets. Examples of potential threats to the digital SCS assets are eavesdropping, interception of emis- sions or sensitive information, IT/OT assets hijacking, network reconnaissance or traffic manipu- lation, data poisoning, data manipulation and all kind of nefarious activity and abuse, such as Denial of Service (DOS), brute force, phishing, social engineering or targeted attacks, as well as malware, identity or data theft or abuse, manipulation of information, or even geolocalization sig- nals spoofing or jamming. p g j g Additionally, possible threats to the non-digital SCS assets include unauthorized access to the premises, vehicles or IT/OT end devices, terrorism, hacktivism, coercion, extortion or corruption of employees or stakeholders, piracy or mafia, sabotage, vandalism, natural disasters, environ- mental damages and theft. g Although in most people’s minds a threat may be related to a deliberate act, there is also the likelihood of unintentional damage, which results from data deletion, information leakage, third party security failure, erroneous administration of IT/OT systems, use of unreliable sources or improper penetration testing. An attacker can take advantage of coincidences like all types of outages and natural or environmental disasters, or even force them to carry out their plan. Sys- tems and devices failures and malfunctions, as well as failure or disruption of service providers are also common threats. Threat scenario in the LNG Transport SCS p A fake email is sent by the supposed IT company that supports and maintains the ICT infrastruc- ture of a shipping company, requesting the downloading and installation of a software that im- proves the performance of their systems. In fact, the email is sent by a group of terrorists who, subsequently, download and execute arbitrary code on the victims’ systems and gain access. As 952690 CYRENE PROJECT PARTNERS 31/05/2021 18 2.2.2.1 Data Protection and Data Security Information privacy is a set of rules that governs the collection and use of personal information. It addresses individuals’ right to decide about the processing of information related to them by a third party. Information privacy is one of the foundational pillars of privacy protection and personal data protection. Closely related to Information Privacy is the concept of Information Security. Information Security is about preserving the ‘security triad’ - confidentiality, integrity and availability of information (Densmore, 2019). There are similarities between privacy and security but there are also factors that distinguish them. Therefore, privacy and security could be seen as supplementary concepts but not complementary. Information integrity is about its authenticity and it relates to accuracy and completeness of personal information. Confidentiality of information is related to limited access to information, whereas availability enables access to information but only to those who are authorized to use this information. To satisfy the standards of the ‘security triad’, appropriate security controls have to be implemented and security incidents should be prevented. In this way, information security preserves information privacy. Differences between privacy and security could be found in the fact that implementing information security does not necessarily preserve information privacy. Namely, information privacy serves to protect specific type of information. However, there is no information privacy without the implementation of information security. Therefore, we can have security without privacy, but we cannot have privacy without security (Densmore, 2019). D2.1 – Supply Chain Analysis and Requirements 2.2.2 Legal framework The pivotal concepts of the CYRENE regulatory end ethical framework are related to data security and data protection. However, there are similar concepts such as privacy and information security. This chapter briefly sheds light on these mentioned concepts but also on regulation and legislation. The chapter provides preliminary facts that are crucial for the development of a regulatory framework specifically designed for this project. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 19 D2.1 – Supply Chain Analysis and Requirements are formally promoted as regulations, but contain principles and general rules. These principles and rules might be specified by legislations adopted by lower legislative instances2. are formally promoted as regulations, but contain principles and general rules. These principles and rules might be specified by legislations adopted by lower legislative instances2. As explained earlier, the scope of this document is to present a specific regulatory framework designed to contribute to the project objectives. Therefore, this document presents specific regulations adapted for a specific purpose. Nevertheless, the document first refers to the principles and rules of various but relevant legislations. In the following sections, these principles and rules are ‘custom-made’ and are presented as the CYRENE regulatory framework2. 2.2.2.2 Regulation and Legislation There is a common mistake with using terms of regulation and legislation in the same sense. They may be similar, but the differences between them should not be neglected. Both regulation and legislation contain provisions with rules, rights, and obligations and it would not be wrong to claim that they regulate certain relations, entities or fields. However, there are at least two distinct lines between these concepts. The first one concerns the respective sources. Whereas legislation usually refers to statutory law enacted by the legislator (the legislative branch of government), regulation is adopted and promoted by entities that are supposed to develop a self-regulatory system (in order to introduce certain rights/obligations). In other words, legislations are acts adopted by a state, whereas regulations might be adopted by companies, industry associations, formal or informal bodies. The second distinctive line concerns the relationship between general and specific. Legislators often adopt rules that are general and applicable in many perspectives. Thus, the concrete application of the rules would only be possible if general rules and principles are specified to be used in a specific context. This process is usually carried out by the development of regulation, or more specifically, self-regulation. However, legislation might be a subset of a regulation. This would be the case when states or other legislative instances are allowed to adopt documents that 952690 CYRENE PROJECT PARTNERS 31/05/2021 20 2.2.2.3 Relevant European Union Law All actions taken by the EU institutions are based on treaties. These are binding agreements between the EU Member States which set out the EU objectives, the rules for the EU institutions, the decision-making processes and the relationship between the EU and the Member States. Treaties are the starting point for the EU Law and are known in the EU as primary law. The body of law derived from the principles and objectives of the treaties is known as secondary law. Secondary law includes regulations, directives, decisions, recommendations and opinions. 952690 CYRENE PROJECT PARTNERS 21 31/05/2021 2 Kosti, N., Levi-Faur, D., & Mor, G. (n.d.). Legislation and regulation: three analytical distinctions. The Theory and Practice of Legislation, 7(3). https://www.tandfonline.com/doi/full/10.1080/20508840.2019.1736369 3 https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:12012P/TXT 2 Kosti, N., Levi-Faur, D., & Mor, G. (n.d.). Legislation and regulation: three analytical distinctions. The Theory and Practice of Legislation, 7(3). https://www tandfonline com/doi/full/10 1080/20508840 2019 1736369 D2.1 – Supply Chain Analysis and Requirements The Charter contains all rights granted by the ECHR. However, the Charter addresses some additional freedoms and rights in order to meet the reality of newly formed issues. One of the newly granted rights relates to the protection of personal data. The Charter ensures that private and family right should be respected granting that ‘Everyone has the right to respect for his or her private and family life, home and communications.’ In addition, Article 8 regulates the protection of personal data by granting that ‘Everyone has the right to the protection of personal data concerning him or her’. In addition, the same article lays down that data processing must be carried out fairly, within the specified purposes, and based on consent or other ground laid down by law. Finally, compliance with the ‘rules shall be subject to control by an independent authority.’ Despite academic critics and polemics (Sloot, 2017), the Charter separated data protection from the protection of personal privacy. The additional significance of the Charter lays in the fact that both rights are classified as fundamental rights. Charter of Fundamental Rights of the European Union The Charter of Fundamental Rights of the European Union (hereinafter referred to as the Charter) brings together the most important personal freedoms and rights granted by the EU Law into one legally binding document. The Charter was announced in 2000 and came into force in December 2009 together with the Treaty of Lisbon3. The purpose of the Charter is to promote human rights within the territory of the EU. Many of the rights existing in the Charter were previously set out in:  The EU Treaties  The European Convention on Human Rights  Case law of the Court of Justice of the European Union  National constitutions The Charter has the same legal power as the EU Treaty. This means that it is superior to the legislation of the Member States. The Charter applies when EU countries adopt or apply a national law to implement an EU directive or when their authorities directly apply an EU regulation. In cases where the Charter does not apply, the protection of fundamental rights is guaranteed under the constitutions or constitutional traditions of EU countries and international conventions they have ratified. The Charter does not extend the scope of the EU to matters which are outside its normal competence. 31/05/2021 31/05/2021 21 4 https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32016R0679 D2.1 – Supply Chain Analysis and Requirements Purpose limitation principle. The purpose limitation principle is embedded in Art 5(1)b of the GDPR. This article foresees that personal data shall be ‘collected for specified, explicit and legitimate purposes and not further processed in a manner that is incompatible with those purposes. Purpose limitation principle. The purpose limitation principle is embedded in Art 5(1)b of the GDPR. This article foresees that personal data shall be ‘collected for specified, explicit and legitimate purposes and not further processed in a manner that is incompatible with those purposes. Data minimization. ‘Personal data must be adequate, relevant and limited to what is necessary in relation to the purposes for which they are processed’. To satisfy the requirements of this principle, data controllers have to pass proportionality and necessity tests. That means data controllers should be able to prove that plans regarding the use of a particular scope and a type of data are reasonable to achieve the specified purpose of data processing. Data accuracy. The GDPR laid down that ‘Personal data must be accurate and, where necessary, kept up to date’. Therefore, ‘every reasonable step must be taken to ensure that personal data that are inaccurate, having regard to the purposes for which they are processed, are erased or rectified without delay’. Storage limitation. Storage limitation principle is about the obligation to keep personal data ‘in a form that permits identification of data subjects for no longer than is necessary for the purposes for which the personal data are processed.’ So, once the purpose of processing is identified, data controllers have to determine the retention period. Integrity and confidentiality. The principles of integrity and confidentiality are embedded in the GDPR provision stating that personal data should be ‘processed in a manner that ensures appropriate security of the personal data, including protection against unauthorized or unlawful processing and accidental loss, destruction or damage, using appropriate technical or organizational measures’. Concerning the security of personal data, data controllers should take into consideration several factors when determining appropriate technical and organizational security measures. Also, this principle should be interpreted in the context of data controllers’ obligation to report a breach of personal data in a relevant way. Accountability principle. The accountability principle is not a new principle in the data protection framework. Nevertheless, the GDPR clearly defines it. In that way, the GDPR significantly contributes to the existing data protection framework. The essence of the principle lays in the data controllers’ obligation to comply with other principles as well as to be able to demonstrate it. 5 https://eur-lex.europa.eu/legal-content/GA/TXT/?uri=CELEX:32016L1148 GDPR The Regulation (EU) 2016/6794 on the protection of natural persons regarding the processing of personal data and on the free movement of such data (hereinafter GDPR) is the core legislative source of the EU Data Protection Law. The GDPR empowers the EU Data Protection Law by promoting a common set of data protection rules that are implementable in all Member States of the EU. The GDPR is a Regulation and unlike directives, regulations are directly applicable under EU law. In other words, there is no need for their transposition into national laws, and there is no need for national implementation. For that reason, the GDPR is of the highest relevance for CYRENE and represents a common framework for protecting personal data in the EU. To define its scope with regards to CYRENE, it should be noted that the GDPR does not apply to the processing of personal data regarding:  activities which fall outside the scope of the EU Law,  activities which fall outside the scope of the EU Law,  certain matters of the EU security and defense policy,  purely domestic activities and  cases of data processing carried out by competent authorities for the purposes of the prevention, investigation, detection or prosecution of criminal offences or the execution of criminal penalties, including the safeguarding against and the prevention of threats to public security.  cases of data processing carried out by competent authorities for the purposes of the prevention, investigation, detection or prosecution of criminal offences or the execution of criminal penalties, including the safeguarding against and the prevention of threats to public security. The GDPR confirms and additionally develops principles promoted by fair information practices. Therefore, the GDPR does not provide novelties concerning promoted principles but rather organizes and specifies them in a different way than it has been the case before. The GDPR promotes six principles, plus the accountability principle that is extracted as the separate one. All of them are relevant in the context of CYRENE. Lawfulness, fairness, and transparency principle. Lawfulness means that the processing of personal data has to be carried out on the basis of one of the six legal grounds enshrined by the GDPR: consent, the performance of a contract, legal obligation, the vital interest of individuals, public interest and the legitimate interest. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 22 D2.1 – Supply Chain Analysis and Requirements collaborate and form a ‘Cooperation Group’ to support and facilitate strategic cooperation and the information exchange. NIS Directive imposes an obligation to the Member States to effectively cooperate on specific cybersecurity incidents and share information about risks via CSIRT Network. Member States should identify the ‘operators of essential services’ within business sectors such as energy, transport, water, banking, financial market infrastructures, healthcare and digital infrastructure. In these sectors, operators should take appropriate security measures and notify serious incidents to the relevant national authority. Article 23 of the directive requires a periodical review of the directive by the European Commission. As a result of the review process, the new legislative proposal has been presented in December 2020. Proposal for ‘NIS 2.0’ contains measures for the improvement of cybersecurity infrastructures, and particularly the resilience and incident response capacities of public and private entities, and competent authorities. The new proposal expands the scope of the directive application and includes medium and large companies. NIS Directive The Directive 2016/1148 on the security of network and information system (hereinafter NIS Directive)5 is the first EU legislative initiative exclusively dedicated to cybersecurity. NIS Directive was adopted and entered into force in 2016, whereas the Member States had to transpose this directive into national laws until November 2018. For the effective implementation of the directive, the Member States had at their disposal the "NIS toolkit". This toolkit provides practical information about the best practices, explanations, interpretation of specific provisions, and how they should work in practice. The general goal of the NIS Directive is to provide legal measures to strengthen the overall level of cybersecurity in the EU. For that purpose, Member States should appropriately equip Computer Security Incident Response Team (CSIRT) and a competent national NIS authority. They should 952690 CYRENE PROJECT PARTNERS 31/05/2021 23 D2.1 – Supply Chain Analysis and Requirements particularly important for the purpose of protecting the availability, authenticity, integrity, or confidentiality of stored, transmitted, or processed data and accompanied functions and services. particularly important for the purpose of protecting the availability, authenticity, integrity, or confidentiality of stored, transmitted, or processed data and accompanied functions and services. The EU Member States should establish national cybersecurity certification authorities. These bodies should monitor and enforce the obligations of manufacturers or providers of ICT products, processes or services. In addition, national cybersecurity certification authorities should handle complaints lodged by natural or legal persons in relation to European cybersecurity certificates. Finally, the European Cybersecurity Certification Group (ECCG) should be established. This body should ensure the consistent application of the European cybersecurity certification framework and monitor the consistent implementation of the cybersecurity program. Also, ECCG should advise and assist the European Commission in matters related to certification frameworks and cybersecurity programs. The Cybersecurity Act complements the GDPR by providing details related to implementation of appropriate security measures. It also works in conjunction with the NIS Directive to protect critical national infrastructure. However, while the NIS Directive applies only to specific operators, the Cybersecurity Act encourages all businesses to invest in cybersecurity. 7 Hijmans, H., & Raab, C. (2018). Ethical Dimensions of the GDPR. In Commentary on the General Data Protection Regulation. Edward Elgar. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3222677 Regulation (EU) 2019/881 g ( ) Regulation (EU) 2019/8816 has replaced Regulation (EU) No 526/2013 (Cybersecurity Act) and it represents the regulatory framework for ENISA (the European Union Agency for Cybersecurity) on information and communications technology cyber-security certification. This Regulation foresees a permanent mandate for the European Union Agency for Network and Information Security (ENISA) and the creation of an EU certification framework for ICT products, processes, and services. It is an undeniable fact that cyberattacks are becoming more sophisticated and they are usually featured by cross-border effects. Therefore, there is an increasing need for coordinated and effective responses as well as appropriate crisis management at the EU level. The goal of Regulation is to empower cybersecurity structures at the EU level by establishing the set of measures that the Member States should develop as well as by strengthening the cooperation among them. The Cybersecurity Act has two major fields focal:  The first one refers to empowering ENISA by making it a permanent agency of the EU  The second one is about establishing a European cybersecurity certification framework to ensure the application of a common certification for information and communications tech- nology (“ICT”) products, processes and services.  The second one is about establishing a European cybersecurity certification framework to ensure the application of a common certification for information and communications tech- nology (“ICT”) products, processes and services. ENISA should assume the key role by assisting the EU Member States (and relevant institutions) to build and enhance capabilities and preparedness to prevent, detect and respond to cyber threats and incidents, in relation to the security of network and information systems. Moreover, ENISA should support the development and enhancement of the Member States and the EU computer security incident response teams (CSIRTs). Finally, ENISA should promote the exchange of best practices between all Member States. The Regulation sets out the cybersecurity certification framework. The goal is to enhance the level of cybersecurity in the Union by providing a mechanism to attest the ICT products, processes and services. This attest should comply with advanced determined security requirements. This is 952690 CYRENE PROJECT PARTNERS 6 https://eur-lex.europa.eu/eli/reg/2019/881/oj 24 31/05/2021 952690 CYRENE PROJECT PARTNERS 31/05/2021 24 D2.1 – Supply Chain Analysis and Requirements Accountability implies that a responsible agent attempts to respect other principles and demonstrates its compliance, even when this is not explicitly required by law (Hijmans & Raab, 2018, 107). Therefore, accountability imposes both - obligation to behave in a certain way; and preparedness to explain the behavior to relevant stakeholders (e.g. public bodies in a form of regulatory instances as well as individuals). Processing of data must be limited to the specified purposes and appropriate security measures should be applied. In cases where measures such as anonymization or pseudonymization are not applied, an explanation is needed. This is necessary (inter alia) to make an informed decision about personal data processing. However, there are situations that individuals cannot decide on processing their personal data due to reasons that override their freedoms and rights. The principle of fairness requires an assessment of how processing will affect individuals. If processing negatively affects an individual, then processing will be unfair. However, even unfair processing could be legitimate (e.g. personal data may be collected by tax authorities about an individual who has not paid taxes). One of the most important ethical issues concerning data processing and protection is transparency. Transparency is not only about providing access to information. It also refers to ways about how information is provided. Delivering info about data processing before processing starts affects individuals’ choice concerning the protection of their personal privacy. Therefore, one of the foundations regarding transparency refers to providing information about data processing in a timely manner. A timely manner is a quite general construction, and its specification is context-based. Transparency means that information about processing activities should be clear, concise, easy to understand and provided in an accessible manner. These requirements are general, and their practical form will depend on a particular context. In practice, that means when data is transferred from one to another country such transfers must comply with the laws of the country in which the data was collected and also facts about the transfer are properly communicated. Also, detailed information on the informed consent procedures related to the processing of personal data must be provided (e.g. in a language and terms understandable by the participants). 2.2.2.4 Ethical issues Law prescribes what must, can or cannot be done. However, ethics goes beyond what is stipulated by law. Law is based on certain ethical values and it supports fundamental rights and freedoms (Hijmans & Raab, 2018, 17). Moreover, ‘laws come and go; the ethics stays’[12]. There is an increasing trend of ethical discussions on data, including personal data. It comes as no surprise due to rapid technological development. New technologies could improve security measures that protect data. However, aspirations towards high security might also misuse information and subsequently jeopardize personal privacy. For instance, the application of a surveillance system in public places might be at cost of our personal privacy. Thus, it would not be wrong for two opposite conclusions to be both correct. Therefore, it is quite important to assess the nature of data intended to be processed as well as the consequences of data processing. Whoever processes data should ensure that relevant legal framework(s) are respected. This is particularly important when sensitive data is processed. Data processing generates accountability of the entities that processes the data. The essence of the accountability principle lies in the obligation to be compliant with all other data protection principles and to be able to demonstrate it. These requirements are part of the legal responsibility, and it is important to underline that accountability goes beyond legal responsibility. Legal responsibility concerns who is supposed to do what, and it may include legal consequences for the performance or non-performance of duties and tasks that are regulated by certain legislation (such as GDPR). 952690 CYRENE PROJECT PARTNERS 25 31/05/2021 7 Hijmans, H., & Raab, C. (2018). Ethical Dimensions of the GDPR. In Commentary on the General Data Protection Regulation. Edward Elgar. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3222677 952690 CYRENE PROJECT PARTNERS 31/05/2021 25 D2.1 – Supply Chain Analysis and Requirements Moreover, if data processing involves profiling, the data subjects shall be informed of the existence of the profiling, its possible consequences, and how the fundamental rights of the research participants will be safeguarded. Entities that process data (and particularly personal data) should designate a Data Protection Officer (DPO) – a person with expert knowledge of data protection law and practices should assist the controller or processor to monitor internal compliance with relevant regulation. DPO may evaluate the ethics risks related to data processing activities. This should include an opinion on whether or not a data protection impact assessment should be conducted. The opinion, the risk evaluation and, if applicable, the data protection impact assessment should be conducted. Therefore, accountability includes a risk-based approach. In other words, whoever processes data should assess risks related to the processing. In that way, ethical judgements are made. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 26 D2.1 – Supply Chain Analysis and Requirements The standard focuses on actively managing and reducing risks. Critical security aspects in the supply chain can include financial aspects, manufacturing, information management and logistics, storage in transit and warehousing of goods. This International Standard is based on the ISO format adopted by ISO 14001:2004 because of its risk-based approach to management systems. However, organizations that have adopted a process approach to management systems (e.g. ISO 9001:2000) may be able to use their existing management system as a foundation for a security management system as prescribed in this International Standard. It is not the intention of this International Standard to duplicate governmental requirements and standards regarding SC security management to which the organization has already been certified or verified to comply with. Verification may be by an acceptable first, second, or third-party organization. This International Standard is based on the methodology known as Plan-Do-Check-Act (PDCA), described as follows. This International Standard is based on the methodology known as Plan-Do-Check-Act (PDCA), described as follows.  Plan: establish the objectives and processes necessary to deliver results in accordance with the organization’s security policy.  Do: implement the processes.  Do: implement the processes.  Check: monitor and measure processes against security policy, objectives, targets, legal and other requirements, and report results.  Act: take actions to continually improve the performance of the security management system.  Act: take actions to continually improve the performance of the security management system. Moreover, this International Standard specifies the requirements for a security management system, including those aspects critical to security assurance of the SC. Security management is linked to many other aspects of business management. Aspects include all activities controlled or influenced by organizations that have an impact on SC security. These other aspects should be considered directly, where and when they have an impact on security management, including transporting these goods along with the SC. Finally, this International Standard is applicable to all sizes of organizations, from small to multinational, in manufacturing, service, storage or transportation at any stage of the production or SC that wishes to: Finally, this International Standard is applicable to all sizes of organizations, from small to multinational, in manufacturing, service, storage or transportation at any stage of the production or SC that wishes to: 1. establish, implement, maintain and improve a security management system; 2. 2.2.3 SC security standards (families of ISO2800x) ISO 280008 was prepared by Technical Committee ISO/TC 8, Ships and marine technology, in collaboration with other relevant technical committees responsible for specific nodes of the Supply Chain. This International Standard has been developed in response to demand from the industry for a security management standard. Its ultimate objective is to improve the security of SCs. It is a high- level management standard that enables an organization to establish an overall SC security management system. It requires the organization to assess the security environment in which it operates and to determine if adequate security measures are in place and if other regulatory requirements already exist with which the organization complies. If security needs are identified by this process, the organization should implement mechanisms and processes to meet these needs. Since SCs are dynamic in nature, some organizations managing multiple SCs may look to their service providers to meet related governmental or ISO SC security standards as a condition of being included in that SC in order to simplify security management, as illustrated in Figure 4. Figure 4 - ISO28000 overview. Figure 4 - ISO28000 overview. This International Standard is intended to apply in cases where an organization’s SCs are required to be managed in a secure manner. A formal approach to security management can contribute directly to the business capability and credibility of the organization. Compliance with an International Standard does not in itself confer immunity from legal obligations. For organizations that so wish, compliance of the security management system with this International Standard may be verified by an external or internal auditing process. 8 https://www.iso.org/standard/44641.html 31/05/2021 952690 CYRENE PROJECT PARTNERS 31/05/2021 27 9 https://www.iso.org/standard/73906.html 10 NIST “Case studies in Cyber Supply Chain Risk Management” (2020). Online available: https://nvlpubs.nist.gov/nistpubs/CSWP/NIST.CSWP.02042020-1.pdf , accessed on 24-2-2021 2.2.4 Risk Management standards (families of ISO2700x) ISO270009 is a set of International Standards that regulate Information Security Management Systems (ISMS). They are used to manage information security risks and controls within an organization. The central principle of the ISO27000 set of standards is to bring information security under overt management control. These standards help organizations to develop and implement a framework for managing the security of information assets as well as to prepare themselves to assess their ISMS. ISO27000 contains vocabulary and requirements for ISMS, Code of practice for information security management, ISMS implementation guidance, measurement for ISMS, information security risk management, and guidelines for ISMS auditing. ISO27000 family of standards defines requirements for an ISMS, provide direct support, and detailed guidance for the Plan-Do-Check-Act (PDCA) processes and requirements. Sector- specific guidelines are also addressed by ISMS family. D2.1 – Supply Chain Analysis and Requirements 2.2.4 Risk Management standards (families of ISO2700x) 9 https://www.iso.org/standard/73906.html assure conformance with stated security management policy; 3. demonstrate such conformance to others; 4. seek certification/registration of its security management system by an Accredited third party Certification Body; 5. make a self-determination and self-declaration of conformance with this International Standard. There are legislative and regulatory codes that address some of the requirements in this International Standard. There are legislative and regulatory codes that address some of the requirements in this nternational Standard. It is not the intention of this International Standard to require duplicative demonstration of conformance. It is not the intention of this International Standard to require duplicative demonstration of conformance. Organizations that choose third-party certification can further demonstrate that they are contributing significantly to SC security. Organizations that choose third-party certification can further demonstrate that they are contributing significantly to SC security. 952690 CYRENE PROJECT PARTNERS 31/05/2021 28 D2.1 – Supply Chain Analysis and Requirements Additional guidelines have been provided by ENISA on hot SC topics, such as IoT security to illustrate indications and good practices according to existing standards and research11. ENISA has also provided a report on guidelines for maritime cybersecurity12 in an attempt to help port operators adopt good practices for cyber risk assessment, regardless of the risk assessment methodology they have chosen to use. Considering the multi-level specificities of the modern ICT SCs (i.e. sectoral, cross-sectoral) SCRM strategies [15] should be used to minimize the existence of vulnerabilities and the potential of loss within a Supply (SC) promoting business continuity and security maintenance. Thus, for managing cybersecurity risks that originate from supply chains, organizations need to understand their supply chains, including multiple layers of sub-suppliers13. According to SCS stakeholder interviews10, SCS operators collaboration can facilitate organizations to avoid some of the common pitfalls, early in the maturity journey10[22]. NIST SP 800-16114 provides guidance to federal agencies on identifying, assessing, and mitigating ICT supply chain risks introducing a multi-tiered, SCRM-specific approach. In principle, choosing an effective method and a proper tool for risk analysis and risk evaluation proves to be complicated. In recent years, a number of concepts, algorithms and tools have evolved from research, specially designed to protect the IT infrastructure and related systems, mostly utilizing quantitative risk assessment methods based on monetary costs [23]. There are remarkable examples of semi-quantitative risk assessment approaches, such as the Fault Tree Events Analysis [24], the OBEST object-based event scenario tree that combines features of event tree analysis and Monte-Carlo discrete event simulation, which is able to perform a deep analysis on the uncertainty subjects [25]. The difficulty to obtain precise and reliable figures for a quantitative risk assessment is the main reason why the German Federal Office of Information Security (BSI) recommends qualitative risk assessment (e.g. based on risk scoring matrices, etc.) instead of quantitative risk assessment, although the need for the latter has clearly been recognized [26]. In Boiko et al. [27] a qualitative research method is presented for analysing the Supply Chain process and identifying the most effective strategies for information support in Supply Chain environments to eliminate cyber risks. 2-2021 12 Cyber Risk Management for Ports - Guidelines for cybersecurity in the maritime sector. ENISA, 2021, [Online]. Available: https://www.enisa.europa.eu/publications/guidelines-cyber-risk-management-for-ports. Accessed: March 15, 2021 13 Boyens, J., Paulsen, C., Bartol, N., Winkler, K., & Gimbi, J. (2021). Key Practices in Cyber Supply Chain Risk Management: Observations from Industry (No. NIST Internal or Interagency Report (NISTIR) 8276). National Institute of Standards and Technology, https://doi.org/10.6028/NIST.IR.8276 14 NIST SP 800-161 (2015). “Supply Chain Risk Management Practices for Federal Information Systems and Organizations“. Online available: http://dx.doi.org/10.6028/NIST.SP.800-161, accessed on 24-2-2021 11 ENISA(2020). “Guidelines for Securing the Internet of Things”. Online available: https://www.enisa.europa.eu/publications/guidelines-for-securing-the-internet-of-things , accessed on 24- 2-2021 2.2.5 SC risk assessment methodologies & Tools Supply Chain risk management (SCRM) can be considered the cross-section of Supply Chain management and risk management [13] aiming to minimize the level of uncertainty on the supply chain performance and provide adequate solutions to undertake [14]. SCRM can be identified as the process of applying strategies to capture, estimate, prioritize and mitigate risks [15]. The literature reviews a variety of SCRM definitions focusing on different aspects [16][17][18][9]. Supply Chain Service performance can be jeopardized by common threats (i.e., traditional cyber attack, cyber piracy, espionage, sabotage, etc.) and new rising threats (i.e., APTs, ransomware, botnet) capable of being supported by multiple sophisticated threat agents. In addition, the advent of emerging technologies (i.e., digital twins, IoT, Swarm Intelligence-based techniques, Big Data, adversarial learning techniques, etc.) has posed novel threats to the SC ecosystem. Taking into account the analysis of the latest cyber threat landscape reports coming from dominant EU cybersecurity standardization bodies [19], it can be deduced that SC sophisticated cyberattacks have become a new emerging alarming scenario. Digital Supply Chains consist of interconnected dispersed nodes, changing dynamically and impeding the chance to adjust to the tremendously evolving threat landscape10. Therefore, a combination of both proactive/preventive approaches to size the robustness and reactive strategies to improve the agility can be utilized to address this challenge [20][21]. 31/05/2021 31/05/2021 29 952690 CYRENE PROJECT PARTNERS 13 Boyens, J., Paulsen, C., Bartol, N., Winkler, K., & Gimbi, J. (2021). Key Practices in Cyber Supply Chain Risk Management: Observations from Industry (No. NIST Internal or Interagency Report (NISTIR) 8276). National Institute of Standards and Technology, https://doi.org/10.6028/NIST.IR.8276 14 NIST SP 800-161 (2015). “Supply Chain Risk Management Practices for Federal Information Systems and Organizations“. Online available: http://dx.doi.org/10.6028/NIST.SP.800-161, accessed on 24-2-2021 D2.1 – Supply Chain Analysis and Requirements Additional complications arise from the need to map the existing infrastructure of an organization onto a fixed terminology with which the chosen risk assessment method works. Additional complications arise from the need to map the existing infrastructure of an organization onto a fixed terminology with which the chosen risk assessment method works. There is a variety of past, well-known, outstanding risk management methods and risk assessment tools, such as the ISO 27001-, 27005- and 31000- compliant “EBIOS” method used by ANSSI, the “OCTAVE” method [28], a priori distribution of subjectively estimated probabilities utilizing the Bayesian approach, the Magerit open methodology for risk management and the Mehari risk analysis method [29]. Most of them adopt the commonly known rule “risk = probability x potential damage” [30]. The “BowTie”15 is a traditional widely used qualitative risk analysis method. The “CORAS” well-known method [31] allows the incorporation of several different risk assessment processes the recognition of the probability of an attack is done a priori to any risk assessment and not automatically. CRAMM [32] is a risk analysis method for all types of information systems and networks, identifying security requirements, detecting contingency requirements and proposing possible solutions. The STORM-RM [33] is a collaborative and multi- criteria risk management ISO27001-based methodology promoting organization users to participate in the various risk assessment and treatment phases combining risk computation with the AHP algorithm. The Collaborative Cyber/Physical Security Management System (CYSM) approach [34] explores the significant criticality of ports’ infrastructure and the complexity of the interrelationships among the internal and external systems and infrastructures. Oliveira et al.[35] provides an extended research on SCRM profile works of the last fifteen years to discover key-players in the SCRM research field and conclude that there is a lack of consensus among authors undertaking a description of SCRM steps. In addition, there is limited work on developing stable SCRM solutions of well-defined process descriptions from a managerial perspective. On this account, there is a compelling need for continuous improvement in the SCRM cyber plane in terms of investigating solutions capable of holistically and dynamically assessing an organization’s exposure to Cyber Supply Chain threats and taking into account cross- functional risks [15]. Nevertheless, the qualitative risk assessment does have the appeal of efficiency and is easy to communicate and explain to stakeholders; however, there are no commonly accepted ratings for safety and security that would apply as a standard. 13 Boyens, J., Paulsen, C., Bartol, N., Winkler, K., & Gimbi, J. (2021). Key Practices in Cyber Supply Chain Risk Management: Observations from Industry (No. NIST Internal or Interagency Report (NISTIR) 8276). National Institute of Standards and Technology, https://doi.org/10.6028/NIST.IR.8276 14 NIST SP 800-161 (2015). “Supply Chain Risk Management Practices for Federal Information Systems and Organizations“. Online available: http://dx.doi.org/10.6028/NIST.SP.800-161, accessed on 24-2-2021 952690 CYRENE PROJECT PARTNERS 30 15 https://www.researchgate.net/publication/298916210_The_bowtie_method_A_review D2.1 – Supply Chain Analysis and Requirements sharp assurance for the risk can be provided [37]. Thus, game theory has gained ground in the field of cryptography and the security of IT infrastructures in the past years. sharp assurance for the risk can be provided [37]. Thus, game theory has gained ground in the field of cryptography and the security of IT infrastructures in the past years. There are various strategies in the literature which give a guaranteed payoff under any behaviour of the adversary, regarding scalar and multi-dimensional payoffs [38][39]. Cox (2005) [40] provides a clear connection between game theory and risk analysis claiming that they are deeply complementary and mutually reinforcing. He considers that risk analysis can support game theory by providing probabilities of different consequences for pairs of attacker-defender strategies. Conversely, game-theoretic methods and concepts can improve current risk analyses of adversarial actions. Rajbhandari and Snekkenes in their research work [41] explain how game theory can interact with the classical risk management. Specifically, they provide a full description on how the steps of the classical management model ISO/IEC 27005 can be mapped and utilized in a game-theoretical model. Responses to SC risks are in a variety of cases supported by payoffs and risk mitigation strategies as already presented indicatively. Ceryno et al in their research work [42] state that modern risk models require validation upon real cases and a deeper risk analysis and risk treatment exploration. They continue that there is a lack of research relying on empirical data obtained from face-to-face interviews of SC stakeholders and a unanimous SCRM definition is not yet provided. Methodologies for dependency modelling, simulation and analysis have been recently categorized in the following broad categories [43]: (i) empirical approaches, (ii) agent-based approaches, (iii) system dynamics-based approaches, (iv) economic theory based approaches, (v) network based approaches, and (vi) others. Empirical methods focus on the impact assessment and/or the risk related with the dependencies between Cis [44] and their potential cascading effects. A limitation of empirical approaches is the difficulty in capturing statistical data related to the likelihood of the examined threats. Network-based approaches [45][46] focus on the flow of products or services exchanged between CIs and try to model this flow based on graphs. Other methods can be categorized as hybrid, including characteristics of both empirical and network-based methods [47]. 16 Bodeau D.,J., McCollum, C., D., Fox, D.,B. (2018) "Cyber Threat Modeling: Survey, Assessment, and Representative Framework", The Homeland Security Systems Engineering and Development Institute (HSSEDI) & MITRE Cooperation. Available online: https://www.mitre.org/sites/default/files/publications/pr_18-1174-ngci-cyber-threat-modeling.pdf (Accessed on 19 March 2021) 17 Nagaraju, Vidhyashree, Lance Fiondella, and Thierry Wandji (2017). “A survey of fault and attack tree modeling and analysis for cyber risk management”. In: 2017 IEEE International Symposium on Technologies for Homeland Security (HST), pp. 1–6.DOI: 10.1109/THS.2017.7943455. URL: http://ieeexplore.ieee.org/document/7943455/ A set of key practices that any organization of any size, scope, and complexity can use to manage cybersecurity risks associated with their supply chains is provided in Cyber Supply Chain Risk Management (C-SCRM) approach10. Ensuring cyber-resilience seems to be a critical capability that needs to be improved in order to successfully identify, assess, and mitigate cyber supply chain risks. This can be achieved through creating explicit collaborative roles, structures, and processes for supply chain, cybersecurity, product security, and physical security functions. Organizational accountability is a principle that is highlighted in this report and could be guaranteed through a C-SCRM program. For this reason, it is critical to know by whom the data and infrastructure are accessible. Security requirements should be communicated not only to suppliers, but also to suppliers’ sub-suppliers. The C-SCRM approach also proposes establishing protocols for vulnerability disclosure and incident notification for better managing cyber supply chain risks. Game theory is the study of mathematical models of conflict and cooperation between intelligent rational decision-makers [36] building strategies between two or more players which aim to maximize their payoff and minimize their loss. The application of game theory (i.e. the Minimax approach) in risk management elegantly aims to presume the attacker’s behavior to be optimal in the most unfavorable way for its victim. The appeal of game-theory lies in the fact that, no matter how the adversary actually behaves, it provides the worst-case scenario and, in this manner, a 952690 CYRENE PROJECT PARTNERS 31/05/2021 31 A key role in the analysis of such various methods, mainly empirical, network-based and hybrid ones has the graph theory. Attack Trees or Attack Graphs approaches are well-established, promoting threat scenarios during the risk assessment process16. In recent years, attack models are a primary tool in risk assessment of SC heterogeneous and interconnected systems (i.e. SCADA, AIS systems). They are conceptual diagrams providing possible conceivable attacks, where the root of the tree illustrates a potential exploit and the leaves provide the various actions to achieve that goal aiming to point out optimal attack paths (kill chains)17. Shin et al. in their respective research work [48] 17 Nagaraju, Vidhyashree, Lance Fiondella, and Thierry Wandji (2017). “A survey of fault and attack tree modeling and analysis for cyber risk management”. In: 2017 IEEE International Symposium on Technologies for Homeland Security (HST), pp. 1–6.DOI: 10.1109/THS.2017.7943455. URL: http://ieeexplore.ieee.org/document/7943455/ 952690 CYRENE PROJECT PARTNERS 32 31/05/2021 D2.1 – Supply Chain Analysis and Requirements 18 https://ec.europa.eu/digital-single-market/en/eu-cybersecurity-act D2.1 – Supply Chain Analysis and Requirements utilize the theory of random graphs to model interdependent networks and provide a theoretical analysis of the cascading effects. Most research work of cascading failures in SC networks refers only to single network models because it is difficult to represent real-world multiple agent network systems of SCs [49]. A remarkable research work on these grounds is the Medusa approach [50][51] proposing multi- order dependencies between stakeholders involved in the maritime supply chain, where cyber threats are estimated in terms of scrutinizing the cascading scenarios modelled by dependency graphs. The efforts described above can be a starting point to leverage research on the impact of risks in supply chains. Notwithstanding, there is still a strong need to investigate methods that focus on SC risk impact analysis and how to facilitate SC stakeholders better comprehend cascading effects of potential attacks in a vertical manner. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 33 3. EU Certification The general definition and requirements (policy, legal, standards, methodologies, technical) regarding the security certification will be reviewed in this section. 3.1 Security Certification - EU requirements 3.1 Security Certification - EU requirements 952690 CYRENE PROJECT PARTNERS 34 31/05/2021 18 https://ec.europa.eu/digital-single-market/en/eu-cybersecurity-act 3.1.2 Security Certification standards addressing EU requirements EU Cybersecurity certification requires formal evaluation of products, services and processes against a defined set of criteria in order to provide assurance that the products comply with specified requirements and standards. Each standard specifies one or more level(s) of assurance (basic, substantial or high), based on the level of risk associated with the intended use of the product, service or process. ISO/IEC 1540819 and ISO/IEC 1804520 are a pair of international standards for IT systems security evaluation and certification. ISO 1706521 International Standard focuses on requirements for the competence, consistent operation and impartiality of the certification bodies evaluating and certifying products, processes and services. CYRENE focuses on creating solid links and significantly influencing a number of initiatives in the areas of cybersecurity, data protection and software standardization. Specifically, CYRENE combines ISO standards in order to build a Conformity Assessment Process for ensuring the security and resilience of Supply Chain services. D2.1 – Supply Chain Analysis and Requirements In the context of the Cybersecurity Act, Supply Chains are recognized as “global” and the introduction of certification schemes should lead to reducing market fragmentation. In the context of the Cybersecurity Act, Supply Chains are recognized as “global” and the introduction of certification schemes should lead to reducing market fragmentation. Furthermore, the Cybersecurity act gives tasks and provides resources to ENISA in order to assist EU member states in dealing with cyber-attacks. This will be accomplished by enhancing the EU Member States with an efficient information sharing through the network of Computer Security Incident Response Teams (CSIRTs) and raising cybersecurity awareness through exercises and trainings. 3.1.1 Cybersecurity Act - The European certification legal instrument The EU Cybersecurity act18 aims to coordinate a number of certification schemes (e.g., ISO 15408/18045, NCSC) in order to boost the digital single market, scale up the response to cyber- attacks, improve cyber resilience and increase the trust for consumers of ICT products, services and processes within the union. The Cybersecurity Act provides a framework for certification schemes based on the following standards: ISO/IEC 15408, ISO/IEC 18045 and ISO/IEC 17065. These complementary standards constitute the basis for all cybersecurity evaluation schemes in the cybersecurity act. According to the cybersecurity act, ENISA, the European Union Cybersecurity Agency, is assigned a central role to establish, support and implement the EU cybersecurity certification framework. The framework indicates the following requirements for each certification scheme:  A certification scheme is designed to achieve a number of security objectives including confidentiality, integrity, availability, accountability, non-repudiation of stored, transmitted and processed data. Design goals of the scheme also include post-incident recovery, business continuity and identifying known vulnerabilities and dependencies.  A certification scheme may specify one or more assurance levels including basic, substantial and high. These assurance levels shall be associated with the risk level related to the intended use of the ICT product, ICT service or ICT process, in terms of the probability and impact of an incident.  A certification scheme may allow for conformity self-assessment to those ICT related products, services and processes that present a low risk corresponding to assurance level ‘basic’.  A certification scheme includes a number of other elements such as: scope (products, services and processes), references to standards (international, European and national standards), evaluation criteria, conditions for marks or labels, rules concerning vulnerability disclosure, validity period, conditions for mutual recognition with third countries.  A certification scheme provides supplementary cybersecurity information. It includes the guidance and recommendations to assist end-users with the secure configuration, availability of cybersecurity-related updates, and a reference to online repositories listing publicly disclosed vulnerabilities that are related to ICT products and services. 31/05/2021 31/05/2021 34 D2.1 – Supply Chain Analysis and Requirements components, which are defined into ISO/IEC 15408-2 and ISO/IEC 15408-3. Finally, it describes the key concepts of protection profiles (PP), it gives guidelines for the specification of Security Targets (ST) and the organization of the components throughout the model. components, which are defined into ISO/IEC 15408-2 and ISO/IEC 15408-3. Finally, it describes the key concepts of protection profiles (PP), it gives guidelines for the specification of Security Targets (ST) and the organization of the components throughout the model. The security functional requirements, which are described in ISO/IEC 15408-1, establish a comprehensive catalogue of security functional components that serve as standard templates. These templates are described analytically in ISO/IEC 15408-2 [53] and are a standard way of expressing the functional requirements for ToEs (Targets of Evaluation). The templates are organized using a hierarchical structure of classes, families and components. ISO/IEC 15408-2, also, provides guidance on specifying custom security requirements, where no suitable predefined security functional templates are in place. Last, the ISO/IEC 15408-3 [54] describes a set of assurance components, which serve as standard templates for expressing and covering ToEs assurance requirements. The assurance components are organized into a set of components, families and classes. Also, ISO/IEC 15408- 3 presents the Evaluation Assurance Levels (EALs), which define a scale for rating ToEs assurance levels. 3.1.2.2 ISO/IEC 18045 ISO/IEC 18045 [55] is a companion standard to the evaluation criteria for IT security defined in ISO/IEC 15408. This standard defines the minimum actions to be performed by an evaluator in order to conduct an ISO/IEC 15408 evaluation, using the corresponding criteria and the evaluation evidence. The target audience for this International Standard is primarily evaluators applying ISO/IEC 15408 and certifiers confirming evaluator actions, i.e. sponsors and/or developers. It is considered as a “companion” document, which is helpful mainly for security professionals, which are involved in evaluating compliance with ISO/IEC 15408. Finally, this standard does not define evaluator actions for certain high assurance ISO/IEC 15408 components, where there is no generally agreed guidance yet. 3.1.2.1 ISO/IEC 15408 ISO/IEC 15408 is a guide for the development, evaluation and/or procurement of IT products with security functionality. It establishes the general concepts of IT product security evaluation, specifying the requirements for the security functions of IT systems and the assurance measures applied to them during a security evaluation. The standard consists in three parts. The first part, i.e., ISO/IEC 15408-1 [52], provides an overview of all parts of ISO/IEC 15408 standard. In more details, it defines the general concepts and the principles of IT security evaluation. It establishes the core concept of the Target of Evaluation (ToE) scheme and refers to the evaluation criteria. It focuses on the basic security concepts necessary for IT products evaluation. Also, it defines the permitted functional operations for the functional and assurance 952690 CYRENE PROJECT PARTNERS 35 31/05/2021 19 https://www.enisa.europa.eu/topics/threat-risk-management/risk-management/current-risk/laws- regulation/rm-ra-standards/iso-iec-standard-15408 20 https://www.enisa.europa.eu/topics/threat-risk-management/risk-management/current-risk/laws- regulation/rm-ra-standards/iso-iec-18045 21 https://www.iso.org/standard/46568.html 952690 CYRENE PROJECT PARTNERS 35 31/05/2021 19 https://www.enisa.europa.eu/topics/threat-risk-management/risk-management/current-risk/laws- regulation/rm-ra-standards/iso-iec-standard-15408 20 https://www.enisa.europa.eu/topics/threat-risk-management/risk-management/current-risk/laws- regulation/rm-ra-standards/iso-iec-18045 21 https://www.iso.org/standard/46568.html 31/05/2021 35 3.1.3 The European Cyber Security Certification Schema (EUCC) 3.1.3 The European Cyber Security Certification Schema (EUCC) Securing network and information systems in the European Union has been deemed as a key objective in an effort to keep the EU online economy functional and secure. ENISA22 contributes to EU cyber policy, enhances the trustworthiness of ICT products, services and processes with cybersecurity certification schemes, cooperates with Member States and EU bodies, and helps Europe prepare for tomorrow’s cyber challenges. The EUCC scheme (Common Criteria based European candidate cybersecurity certification scheme) looks into the certification of ICT products cybersecurity, based on the Common Criteria, the Common Methodology for Information Technology Security Evaluation, and corresponding standards, respectively, ISO/IEC 15408 and ISO/IEC 18045. This scheme will improve the European Union Internal Market conditions for ICT products, and as a result also have positive effects for ICT services and ICT processes relying on such products. The purpose of the EU cybersecurity certification (EUCC) framework is to establish and maintain trust and security in cybersecurity products, services and processes. Drawing up cybersecurity certification schemes at EU level aims at providing criteria to carry out Conformity Assessments to establish the degree of adherence of products, services and processes against specific requirements. Users and service providers alike, need to be able to determine the level of security assurance of the products, services and processes they procure, make available or use. The EU cybersecurity certification framework lays down the procedure for the creation of EU cybersecurity certification schemes, covering ICT products, services and processes. Each scheme will specify one or more level(s) of assurance (basic, substantial or high), on the basis of the level of risk associated with the envisioned use of the product, service or process. It serves the purpose of providing notice and assurance to users about the level of conformity against stated requirements. EU cybersecurity certification schemes serve as the vehicle to convey such requirements from the EU policy level to the industry service provision level and further to the users and conformity assessment bodies. Cybersecurity certification requires the formal evaluation of products, services and processes by an independent and accredited body against a defined set of criteria, standards, and the issuing of a certificate indicating conformance; as such cybersecurity certification plays a key role in increasing trust and security in products, services and processes. 952690 CYRENE PROJECT PARTNERS 37 31/05/2021 22 ENISA EUCC Cybersecurity Certification: https://www.enisa.europa.eu/publications/cybersecurity- certification-eucc-candidate-scheme D2.1 – Supply Chain Analysis and Requirements 3.1.2.3 ISO/IEC 17065 The overall goal of certifying products, processes or services is to give confidence to all interested parties that a product, process or service fulfills specified requirements. ISO17065 standard [56] contains requirements for the competence, consistent operation and impartiality of product, process and service certification bodies. Certification bodies operating under this International Standard do not have to offer all types of products, processes and services certification. In this International Standard, the term “product” can be read also as a “process” or “service”. This International Standard specifies requirements that should be met to ensure that certification bodies operate certification schemes in a competent, consistent, and impartial manner. This International Standard can be used as a criteria document for accreditation or peer assessment or designation by governmental authorities, scheme owners and others. The requirements should be considered as general criteria for certification bodies operating product, process, or service certification schemes. Of course, they may have to be adjusted and/or empowered when specific industrial or other sectors make use of them. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 36 D2.1 – Supply Chain Analysis and Requirements functionalities. Where any such evaluation activities are not appropriate, substitute evaluation activities with equivalent effect shall be undertaken. A European cybersecurity certificate that refers to assurance level ‘high’ shall provide assurance that the ICT products, ICT services and ICT processes for which that certificate is issued meet the corresponding security requirements, including security functionalities, and that they have been evaluated at a level intended to minimise the risk of state-of- the-art cyberattacks carried out by actors with significant skills and resources. The evaluation activities to be undertaken shall include at least the following: a review to demonstrate the absence of publicly known vulnerabilities; testing to demonstrate that the ICT products, ICT services or ICT processes correctly implement the necessary security functionalities at the state of the art; and an assessment of their resistance to skilled attackers, using penetration testing. Where any such evaluation activities are not appropriate, substitute activities with equivalent effect shall be undertaken. A European cybersecurity certification scheme may specify several evaluation levels depending on the rigor and depth of the evaluation methodology used. Each of the evaluation levels shall correspond to one of the assurance levels and shall be defined by an appropriate combination of assurance components. Certification under this scheme shall cover the assurance levels ‘substantial’ and ‘high’ of the CSA. A user of certified products or an applicant to certification shall decide against which security objectives he/she decides to evaluate the ICT product(s) and select the applicable requirements, either in a Protection Profile or a Security Target of the individual ICT product. ENISA may provide associated guidance for this selection, based on risk assessment methods or tools. 3.1.3 The European Cyber Security Certification Schema (EUCC) As per Article 52.6 A European cybersecurity certificate that refers to assurance level ‘substantial’ shall provide assurance that the ICT products, ICT services and ICT processes for which that certificate is issued meet the corresponding security requirements, including security functionalities, and that they have been evaluated at a level intended to minimize the known cybersecurity risks, and the risk of incidents and cyberattacks carried out by actors with limited skills and resources. The evaluation activities to be undertaken shall include at least the following: a review to demonstrate the absence of publicly known vulnerabilities and testing to demonstrate that the ICT products, ICT services or ICT processes correctly implement the necessary security 31/05/2021 37 D2.1 – Supply Chain Analysis and Requirements manufacturing and transportation sector, those use cases from the FISHY project may be used as external Supply Chain pilot scenarios. manufacturing and transportation sector, those use cases from the FISHY project may be used as external Supply Chain pilot scenarios. Most companies developing ICT solutions require the integration or synergy of other ICT components developed by third parties. This can represent a high security risk, making the verification of potential vulnerabilities more difficult. The EU-funded BIECO project24 is working on a holistic framework composed of a set of tools and methodologies that will address the challenges related to vulnerability and risk management, resilience and auditing of complex systems on the level of the ICT supply chain. On the other side, CYRENE will improve the trust of the consumer through the development of an innovative certification schema and a clear and structured Conformity Assessment across all levels of a Supply Chain provision. Cloud computing is an essential element of innovative economies. Despite trust-building efforts, the adoption of cloud computing is limited. A lack of security and transparency is the reason for the slow uptake. European cloud service providers (CSPs) still face multiple challenges for certifying their services. The EU-funded MEDINA project25 will work to counter this trend. It will propose a framework for achieving a continuous audit-based certification for cloud service providers, complying with the EU Cybersecurity Act. The project will also address the definition and assessment of technical and organizational measures, security testing, machine-readable certification language and audit evidence management. Medina framework will be based on European Cybersecurity Certification Scheme for Cloud Services (EUCS). Currently certification schemes are expressed using natural language so MEDINA proposes to transform this certification language into a machine-readable expression. Results of this effort may be utilized on the schema which will be developed inside the CYRENE approach and which is also based on EUCC as that EUCS schema. Parallel with the demand for autonomous Cyber-Physical Systems (CPSoS), grows the need for advanced certification mechanisms that can improve their security posture without compromising their safety. Existing validation methods require exhaustive offline testing of every possible state scenario prior to fielding the system. The EU-funded ASSURED project26 is introducing an innovative, formally verified runtime assurance framework for securing CPS Supply Chains. 3.1.4 R&D certification projects and EU initiatives The European Commission declared cybersecurity as a top priority of the digital and connected Europe and cybersecurity presents an important part of the Commission’s research and innovation funding framework programmes, Horizon 2020 and its successor Horizon Europe. CYRENE will make a direct contribution to the strengthening of the EU’s cybersecurity capacity through collaboration with the winning pilot projects of the 2018 and 2019 Horizon 2020 cybersecurity call. In this section, we have listed several projects dealing with various aspects of cybersecurity in supply chains. FISHY23 is one of the projects aiming to design and develop a solution for cyber resiliency provisioning supporting security assurance and certification management, trust management, data and privacy management and the proper orchestration of their related functional components. An evidence-based security assurance and certification methodology identifying security claims and metrics will be developed which draws similarity to the CYRENE approach. FISHY will make validation and demonstration of the framework on three use cases from different sectors, including agriculture, manufacturing and transportation. Taking into account that CYRENE will also validate its Conformity Assessment process on pilot use cases dealing with the 23 https://fishy-project.eu/ 952690 CYRENE PROJECT PARTNERS 31/05/2021 38 D2.1 – Supply Chain Analysis and Requirements CONCORDIA27 addresses the current fragmentation of security competence by networking diverse competencies into a leadership role via a synergistic agglomeration of a pan-European Cybersecurity Center. CONCORDIA is trying to build a community of strong cooperation between all stakeholders, understanding that all stakeholders have their KPIs, bridging among them, and fostering the development of IT products and solutions along the whole supply chain. Technologically, it projects a broad and evolvable data-driven and cognitive E2E Security approach for the ever-complex ever-interconnected compositions of emergent data-driven cloud, IoT, and edge-assisted ICT ecosystems. CONCORDIA27 addresses the current fragmentation of security competence by networking diverse competencies into a leadership role via a synergistic agglomeration of a pan-European Cybersecurity Center. CONCORDIA is trying to build a community of strong cooperation between all stakeholders, understanding that all stakeholders have their KPIs, bridging among them, and fostering the development of IT products and solutions along the whole supply chain. Technologically, it projects a broad and evolvable data-driven and cognitive E2E Security approach for the ever-complex ever-interconnected compositions of emergent data-driven cloud, IoT, and edge-assisted ICT ecosystems. The SPARTA28 proposal brings together a unique set of actors at the intersection of scientific excellence, technological innovation, and societal sciences in cybersecurity. Strongly guided by concrete and risky challenges, it will set up unique collaboration means, leading the way in building transformative capabilities and forming world-leading expertise centers. Through innovative governance, ambitious demonstration cases, and active community engagement, SPARTA aims at re-thinking the way cybersecurity research is performed in Europe across domains and expertise, from foundations to applications, in academia and industry. CYRENE will collaborate with CONCORDIA and SPARTA for knowledge transfer as well as to strengthen the EU's cybersecurity capacity. CyberSec4Europe29 aims to boost defenses within the vertical sectors of digital infrastructure, finance, government, transport, health, and smart cities. The project utilizes practical experience gained during CyberSec4Europe to develop a specialized roadmap and recommendations for the implementation of network competence. CyberSec4Europe project has identified seven key research and innovation demonstration cases covering a wide spectrum of prominent research areas in both the public and private sectors. Among the demonstration cases are Supply Chain security assurance and maritime transport which are of special interest to CYRENE. The main objective of the project is to develop highly automated middleware for the secure configuration, management of edge devices, processes and safety-critical software components. The ASSURED project will identify and implement a reactive, runtime risk assessment model which will enable the dynamic assessment and forecast of individual, cumulative and propagated risks, taking into account the representation of assets along with their dependencies, the associated threats and vulnerabilities and the potential cascading effects. The ASSURED framework will mainly focus on certification of supply chains including IoT devices while CYRENE will take into consideration certification of individual devices but also, certification of whole infrastructure and assets of the Supply Chain as well certification of business perspectives of Supply Chain. 952690 CYRENE PROJECT PARTNERS 39 31/05/2021 24 https://www.bieco.org/ 25 https://cordis.europa.eu/project/id/952633 26 https://www.project-assured.eu/ 31/05/2021 31/05/2021 39 D2.1 – Supply Chain Analysis and Requirements generic IoT framework for real-time monitoring of the traffic exchanged through heterogeneous IoT platforms within the IoT network. The aim of the SerIot platform is to recognize suspicious patterns, evaluate them and finally to decide on the detection of a security leak, privacy threat and abnormal event detection, while offering parallel mitigation actions that are seamlessly exploited in the background. Large-scale pilots can test SerIoT technology in various use cases including intelligent transport and surveillance, flexible manufacturing within Industrie 4.0 and other emerging domains such as food chain & logistics, m-Health (both at Home & in Hospitals business scenarios) and energy (smart grid). CYRENE will consider security solutions developed in SerIoT, specifically regarding the protection of IoT devices and communications, as these are important sub-components of the CYRENE Conformity Assessment framework. In addition, there exists a set of EU bodies and initiatives related to cybersecurity improvement and certification schemes development, that are of interest. DIGITALEUROPE32 represents a regulatory environment, the leading trade European association, that concerns digitally transforming industries. Its goal is to contribute to both the development and implementation of relevant EU policies, as well as to shape industry policy positions on relevant legislative matters. It implies a wide variety of businesses, corporations and national trade associations and partnerships with European institutions. One of its policy areas is cybersecurity, with a focus on the proposed Cybersecurity Act, with the aim to create a harmonized EU market for cybersecurity certification schemes, and on the implementation of the Directive on the security of network and information systems - the NIS directive33. The NIS directive is the first representative of EU cybersecurity legislation, with the aims to enhance cybersecurity across Europe and create and strengthen a Computer Security Incident Response Team (CSIRT) Network. The NIS directive is a central deliverable within the EU Cyber Security Strategy, that tends to harmonize a framework for evolving of three different aspects of cybersecurity. The Digital Agenda for Europe (DAE)34 defined the key enabling role that the use of ICTs had to play in order to make Europe succeed in its goals. The Digital Market Strategy is built on three pillars: providing better access for consumers and businesses to digital goods and services across Europe, creating the right conditions for digital networks and services to flourish, and maximizing the growth potential of the digital economy. ECHO30 is one of the projects supported by the European Commission with the objective of connecting and sharing knowledge across multiple domains to develop a common cybersecurity strategy for Europe. The project will develop a European Cybersecurity ecosystem, to support secure cooperation and development of the European market, as well as to protect the citizens of the European Union against cyber threats and incidents. One of ECHO’s main objectives is the development of ECHO Security Certification Scheme: Development of sector specific security certification needs within the EU Cybersecurity Certification Framework from ENISA. CYRENE will make key advances comparing to ECHO. Specifically, it will contribute to a comprehensive cybersecurity conformity assessment framework for SCs viewed as a whole, i.e., a complex system with business, infrastructure, and individual devices levels. The secure SerIoT platform is a key step that can be used to implement secure IoT platforms and networks anywhere and everywhere. The SerIoT project31 develops, implements and tests a 952690 CYRENE PROJECT PARTNERS 40 31/05/2021 27 https://www.concordia-h2020.eu/ 28 https://www.sparta.eu/ 29 https://cybersec4europe.eu/ 30 https://echonetwork.eu/ 31 https://seriot-project.eu/ 31/05/2021 31/05/2021 40 The European Cyber Security Organization (ECSO)35 is a fully self-financed non-for-profit organization. ECSO is the privileged partner of the European Commission for the implementation of the Cybersecurity Public-Private Partnership (PPP), and it also unites European Cybersecurity stakeholders. The main aims of ECSO include the coordination of the development of the European Cybersecurity Ecosystem, the support to protecting European Digital Single Market and the contribution to the advancement of European digital sovereignty and strategic autonomy. ECSO has collaborations with different European Agencies and Bodies, such as ENISA. 952690 CYRENE PROJECT PARTNERS 41 31/05/2021 32 https://www.digitaleurope.org/ 33 https://enisa.europa.eu/topics/nis-directive 34 https://www.europarl.europa.eu/factsheets/en/sheet/64/digital-agenda-for-europe 35 https://ecs-org.eu/ 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 31/05/2021 41 D2.1 – Supply Chain Analysis and Requirements CEN-CENELEC-ETSI Cyber Security Coordination Group (CSCG)45 is dedicated to provide strategic advice in the field of IT security, Network and Information Security (NIS) and cybersecurity (CS). It was created in 2011. In 2016, it was converted to CEN-CENELEC Focus Group on Cybersecurity. The aim is to prepare a European roadmap on cybersecurity standardization, while providing active support on global initiatives on cybersecurity standards. CEN-CENELEC-ETSI Cyber Security Coordination Group (CSCG)45 is dedicated to provide strategic advice in the field of IT security, Network and Information Security (NIS) and cybersecurity (CS). It was created in 2011. In 2016, it was converted to CEN-CENELEC Focus Group on Cybersecurity. The aim is to prepare a European roadmap on cybersecurity standardization, while providing active support on global initiatives on cybersecurity standards. The aim is to prepare a European roadmap on cybersecurity standardization, while providing active support on global initiatives on cybersecurity standards. 45 https://www.cencenelec.eu/standards/Sectorsold/DefenceSecurityPrivacy/Security/Pages/Cybersecurity.a spx 46 https://sogis.org/ D2.1 – Supply Chain Analysis and Requirements Regarding Public Private Partnerships (PPPs), the representatives that are relevant as collaborators include: Big Data Value Association (BDVA)36, ARTEMIS industry association37 and ECSEL Joint association38. Regarding Public Private Partnerships (PPPs), the representatives that are relevant as collaborators include: Big Data Value Association (BDVA)36, ARTEMIS industry association37 and ECSEL Joint association38. European Competence Network of Cybersecurity Centers of Excellence39 strives to retain and develop the cybersecurity technological and industrial capacities of EU. Its goals include securing EU Digital Single Market and strengthening and sustaining the cybersecurity competence of Europe. It comprises four EU pilot projects: CONCORDIA, ECHO, SPARTA and CyberSec4Europe, with the aim to prepare the European Cybersecurity Competence Network. The ENISA40 is oriented towards achieving a high common level of cybersecurity across Europe. ENISA contributes to EU cyber policy and cybersecurity certification schemes, and also plays a key role in supporting the collaboration between cybersecurity stakeholders and institutions and agencies. The EU Cybersecurity Act41 establishes an EU-wide cybersecurity certification framework, and also strengthens the role of ENISA, as it grants a permanent mandate to the agency. ENISA has also announced the creation of the Stakeholders Cybersecurity Certification Group (SCCG)42. Its aim is to focus on strategic issues regarding cybersecurity certification, to assist in preparation of the Union rolling work programme and also to create market-driven certification schemes. The regulation on electronic identification and trust services for electronic transactions in the internal market - eIDAS Regulation (Regulation (EU) N°910/2014)43 provides a predictable regulatory environment for enabling secure and seamless electronic interactions. It creates a European internal market for electronic trust services. The GSMA IoT Security Guidelines and Assessment44 represents a European standard organization. It is oriented towards a set of IoT Security Guidelines, supported by an IoT Security Assessment scheme. It promotes best practices for the security of IoT services and a mechanism for security measures evaluation. 952690 CYRENE PROJECT PARTNERS 42 31/05/2021 36 https://www.bdva.eu/ 37 https://artemis-ia.eu/ 38 https://www.ecsel.eu/ 39 https://cybercompetencenetwork.eu/ 40 https://enisa.europa.eu 41 https://ec.europa.eu/digital-single-market/en/news/eu-cybersecurity-act-brings-strong-agency- cybersecurity-and-eu-wide-rules-cybersecurity 42 https://ec.europa.eu/digital-single-market/en/stakeholder-cybersecurity-certification-group 43 https://ec.europa.eu/futurium/en/content/eidas-regulation-regulation-eu-ndeg9102014 44 https://www.gsma.com/iot/iot-security/iot-security-guidelines/ 42 D2.1 – Supply Chain Analysis and Requirements CEN-CENELEC-ETSI Cyber Security Coordination Group (CSCG)45 is dedicated to provide strategic advice in the field of IT security, Network and Information Security (NIS) and cybersecurity (CS). It was created in 2011. In 2016, it was converted to CEN-CENELEC Focus Group on Cybersecurity. D2.1 – Supply Chain Analysis and Requirements Various examples of certificates issued by a National Authority with claims of compliance against Common Criteria may be found online47. One of them relates to mobile ID software solution developed by company Thales48. This software enables citizens to securely log on to public and private eServices and to smoothly prove who they are online while guaranteeing data protection and privacy. The Gemalto Mobile ID software has demonstrated a level of resistance to the most advanced security penetration tests against mobile applications. Security tests on the Gemalto mobile ID software were performed by the internationally renowned testing laboratory “Brightsight” under the supervision of the NSCIB (Netherlands Scheme for Certification in the Area of IT Security), in cooperation with of The Netherlands Ministry of Interior and Kingdom Relations. 3.1.5 Examples of Certificates A certificate contains the most relevant information for the identification of the product and the assurance level obtained. It should include a unique identifier established by the issuer of the certificate, information related to the certified ICT product and its manufacturer or provider, information related to the evaluation and certification of the ICT product. The certificate is only related to the cybersecurity certification requirements of the product at the moment of issuance of the certificate. It is not related to the product itself. It only expresses that the cybersecurity related material and information of the product meets the requirements of this certification related information. There may exist different options to claim that a product, system or services complies with cybersecurity certification requirements. Some examples of certificates can be:  self-declaration without any assessment,  self-declaration based upon a self-assessment or a voluntary third-party assessment,  self-declaration based upon a self-assessment or a voluntary third-party assessment,  accredited certificate based upon a third-party assessment (in-house or extern  certificate issued by a National Authority based upon a third-party assessmen ate issued by a National Authority based upon a third-party assessment (external) Common Criteria certifications enable an objective evaluation to validate that a particular product or system satisfies a defined level of robustness. It not only provides assurance that the process of specifying and implementing a secure solution has been rigorously conducted, but also that the solution has reached the expected level of trust for final use. In order to obtain Common Criteria certification, vendors have to complete several steps. First, they must provide a Security Target description including an overview of the product and its security features as well as an evaluation of potential security threats and a self-assessment. Second, organizations must find an independently-licensed laboratory to evaluate their product and determine if it meets security properties to a satisfactory level. If the product passes the evaluation, certification of the security properties of the product is issued by various Certificate Authorizing Schemes. These certificates are recognized by all the members of the CCRA and groups such as SOG IS46. 45 952690 CYRENE PROJECT PARTNERS 31/05/2021 43 47 https://www.commoncriteriaportal.org/products/ 952690 CYRENE PROJECT PARTNERS 44 31/05/2021 p p g p 48 Gemalto Advanced Whitebox PKI SDK for Android v1.0.1.300, https://www.commoncriteriaportal.org/files/epfiles/Certification%20Report%20NSCIB-CC-230855-CR.pdf 49 https://ec.europa.eu/growth/single-market/goods/building-blocks/conformity-assessment_en D2.1 – Supply Chain Analysis and Requirements  Security by design – the SC provider shall design and pre-configure the delivered product so as that functionalities are based on well-established security practices and are reduced to the strict minimum required for system operations;  Security by design – the SC provider shall design and pre-configure the delivered product so as that functionalities are based on well-established security practices and are reduced to the strict minimum required for system operations;  Least privilege – the SC provider shall design and pre-configure the product according to the least privilege principle, whereby administrative rights are only used when absolutely necessary, sessions are technically separated and all accounts will be manageable  Strong authentication – the SC shall provide and support strong authentication mechanisms for all accounts. If authentication is unsuccessful the product shall not allow any user specific activities to be performed;  Strong authentication – the SC shall provide and support strong authentication mechanisms for all accounts. If authentication is unsuccessful the product shall not allow any user specific activities to be performed;  Asset protection – the SC shall provide an adequate level of protection for critical information assets during storage and transmission; ection – the SC shall provide an adequate level of protection for critical information ing storage and transmission;  Supply chain integrity – the supply chain provider should provide means to ensure that the SC is genuine, cannot be tainted during operation, and its integrity is warranted throughout the SC’s lifecycle. Currently this requirement can be technically fulfilled only partly;  Documentation transparency – the SC provider shall offer comprehensive and understandable documentation about the overall design of the SC, describing its architecture, functionalities and protocols, their realisation in hardware or software components, the interfaces and interactions of components with each other and with internal and external services, in order to be able to implement and use the product in the most secure way possible. 3.2.2 Target of Evaluations (ToE) - Security & Assurance Requirements 3 2 2 1 ToE Description 3.2.2 Target of Evaluations (ToE) - Security & Assurance Requirements 3.2.2.1 ToE Description 3.2.1 Basic Concepts and Requirements The basic concept of the Supply Chain Conformity Assessment process is based on the general frame, within the scope of the conformity definition, which contains several abstract categories based on the EU Commission single market for goods regulation49 . In order for a product to be placed in the market, several conformity assessment steps need to be followed. These steps include a demonstration that all of the legislative requirements are met by the product testing, inspection and certification of the product, and making sure that the procedure for each product is specified in the applicable product legislation. The same steps are also required for SC, not from a product perspective, but that of a collection of processes, products and services. CYRENE will evolve conformity assessment processes by meeting the same steps having a supply chain as a constant instead of an individual product, and based on the steps taken, new requirements will derive from the entities involved. A preliminary step in that procedure is to identify the non-specific requirements, initially described in the first phase of the CYRENE. As proposed, the CYRENE requirements phase is building upon the proposed baseline security requirements that are included in the ENISA report [9][9]. Such security requirements are: (a) Security by design; (b) Least privilege (c) Strong authentication (d) Asset protection (e) Supply chain integrity (f) Documentation transparency; (g) Quality management (h) Service continuity (i) Conformance to law (j) initial planning of operational and technical measures and controls. Following the abstract requirement sections, a more detailed view of the processes should contain the following specifications. 44 Providing one or more use case scenario(s), can be really helpful for the documentation transparency procedure;  Quality management – the SC provider shall be able to provide evidence that a managed security by design approach has been adopted, including documented secure software development, quality management and information security management processes;  Service continuity – the SC provider shall guarantee support throughout the agreed lifetime of the product such that the system can work as agreed and is secure;  Conformance to law – the SC provider shall accept that all contracts (including those with subcontractors) are conforming to the legal requirements in place;  Data usage restriction – the SC provider shall explicitly declare, justify and document, context and purpose wise, all data collection and processing activities that take or may take place, including relevant legal obligations stipulating them.  Data usage restriction – the SC provider shall explicitly declare, justify and document, context and purpose wise, all data collection and processing activities that take or may take place, including relevant legal obligations stipulating them. CYRENE will take into consideration the aforementioned general requirements and extend them to provide more specific requirements for conformity assessments which will be carried out in deliverable D2.2 “A Cybersecurity Certification proposed Scheme for Supply Chain Services”. CYRENE will take into consideration the aforementioned general requirements and extend them to provide more specific requirements for conformity assessments which will be carried out in deliverable D2.2 “A Cybersecurity Certification proposed Scheme for Supply Chain Services”. D2.1 – Supply Chain Analysis and Requirements Conformity assessment of the ToE is defined as the procedure followed to evaluate whether spec- ified requirements relating to the ToE have been fulfilled50. Thus, according to ISO/IEC 15408-151 and ETSI TS 102 165-1 v5.2.3 (2017-10)52, it is required to clearly define what the ToE is and specify its security aspects. ToE shall be the ICT product (equipment, device, asset, process or service) as a whole or the elements of the ICT product. As far as ISO/IEC 15408 is concerned, the precise relation between the ToE and any IT products is important in only one aspect: the evaluation of a ToE containing only a part of an IT product should not be misrepresented as an evaluation of the entire IT prod- uct. For the purposes of this project, ToE is defined as an interconnected set of resources and pro- cesses that begins with the sourcing of raw material and extends through the delivery of products or services to the end-user across the modes of transport. Thus, in the case of CYRENE, a ToE is a Supply Chain Service (SCS) that may involve many SCS business partners (vendors, manu- facturing facilities, logistics providers, internal distribution centers, distributors, wholesalers, au- thorities and other entities) that contribute to reach the end user8 (provision of the SCS). 3.2.2.1 ToE Description In this section we provide the basic concepts and interrelation of the terms which are necessary for certification processes. The specification of these terms in the CYRENE will be provided in D.2.2. As shown in the Glossary (Appendix A – Glossary and Examples ), according to the ISO15408 a ToE is a set of software, firmware, hardware and/or process which is subject to a security evalu- ation in which it is assessed against security requirements (conformity assessment). 952690 CYRENE PROJECT PARTNERS 31/05/2021 45 3.2.2.2 Security environment The security environment includes all the security aspects of the environment in which the asset is intended to be used. For the purposes of this project, the security environment contains any aspects related to the security of the processes, assets, techniques, and technologies associated with the ToE, i.e. the supply chain service53. The key elements of the security environment are the following51:  Security assumptions: the intended use of the implementation of the ToE;  Assets: the assets that are to be protected, i.e., the ToE itself; the components that com- prise the ToE and all the assets with which the ToE under analysis will interact with;  Threats and threat agents: a set of threats (threat scenarios) that are relevant to operation of the ToE; the threat agents that will be used to enact the identified thre  Organizational security policies: a security policy is expressed by a set of controls that address all the security functional requirements to be implemented by a ToE. In our case the above issues will be specified in D.2.2 for the CYRENE case where the ToE is a SCS. 50 https://eur-lex.europa.eu/eli/reg/2019/881/oj 51 https://www.iso.org/standard/50341.html 52 https://www.etsi.org/deliver/etsi_ts/102100_102199/10216501/05.02.03_60/ts_10216501v050203p.pdf 53 ENISA report (2015) “Supply Chain Integrity: An overview of the ICT supply chain risks and challenges, and vision for the way forward”, v.1.1, August 2015. Online available: https://www.enisa.europa.eu/publications/sci-2015 50 https://eur-lex.europa.eu/eli/reg/2019/881/oj 51 https://www.iso.org/standard/50341.html 52 https://www.etsi.org/deliver/etsi_ts/102100_102199/10216501/05.02.03_60/ts_10216501v050203p.pdf 53 ENISA report (2015) “Supply Chain Integrity: An overview of the ICT supply chain risks and challenges, and vision for the way forward”, v.1.1, August 2015. Online available: https://www.enisa.europa.eu/publications/sci-2015 952690 CYRENE PROJECT PARTNERS 46 31/05/2021 52 https://www.etsi.org/deliver/etsi_ts/102100_102199/10216501/05.02.03_60/ts_10216501v050203p.pdf 53 ENISA report (2015) “Supply Chain Integrity: An overview of the ICT supply chain risks and challenges, and vision for the way forward”, v.1.1, August 2015. Online available: https://www.enisa.europa.eu/publications/sci-2015 53 ENISA report (2015) “Supply Chain Integrity: An overview of the ICT supply chain risks and challenges, and vision for the way forward”, v.1.1, August 2015. Online available: https://www.enisa.europa.eu/publications/sci-2015 46 31/05/2021 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 3.2.2.4 Security requirements The distinction between security objectives and security requirements is of great importance. An objective is the expression of what a security system should be able to do in very broad terms while a requirement is a more detailed specification of how an objective is achieved. More than one requirement could be fulfilled in order to meet one objective. In ETSI TVRA methodology, indicative examples are presented in order to better apprehend this distinction between these two similar terms. The security requirements consist of two categories of requirements: a) the security functional requirements (SFRs) a) the security functional requirements (SFRs) b) the security assurance requirements (SARs) b) the security assurance requirements (SARs) 3.2.2.3 Security objectives A set of security objectives need to be satisfied by the ToE in response to the defined security problem. The security objectives that are to be fulfilled by a ToE should be clearly defined and evaluated in the conformity assessment process. Different definitions for the security objectives have been presented in the bibliography. Based on ISO 28000:2007, a security objective is defined as a specific outcome or achievement required for security in order to comply with the security management policy. It is essential that such out- comes are linked either directly or indirectly to the provision of products, or services delivered by the overall business to its customers or end-users. The ETSI TVRA methodology defines the security objectives of both the asset and its environment. These objectives are expected to cover the assumptions, identified threats and policies that need to be addressed as described in 3.2.2.2 Security environment. An indicative example of security objectives to be set for the SCS-ToE, as defined in this project, could be to ensure the integrity of all SCS processes or the authenticity of the SCS data ex- changed between the SCS-business partners. Security Functional Requirements (SFR) Security functional requirements are a set of requirements specified in the basic security standard and an indication of where in the standard the detailed requirement can be found. In CC, SFRs are defined as the translation of the security objectives for the ToE into a standardized language. The implementation of functional requirements addresses threats of counterfeited or tainted prod- ucts and components. 3.2.3 Methodologies for CAs In order to apply the Threat Vulnerability Risk Analysis (TVRA), a specific method has been developed, which consists of a systematic re-evaluation of undesirable events that need to be prevented in a given system. In order to succeed in doing this evaluation, one needs to identify the assets that compose the system and the associated weaknesses, as well as the threats and the threat agents that might attack the system. After that, the procedure to follow includes a modelling of the likelihood and the impact of the potential attacks on the system’s vulnerabilities so as to determine the risk to which the system is exposed. A system includes assets such as physical, human and logical ones. Those identified assets may have weaknesses that can potentially be attacked by threat agents who enact a threat. This results into breaking some of the initially defined security objectives. When the TVRA is applied, several countermeasures are put in place, whose goal is to protect the assets against threats related to vulnerabilities, by reducing the risk. The TVRA method repeatedly identifies the assets of a given system and the relationships between them. For each one of the assets, a weakness might be established and an assessment of how practical an attack is to be mounted as well as its potential resulting risk. The method process is explained in 10 steps, as follows: 1. Identification of the Target of Evaluation (ToE) resulting in a high-level description of the main assets of the ToE and the ToE environment and a specification of the goal, purpose and scope of the TVRA At first, it is essential to clearly define the scope, purpose and goal of the analysis. The Target of Evaluation (ToE) and its environment must be described, representing a “system under standardization”. This description might as well include information of the system architecture, its relevant applications, information flows and possible attack surfaces. 2. Identification of the objectives resulting in a high-level statement of the security aims and issues to be resolved What matters at this stage is to identify the security objectives in terms of user protection to be given. If those objectives are unknown, it is highly difficult to come up with security requirements and the TVRA would not be fruitful. D2.1 – Supply Chain Analysis and Requirements could allow to testing if attackers are able to violate the SFRs. In particular, the Vulnerability as- sessment class addresses the possibility of exploitable vulnerabilities introduced in the develop- ment or the operation of the ToE. The assessment of development vulnerabilities is covered by the AVA_VAN assurance family. could allow to testing if attackers are able to violate the SFRs. In particular, the Vulnerability as- sessment class addresses the possibility of exploitable vulnerabilities introduced in the develop- ment or the operation of the ToE. The assessment of development vulnerabilities is covered by the AVA_VAN assurance family. y Cases where the security problem description mentions threats where the threat agent is very capable, and a low (or no) Vulnerability analysis (AVA_VAN) are included in the SARs. Security Assurance Requirements (SAR) Based on CC, SARs provide a description of how assurance is to be gained that the ToE meets the SFRs [ISO/IEC 15408-1:2009 (CC)]. Evaluation Assurance Level (EAL) is a scale for meas- uring assurance for component ToE. In ETSI TVRA methodology, asset security assurance re- quirements provide an indication of the EAL that an implementation of the base security standard could be expected to meet. p From the Evaluation service level summary as specified in ISO/IEC 15408, Vulnerability Analysis is the assurance class that will be used in this project. This assessment deals with threats and 952690 CYRENE PROJECT PARTNERS 31/05/2021 47 3.2.3 Methodologies for CAs For most of the telecommunication services, there is a series of technical security issues in which these objectives can be categorized into:  Fraud charging  Privacy protection  Service availability guarantee 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 48 D2.1 – Supply Chain Analysis and Requirements 3. Identification of the functional security requirements, derived from the objectives from step 2 Based on the system objectives identified in step 1, this step aims at identifying the functional security requirements that can be either security or assurance requirements. For this step it might be wise to use the ISO/IEC 15408-2 [i 28] requirements 3. Identification of the functional security requirements, derived from the objectives from step 2 Based on the system objectives identified in step 1, this step aims at identifying the functional security requirements that can be either security or assurance requirements. For this step, it might be wise to use the ISO/IEC 15408-2 [i.28] requirements. Based on the system objectives identified in step 1, this step aims at identifying the functional security requirements that can be either security or assurance requirements. y q y q s step, it might be wise to use the ISO/IEC 15408-2 [i.28] requirements. 4. Systematic Inventory of the assets as refinements of the high-level asset descriptions from step 1 and additional assets as a result of steps 2 and 3 For step 4, the assets are classified and it is vital to identify the nature of the assets that the system includes as well as the complexity of the technology comprised in their construction. The assets are identified in three categories:  physical assets, such as equipment  human assets and  logical assets (information stored in and handled by the physic  logical assets (information stored in and handled by the physical assets) We can consider that an asset is at risk when a weakness related to it exists and a viable threat is present. The gravity of the vulnerability will depend on the value that is attributed to the asset and the probability of the weakness to be abused by a threat. The evaluation must be repeated until no assets can be identified. The relations of the asset have to be identified in what concerns its affiliation to the system and to other assets (parent-child- sibling relationships that might exist). What really is important is to calculate the impact on the system after a successful attack. The asset impact can be of different level of severity (low, medium, high) according to the harm the attack has on the system, as seen in Table 1. Impact Explanation Value Low The concerned party is not harmed very strongly; the possible damage is low. 1 Medium The threat addresses the interests of providers/subscribers and cannot be neglected. 2 High A basis of business is threatened and severe damage might occur in this context. 3 Table 1 - Impact on assets after a successful attack. D2.1 – Supply Chain Analysis and Requirements A successful attack needs a well-elaborated attack method. A successful attack method needs to be practical and in order to evaluate its practicality, the following factors shall be analyzed in order to determine the weight of the attack potential that is needed to take advantage of a vulnerability: • System knowledge. • Time. • Time. • Expertise. • Opportunity. • Equipment. Threat agents need to be identified as well. Threat agents consist of entities that act maliciously on the system’s assets. The extent to which a threat agent is motivated and capable of mounting a successful attack, differs according to the agent. The threat agent’s capability combined with their motivation, gives us the level of the threat. We define capability of the threat agent, the level of technical sophistication of the threat, and motivation the extent to which the agent wants to attack and compromise a given asset or a group of them. Table 2 shows the mapping of both: Motivation Capability Very Little Little Limited Significant Formidable Very low (indifferent) Negligible Negligible Low Low Low Low (curious) Negligible Negligible Low Low Moderate Medium (interested) Negligible Low Moderate Severe Severe High (committed) Low Low Moderate Severe Critical Very high (focused) Low Moderate Severe Critical Critical Table 2 - Mapping of motivation and capability of threat agents. Table 2 - Mapping of motivation and capability of threat agents. 6. Quantifying the occurrence likelihood and impact of the threats The attack factors mentioned in step 5 shall be summed (i.e., Time + Expertise + Knowledge + Opportunity + Equipment) to obtain an overall attack potential rating. The attack potential value is then mapped to a vulnerability rating. The vulnerability rating is thereafter combined with the threat level to obtain the occurrence probability/likelihood. 7. Establishment of the risks As said above, for every asset of the system, the vulnerabilities are identified after having determined their weaknesses and related threats. The probability of each vulnerability is calculated as described in step 6. One shall also calculate the risk associated to each vulnerability and for this, the impact of intensity, and classification of risk, have to be considered. 8. Identification of countermeasures framework (conceptual) resulting in a list of alternative security services and capabilities needed to reduce the risk 8. 3. Identification of the functional security requirements, derived from the objectives from step 2 Based on the system objectives identified in step 1, this step aims at identifying the functional security requirements that can be either security or assurance requirements. For this step it might be wise to use the ISO/IEC 15408-2 [i 28] requirements Impact Explanation Value Low The concerned party is not harmed very strongly; the possible damage is low. 1 Medium The threat addresses the interests of providers/subscribers and cannot be neglected. 2 High A basis of business is threatened and severe damage might occur in this context. 3 Table 1 - Impact on assets after a successful attack. 5. Identification and classification of the vulnerabilities in the system, the threats that can exploit them, and the unwanted incidents that may result The identification process begins with the determination of the weaknesses and the process of locating them, what threats could take advantage of the weaknesses and what is the potential harm due to these attacks. A vulnerability within the system occurs only when a threat can be associated to a weakness. A Weakness is a potential point of attack. Nevertheless, all weaknesses do not provide the possibility of viable attacks. The weaknesses on which a realistic attack can be done, are considered to be vulnerabilities. Vulnerabilities are the potential attack surfaces that need to be identified and examined Vulnerabilities are the potential attack surfaces that need to be identified and examined. Vulnerabilities are the potential attack surfaces that need to be identified and exa 952690 CYRENE PROJECT PARTNERS 31/05/2021 49 952690 CYRENE PROJECT PARTNERS ication of detailed requirements for the security services and capabilities 10. Specification of detailed requirements for the security services and capabilities from step 9 During this final step, security requirements are to be identified both for the assets and their environment, where applicable. Functional security requirements identified in step 3 and the security services and capabilities of the countermeasures and security requirements identified in step 8 and analyzed in step 9, are used and refined at this stage. Guidelines for the specification of detailed requirements are given in ETSI TR 187 011 [i.2]. Identification of countermeasures framework (conceptual) resulting in a list of alternative security services and capabilities needed to reduce the risk 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 50 Given the fact that there might exist various alternative countermeasures, they have to be at first identified and then compared to each other in order to choose the most beneficial in terms of cost, and overall impact. Every new countermeasure and its associated physical asset, bring new vulnerabilities to the system and for this the TVRA shall be applied including the countermeasures in the Given the fact that there might exist various alternative countermeasures, they have to be at first identified and then compared to each other in order to choose the most beneficial in terms of cost, and overall impact. 9. Countermeasure cost-benefit analysis (including security requirements cost- benefit analysis depending on the scope and purpose of the TVRA) to identify the best fit security services and capabilities amongst alternatives from step 8 As stated above, there might be a number of possible countermeasures and for this, an analysis must be conducted in order to choose the most beneficial of them. The chosen countermeasures need to mitigate the attack measures that result in added security and attack protection. Other than that, countermeasures are evaluated according to the standards design, their implementation, the operation, their regulatory impact, and the related market acceptance. 10. Specification of detailed requirements for the security services and capabilities from step 9 During this final step, security requirements are to be identified both for the assets and their environment, where applicable. Functional security requirements identified in step 3 and the security services and capabilities of the countermeasures and security requirements identified in step 8 and analyzed in step 9, are used and refined at this stage. Guidelines for the specification of detailed requirements are given in ETSI TR 187 011 [i.2]. D2.1 – Supply Chain Analysis and Requirements Security Countermeasures are basically new assets added to the system aiming to reduce the calculated risk the system might go through. In a few words, countermeasures are put in place in order to reduce the probability of an attack and/or its impact. Security countermeasures are mainly logical assets but can also be human or physical. Given the fact that there might exist various alternative countermeasures, they have to be at first identified and then compared to each other in order to choose the most beneficial in terms of cost, and overall impact. Every new countermeasure and its associated physical asset, bring new vulnerabilities to the system and for this, the TVRA shall be applied including the countermeasures in the ToE. Security Countermeasures are basically new assets added to the system aiming to reduce the calculated risk the system might go through. In a few words, countermeasures are put in place in order to reduce the probability of an attack and/or its impact. Security countermeasures are mainly logical assets but can also be human or physical. Given the fact that there might exist various alternative countermeasures, they have to be at first identified and then compared to each other in order to choose the most beneficial in terms of cost, and overall impact. Every new countermeasure and its associated physical asset, bring new vulnerabilities to the system and for this, the TVRA shall be applied including the countermeasures in the ToE. Security Countermeasures are basically new assets added to the system aiming to reduce the calculated risk the system might go through. In a few words, countermeasures are put in place in order to reduce the probability of an attack and/or its impact. Security countermeasures are mainly logical assets but can also be human or physical. Given the fact that there might exist various alternative countermeasures, they have to be Security Countermeasures are basically new assets added to the system aiming to reduce the calculated risk the system might go through. In a few words, countermeasures are put in place in order to reduce the probability of an attack and/or its impact. Security countermeasures are mainly logical assets but can also be human or physical. 54 https://www.iso.org/standard/73029.html D2.1 – Supply Chain Analysis and Requirements the different categories of conformity assessment bodies and their activities, (Table 3 - Conformity standards). ISO/IEC 17021:200656 contains standards and criteria for the competence, consistency and impartiality of audit and certification of all forms of managements systems (e.g., quality managements systems or environmental managements systems), as well as the bodies that provide these services. Certification bodies adhering to this International Standard are not expected to provide certification for all forms of management systems. Management system certification is a third-party conformity testing practice. As a result, third-party conformity testing bodies conduct this task. ISO/IEC 17024:201257 contains principles and requirements for a body certifying persons against specific requirements and includes the development and maintenance of a certification scheme for persons. ISO/IEC 17020:201258 indicates requirements for the competence of bodies performing review and for the impartiality and consistency of their inspection activities. Conformity assessment body Standard or guide Conformity assessment body Standard or guide Testing and Calibration laboratories ISO/IEC 17025:2005 Certification bodies for: - product certification - management system certification - certification of persons Inspection of bodies ISO/IEC Guide 17067:2013 ISO/IEC 17021:2006 ISO/IEC 17024:2012 ISO/IEC 17020:2012 Table 3 - Conformity standards. In the context of CYRENE, standards shown in Table 3 - Conformity standards that concentrate on the quality and management (and not security) will be taken into account in Cyrene proposal for the SCS security certification scheme (that will be based on the ISO15408). the different categories of conformity assessment bodies and their activities, (Table 3 - Conformity standards). ISO/IEC 17021:200656 contains standards and criteria for the competence, consistency and impartiality of audit and certification of all forms of managements systems (e.g., quality managements systems or environmental managements systems), as well as the bodies that provide these services. Certification bodies adhering to this International Standard are not expected to provide certification for all forms of management systems. Management system certification is a third-party conformity testing practice. As a result, third-party conformity testing bodies conduct this task. ISO/IEC 17024:201257 contains principles and requirements for a body certifying persons against specific requirements and includes the development and maintenance of a certification scheme for persons. ISO/IEC 17020:201258 indicates requirements for the competence of bodies performing review and for the impartiality and consistency of their inspection activities. and for the impartiality and consistency of their inspection activities. 3.2.4 Standards for CAs Overtime, ISO, and IEC have developed many standards that may help to set up a proper conformity assessment system. The website of ISO provides an overview of international standards and guides for conformity assessment. There are international and also EU specific standards that the CYRENE conformity assessment procedure will be based upon. ISO/IEC 17000:202054 provides the vocabulary and general principles for international conformity assessment based on a functional approach. Any form of conformity assessment reflects the following functions: selection, determination, review, and attestation. ISO/IEC Guide 17067:201355 recommends good practices for all elements of conformity assessment, including normative documents, bodies, systems, schemes, and results. It is intended for use by individuals and bodies who wish to provide, promote, or use ethical and reliable conformity assessment services. Next, international standards have been developed for 952690 CYRENE PROJECT PARTNERS 51 31/05/2021 54 https://www.iso.org/standard/73029.html 55 https://www.iso.org/standard/55087.html 31/05/2021 31/05/2021 51 D2.1 – Supply Chain Analysis and Requirements 3.2.5 Conformance monitoring This paragraph deals with measures put in place in order that a certified system/product can still be considered as certified over time, after the initial acquisition of the certificate. Up to 2020 (where the European Certification Scheme was proposed by ENISA), there was not an official procedure or set of requirements established by the SOG-IS scheme to that end. Such a mechanism will thus have to be officially developed and established. The work can be based on existing procedures related to the approach of maintenance in the SOG-IS scheme. Until a monitoring mechanism is established, this process can be initiated by the risk owner as a way of compliance monitoring by conducting voluntary assessments. There is although a proposed approach by the SOG-IS MRA participants who suggest the following: There is although a proposed approach by the SOG-IS MRA participants who suggest the following:  the Conformity Assessment Bodies (CABs) to establish a validity period of 5 years for each certificate  the CABs to work on evaluation methods so that the initial certificate can retain some of its validity on future updated versions where it can be proven that the updates were carried out according to a set of pre-defined requirements  Manufacturers/developers to:  Manufacturers/developers to: Conformity assessment body Standard or guide Conformity assessment body Standard or guide Testing and Calibration laboratories ISO/IEC 17025:2005 Certification bodies for: - product certification - management system certification - certification of persons Inspection of bodies ISO/IEC Guide 17067:2013 ISO/IEC 17021:2006 ISO/IEC 17024:2012 ISO/IEC 17020:2012 Table 3 - Conformity standards. Table 3 - Conformity standards. Table 3 - Conformity standards. In the context of CYRENE, standards shown in Table 3 - Conformity standards that concentrate on the quality and management (and not security) will be taken into account in Cyrene proposal for the SCS security certification scheme (that will be based on the ISO15408). 952690 CYRENE PROJECT PARTNERS 52 31/05/2021 56 https://www.iso.org/standard/29343.html 57 https://www.iso.org/standard/52993.html 58 https://www.iso.org/standard/52994.html#:~:text=ISO%2FIEC%2017020%3A2012%20specifies,to%20an y%20stage%20of%20inspection 31/05/2021 52  Manufacturers/developers to: o Carry out impact analyses of all modifications added to the product or the system over its lifecycle o Monitor common vulnerabilities and exposures (CVEs) that may apply to the system and submit an impact analysis when necessary, to the competent CAB o Showcase the actions taken in order to maintain levels of security, also reporting to the competent CAB o Manage customer complaints and keep record of corrective actions  the CABs to periodically review issued certificates based on a security assessment of an ITSEF (Information Technology Security Evaluation Facility) which considers the impact analyses of the accumulated changes since the latest evaluation carried out  the CABs to withdraw certificates in case there is evidence that the commitments/requirements are not respected or in case a security assessment has failed and no corrections can be applied. 952690 CYRENE PROJECT PARTNERS 31/05/2021 53 31/05/2021 D2.1 – Supply Chain Analysis and Requirements 4. Methodology This chapter gives an overview of the methodology followed for the elicitation and analysis of requirements as well as their validation with the project’s Advisory Boards. Figure 5 summarizes the requirements engineering process. Figure 5 summarizes the requirements engineering process. g q g g p Figure 5 - Overview of requirements elicitation and validation process. Figure 5 - Overview of requirements elicitation and validation process. The first column on the left includes the investigation of the Standards (SotA) by means of literature overview and interviews to experts and stakeholders. This process, that leads to the elicitation of the three CYRENE ToEs, is described in paragraph 4.1. The validation of requirements is performed by involving three different Advisory Boards (User, External and Ethical) and then leads to the finalization of Cyrene requirements. In the next two subsections, the methodology used for requirements elicitation and validation is better explained. 4.1 Methodology for requirements elicitation The methodology used for the requirements elicitation is summarized in Figure 6 and described below. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 54 D2.1 – Supply Chain Analysis and Requirements Figure 6 - Overview of the methodology for requirements elicitation . D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements Different iterations of these three steps are usually performed in order to identify other scenarios and adding more details in order to better understand the AS-IS situation and to validate requirements. In particular, in CYRENE, the elicitation of the three ToE requirements (business, technical, sectorial, as will be detailed in chapter 5) has been made involving end users, technical staff, operators and managers from the pilot partners, by means of interviews and one-to-one meetings. The conclusion of the iterative process (presentation and questions as shown above, feedback, dedicated meetings and deep dives) then led to the “High-level Requirements” step. The business goals emerged from the previous phases are analysed here, in order to list the business needs of the final users. Moreover, matching the results of this analysis with the inputs and feedbacks of the discussion points mentioned above, it is possible to extract the high-level requirements, translating the business needs into relevant functionalities of the system. Figure 6 - Overview of the methodology for requirements elicitation . The requirements elicitation process has been performed during the first eight months of the project. It started with the “Industry overview” step. The general situation regarding the outlook of the industry (e.g.: trends in cybersecurity in IoT, etc.) is examined, through an analysis of the company strategic agenda, the industry outlook as hypothesised by consultancy firms or think tanks (e.g.: Gartner, Allied Market Research) and through dedicated interviews with key stakeholders providing their specific expertise (e.g.: the impact of connected cars on cybersecurity). This results in an overview of the existing services in the Industry and a forecast of their evolution and the required systems. After this first result, there are two other steps, where the process is iterated several times, before arriving to a definition of high-level requirements. During these phases, end-users, technical staff, operators and managers are involved. The “Scenarios in the selected industry” steps narrows the analysis to the specific cybersecurity scenarios in Supply Chains, and to the processes managed by the stakeholders involved. Then, in the “Description of scenarios” step, the AS-IS situation is presented, describing the actions performed by the people involved during their daily activities, and which tools they use to face the cybersecurity issues encountered. In particular, as it will be better described in the Chapter CYRENE Conformity Assessment , Business processes, actors, and infrastructures involved in the considered scenarios are described in detail and modelled. The last step of this iterative process is the one that sets the ground for the elicitation of the requirements and is called “Contribution of CYRENE to the scenario”. In fact, this phase is the one when the objectives of the project are presented to the stakeholders, trying to match the user needs with CYRENE layers that could satisfy these needs. 952690 CYRENE PROJECT PARTNERS 31/05/2021 55 D2.1 – Supply Chain Analysis and Requirements 4.3 CYRENE Questionnaire for enriching the requirements with feedback collected by external stakeholders 4.3 CYRENE Questionnaire for enriching the requirements with feedback collected by external stakeholders CYRENE organised on 31st of March 2021 the 1st CYRENE Workshop. Its preparation was taken care by the Privanova (PN) Team. During the workshop, the partners presented an overview of the project, the security aspects of supply chains in general and also grounded their presentations with the Supply Chains (SC) as Targets of Evaluation (ToEs). Except for the discussion and collection of feedback by the Advisory Board members, the consortium shared the CYRENE Questionnaire as well, in order to be filled within a defined time frame. The CYRENE Questionnaire was a campaign which was held between the 31st of March 2021 and finalised on the 10th of May 2021. Some high level insights of the questionnaire on the infographic dimension of the 23 total collected results include that 69,5% of the participants are private companies, while 4,4% are non-profit organisations and 26,1% are public authorities. Among the private companies, 4,3% are coming from the automobile sector, while the public and non-profit sectors are representing port authorities. The majority of the companies are representing large IT companies, supply chain providers, software houses, system integrators and technology providers, maritime ICT domain, healthcare service providers and consultancy services. The rest of the participants represent research organisations and universities. Among the participants, 30% are coming from small companies, 21% from medium ones and 49% from large ones. Last but not least, the professional background of the participants is dealing with the OT and IT systems and devices surveillance services, project management, blockchain and B2B platforms, chief technology operation, ICT security policy making, systems maintenance and security automation operations, GDPR and ISO27001 compliance, cybersecurity services and research activities on cybersecurity certification, testing, evaluation and mitigation actions. 4.2 Methodology for requirements validation After the previous process, that led to listing the ToEs’ requirements, the requirements were validated through the three Advisory Boards: User Advisory Boards, Ethics Advisory Boards and External Advisory Boards. The validation followed the organization of a workshop at the end of month 6 of the project, where the members of the Advisory Boards were invited. Details and results related to the workshop material, organization and results are better explained in Appendix B – Validation of CYRENE ToEs. Project material was shared with the Advisory Boards some days before the event, including a first version of the current deliverable, project presentations and a questionnaire. In particular, the questionnaire (see more details in the Appendix B – Validation of CYRENE ToEs), addresses mainly the following aspects:  “How ICT threats impact on your daily work?”  “How do you face the problems you encounter?”  “Which tools/measures are you using to identify, monitor and mitigate risks The workshop was structured in the following way: The workshop was structured in the following way: 1. Presentation of CYRENE: slides were presented to the participants in order to introduce them to project main objectives. 2. The discussion started then from these points in order to have feedbacks from the Advi- sory Boards on the requirements and their validation. At the end of the workshop, all participants were asked to give their answers to the questionnaire. At the end of the workshop, all participants were asked to give their answers to the questionnaire. Moreover, the questionnaire was also submitted to wider audience of stakeholders contacted by the project partners, in order to have additional feedback. Moreover, the questionnaire was also submitted to wider audience of stakeholders contacted by the project partners, in order to have additional feedback. 56 31/05/2021 952690 CYRENE PROJECT PARTNERS 56 4.3.1 Feedback from the questionnaire In this paragraph, the most significant results of the questionnaire are presented. In fact, some relevant questions asked to the stakeholders are shown with the related infographic description including tables and graphs and then the results are summarized. Figure 7 depicts the statistics of the question “In case you perform any tasks related to ICT Systems Security, please specify the cybersecurity activities and their frequency that are applicable to your organisation.” 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 57 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 58 31/05/2 952690 CYRENE PROJECT PARTNERS 31/05/2021 58 D2.1 – Supply Chain Analysis and Requirements Figure 7 - Cybersecurity activities frequency. D2.1 – Supply Chain Analysis and Requirements Figure 7 - Cybersecurity activities frequency. Figure 7 - Cybersecurity activities frequency. Table 4 summarizes the statistics of the question “Please identify the set of Supply Chain (SC) Services that are critical for your organisation in terms of business importance and scale them accordingly ” Table 4 summarizes the statistics of the question “Please identify the set of Supply Chain (SC) Services that are critical for your organisation in terms of business importance and scale them accordingly.” Supply chain service Total score (max score is 115) Solutions provider 63 Components/Peripherals provider 61 Third party services 60 Transportation 57 Public Authority 56 Local / Global Agent's services 54 Outbound Logistics 52 Inbound Logistics 49 Warehouse 44 Retailer 34 59 D2.1 – Supply Chain Analysis and Requirements Table 4 - Business importance of SCS. Table 5 summarizes the statistics of the question “What Security Standards and proven guidelines has your organization adopted?” Standards Number of respondents ISO / IEC 27001 11 ISO 9001 8 I do not know / It does not concern 7 ISO 28001 4 NIST Framework for the Improvement of Critical Cybersecurity Infrastructures 3 ISO / IEC 27002 3 ISO / IEC 27035 3 ISO / IEC 15408 1 ISO / IEC 27005 1 ISO / IEC 18045 1 NIST SP800-30 1 NIST SP800-61 1 Table 5 - Security standards and guidelines adopted. rizes the statistics of the question “What Security Standards and proven our organization adopted?” Table 5 - Security standards and guidelines adopted. Figure 8 summarizes the results of the question “Are you aware of the EU Cybersecurity Certification Framework for ICT products and services?” Figure 8 - Awareness of EU Cybersecurity Certification Framework for ICT products and services. 8 - Awareness of EU Cybersecurity Certification Framework for ICT products and services. 952690 CYRENE PROJECT PARTNERS 60 D2.1 – Supply Chain Analysis and Requirements Figure 9 summarizes the result of the question “How does your organization address the following cybersecurity issues in the Supply Chain Services you are involved in?” Figure 9 summarizes the result of the question “How does your organization address the following cybersecurity issues in the Supply Chain Services you are involved in?” Figure 9 - Measures to address cybersecurity issues. Figure 9 - Measures to address cybersecurity issues. Figure 10 summarizes the results of the question “Does your organization provide an effective cybersecurity management plan for the Supply Chain Services you are involved in?” 952690 CYRENE PROJECT PARTNERS 31/05/2021 61 upply Chain Analysis and Requirements Figure 10 - Effective cybersecurity management plan applied to SCS. D2.1 – Supply Chain Analysis and Requirements Figure 10 - Effective cybersecurity management plan applied to SCS. Figure 10 - Effective cybersecurity management plan applied to SCS. Figure 11 summarizes the results of the question “Are you aware of the content of the security standards and best practices that your organization has adopted and implemented?” Figure 11 - Awareness of security standards and best practices adopted. Figure 11 - Awareness of security standards and best practices adopted. Figure 12 summarizes the result of the question “Choose the Security Procedures and Policies that apply to your organization” Figure 12 summarizes the result of the question “Choose the Security Procedures and Policies that apply to your organization” Figure 12 - Security procedure - Number of respondents. 952690 CYRENE PROJECT PARTNERS 62 D2.1 – Supply Chain Analysis and Requirements Figure 13 summarizes the results of the question “Does your organization comply with the legal and regulatory principles and EU directives regarding the security of Supply Chain Services and the protection of Personal Data?” D2.1 – Supply Chain Analysis and Requirements Figure 13 summarizes the results of the question “Does your organization comply with the legal and regulatory principles and EU directives regarding the security of Supply Chain Services and the protection of Personal Data?” Figure 13 - Compliance with legal and regulatory principles and EU directives. Figure 13 - Compliance with legal and regulatory principles and EU directives. Figure 14 summarizes the results of the question “Have you experienced any cybersecurity issue in the last 3 years?” Figure 14 - Experience of cybersecurity issues in last years. Figure 14 - Experience of cybersecurity issues in last years. Figure 15 summarizes the results of the question “Regarding potential security incidents to the services, information systems, and infrastructure used by your organization, please evaluate what is the probability to experience a threat in the future.” 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 63 D2.1 – Supply Chain Analysis and Requirements Figure 15 - Proba Figure 15 - Probability of threats in the future. Figure 15 - Probability of threats in the future. 952690 CYRENE PROJECT PARTNERS 64 31/05/2021 64 D2.1 – Supply Chain Analysis and Requirements Figure 16 summarizes the results of the question “To whom do you report for issues related to risks and security threats and how often?” Figure 16 summarizes the results of the question “To whom do you report for issues related to risks and security threats and how often?” s s a d secu ty t eats a d o o te Figure 16 - Addressee of risks and security threats. Figure 16 - Addressee of risks and security threats. Figure 17 summarizes the results of the question “What are the most important tools (e.g. human or technical resources, processes, etc.) required for secure service delivery?” (maximum score is 115). 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 65 D2.1 – Supply Chain Analysis and Requirements Figure 17 - Relevant tools for secure service delivery. Figure 18 summarizes the results of the question “Which of the following ICT Systems security measures apply to your day-to-day operations?” easu es app y to you day to day ope at o s pp y y y y p 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 66 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 67 D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 68 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 90 CYRENE PROJECT PARTNERS 69 31/05/2021 Figure 18 - ICT Systems security measures applied to daily operations. Figure 18 - ICT Systems security measures applied to daily operations. Figure 18 - ICT Systems security measures applied to daily operations. 952690 CYRENE PROJECT PARTNERS 69 31/05/2021 31/05/2021 69 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements - About half of the people interviewed have knowledge about EU Cybersecuri - About half of the people interviewed have knowledge about EU Cybersecurity Certification Framework for ICT products and services. - About half of the people interviewed have knowledge about EU Cybersecurity Certification Framework for ICT products and services. - Most of the people declares they usually utilize best practices to solve the listed cybersecurity issues they can encounter in the SC, from Business continuity issues to Attacks in cyberspace. - Most of the people declares they usually utilize best practices to solve the listed cybersecurity issues they can encounter in the SC, from Business continuity issues to Attacks in cyberspace. y p - 50% of people think their companies provide an effective cybersecurity management plan for the Supply Chain Services they are involved in. - 50% of people think their companies provide an effective cybersecurity management plan for the Supply Chain Services they are involved in. - The majority in fact knows company best practices to be used in these cases and the most commonly used Security Procedures and Policies. Most utilized Security Procedures and Policies in organizations are Malware Detection, Backup and Disaster Recovery, Information security and Access Control, as from Figure 12 - Security procedure - Number of respondents. - From Figure 13 - Compliance with legal and regulatory principles and EU directivesFigure 13, 70% of people answer that his/her company follows the EU directives regarding the security of Supply Chain Services and the protection of Personal Data, while most of the rest are not aware. - In general, people think that the most probable threats are the following: uncontrolled access to the systems, intrusion of software, unintentional malfunction and technical system failure. - In general, people think that the most probable threats are the following: uncontrolled access to the systems, intrusion of software, unintentional malfunction and technical system failure. y - In case of risks, most of them reports to the information security officer. - The tools that are considered as the most important for secure service delivery are, from an organizational and individual point of view, the awareness, the trainings and the collaborative means, mitigation skills, risk assessment and experienced human resources and, as technical tool, the intrusion detection mechanisms, as from Figure 17. D2.1 – Supply Chain Analysis and Requirements Figure 19 summarizes the results of the question “Do you perform periodic audits to inspect user activity on the Organization's network?” Figure 19 - Perform of periodic audits. Figure 19 - Perform of periodic audits. Figure 20 summarizes the results of the question “Has an external certified analyst been assigned the analysis and evaluation of threats and vulnerabilities, as well as the execution of penetration tests (vulnerability scanning / penetration testing) in the infrastructure of your Organization? If so, how often do you carry out such checks and when was the last time?” Figure 20 - Analysis and evaluation of infrastructure security performed by external certified analyst. Figure 20 - Analysis and evaluation of infrastructure security performed by external certified analyst. 4.3.1.1 Conclusions from the questionnaire The answers to the questionnaire give us interesting information regarding the security in Supply Chain and in general in the organizations involved. The main results that we can extract and that have been presented as graphs are summarized as follows: - The frequency for cybersecurity activities is very heterogeneous and very different from one participant to another. - The frequency for cybersecurity activities is very heterogeneous and very different from one participant to another. - All listed SCSs are considered critical, in terms of business importance, in particular the solutions providers, the components/Peripherals providers and the third-party services, as from Table 4. - All listed SCSs are considered critical, in terms of business importance, in particular the solutions providers, the components/Peripherals providers and the third-party services, as from Table 4. - Most of the participant states that their companies have adopted ISO / IEC 27001 and ISO 9001 hil t f th d ’t h t t l k l d if th t d d d t d - Most of the participant states that their companies have adopted ISO / IEC 27001 and ISO 9001, while most of them doesn’t have total knowledge if other standards are adopted. - Most of the participant states that their companies have adopted ISO / IEC 27001 and ISO 9001, while most of them doesn’t have total knowledge if other standards are adopted. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 70 D2.1 – Supply Chain Analysis and Requirements - ICT Systems security measures listed are applied to day-to-day operations from the majority and in one case from all of the participants to the questionnaire. - ICT Systems security measures listed are applied to day-to-day operations from the majority and in one case from all of the participants to the questionnaire. j y p p q - Only a slight majority performs periodic audits to inspect user activity on the organization's network. - Only a slight majority performs periodic audits to inspect user activity on the organization's network. - 56% of people declare “Not applicable” as answer to the question related to the analysis and evaluation of infrastructure security performed by external certified analyst. - 56% of people declare “Not applicable” as answer to the question related to the analysis and evaluation of infrastructure security performed by external certified analyst. To conclude, even considering the limitations of the sample, it is important to stress that: To conclude, even considering the limitations of the sample, it is important to stress that:  An assessment that involves all the SCSs providers is needed, since they are all considered important from a business point of view for the organization. Moreover, this implies that the communication between all the participants of the SC should also be secure, in order to ensure integrity and confidentiality of all the data transmitted, and also that the actors involved in the communication are authorized.  The people involved have no deep knowledge of security standards and policies adopted by their organization nor all of them perform cybersecurity activities frequently. As a result, it is necessary to provide a software solution that supports end-users in their daily operations, allowing auditing all the activities of the SC, identifying vulnerable or compromised assets, performing an analysis of the risks of threats and an impact assessment. All these features will support the decision-making process, by proposing and evaluating different mitigation strategies, therefore being very relevant to the Conformity self-Assessment of ICT related products, services and processes. 952690 CYRENE PROJECT PARTNERS 31/05/2021 71 5. CYRENE Conformity Assessment Within a Conformity Assessment (CA) process, the Target of Evaluation (ToE), as mentioned in 3.2, is considered as “a set of software, firmware, hardware and/or process possibly accompanied by guidance” (ISO/IEC 15408-1:2009, see Appendix A – Glossary and Examples. The CYRENE ToE is the SCS and the CYRENE CA methodology (that will be described in D3.1 “Conformity Evaluation Process & Multi-Level Evidence-Driven Supply Chain Risk Assessment”) is oriented to evaluate the SCS-ToE against the security requirements, i.e., the CYRENE CA aims to examine whether specified security requirements related to a given SCS have been fulfilled. CYRENE CA methodology applies to every SCS in any sector. The CYRENE CA methodology will give the opportunity to SC stakeholders to evaluate their SCSs under all or one of the three different perspectives: the SC business view, the technical view and the sectorial view. To illustrate this, we will develop the CYRENE CA methodology in the scope of three SCS- ToEs; namely, ToE I: CYRENE business SC, ToE II: CYRENE technical SC, ToE III: CYRENE sectoral SCS, which will be thoroughly analysed in the coming sections. This is carried out in terms of decomposing the SCS to its generic components: SCS processes, business partners involved in the provision of the SCS and highlight the infrastructure they utilize to execute their tasks within the SCS performance. For each ToE, the CYRENE CA methodology can be applied capturing the different aspects of the SCS under the particular viewpoint and examining only the SCS components that matter in this viewpoint. SCS-ToE I (business view) will be evaluated to the identification, analysis, assessment and mi- gration of process-related threat scenarios that concern the SCS-ToE I. It views the SCS under evaluation from a business perspective, entailing SCS processes, business partners information exchange, business logic, etc. to assess their conformity according to the specific ToE I require- ments, described in section 5.2.1. Thus, in this ToE the business environment enfolds the SCS processes and business partners participating in these processes. Therefore, the components of the SCS-ToE I under evaluation here are all SCS processes, business partners, data that operate in the provision of the underlined SCS. Digital assets are out of the current scope of the evaluation and will not be subjected to this ToE I. D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 72 D2.1 – Supply Chain Analysis and Requirements These three differently adopted views reflect the following CYRENE SCS Circles of consideration which have been described in section 2.1. CYRENE adopts three different views in order to cap- ture the entire SCS ecosystem, since the SCS usually are described either from a business per- spective or a technical perspective or a snapshot of the entire SCS. The CYRENE conformity assessment methodology and the multi-level evidence-driven supply chain risk assessment of CYRENE will be thoroughly described and analysed in D3.1 and is de- signed in order to assess any SCS independently of the viewpoint. CYRENE will undertake as an example of its SCS-ToE the Vehicle Transform Service (VTS) and it will assess it by adopting the three (3) different views described above, as it will be presented in the next section. The “Vehicle Transport Service” is a complex SCS, including critical sectors, such as the Automotive Industry and the Maritime Transport Industry. It has been selected be- cause the industries involved impose a high financial and business impact for the EU. The current section targets at identifying the three ToEs and recognizing their business environ- ment (section 5.1). Technical specifications and further analysis of ICT infrastructures and assets of the VTS that may be included in ToE II and ToE III and will be further analysed in D3.1. Once the business environments of the three ToEs are identified, the requirements for self-conformity assessing the CYRENE ToEs are presented (section 5.2). 5. CYRENE Conformity Assessment SCS-ToE II (holistic technical view) provides a technical-asset interdependent view of the SCS and is set to detect and analyze threats, identify and mitigate risks, examining the SCS processes, the business partners involved and the ICT infrastructure together with the individual SCS assets. The latter are hosted and operated by the business partners and are necessary for the provision of the SCS. Thereby, it focuses on assessing the conformity of these SCS components with re- spect to the ToE II requirements that are specified in section 0. The components of the SCS-ToE II considered under this type of evaluation are: SCS processes, business partners, data and all SCS assets (digital and physical) that participate in the provision of the entire SCS. SCS-ToE III (individual technical view-snapshot) considers a sector-specific view of the SCS. The boundary of the SCS-ToE III is within the scope of one of the business partners involved in the SCS. The components of the SCS-ToE III considered under this evaluation here are the pro- cesses and SCS assets that one of the business partners host and operate in order to participate in the entire SCS. 952690 CYRENE PROJECT PARTNERS 31/05/2021 73 Identification and description of SCS business processes of the ToE.  Identification and description of SCS business processes of the ToE. In this section a brief description of each identified business processes of the SCS (the VTS) along with the business goal is provided. The process description follows the table format shown in Table 6: In this section a brief description of each identified business processes of the SCS (the VTS) along with the business goal is provided. The process description follows the table format shown in Table 6: 5.1 Targets of Evaluations (ToE) CYRENE has adopted as an example of a SCS-ToE the Vehicle Transport Service (VTS) which is a SCS incorporating different business sectors and industries (e.g., maritime, automotive, transport, tourism). CYRENE described the VTS-SCS from three different angles (as mentioned in the previous sec- tion): VTS-ToE I: business SC, VTS-ToE II: technical SC, VTS-ToE III: sectoral SC. In sec- tions 5.1.1, 5.1.2, 5.1.3 , all these three (3) different views will be analysed and illustrated in de- tail. The Vehicle Transport SCS is considered as a massively complex system with numerous players for the manufacturing, shipment and delivery of various types of vehicles. It supports composite processes (i.e., domestic and international transportation, communications and information tech- nology, warehouse management, order and inventory control etc.), which enfolds an aggregation of industry sectors, such as maritime transport and automobile industry. It includes several inter- actions and tasks among the various entities engaged (stakeholders and actors) having different goals and requirements entailing vehicles manufacturing and storage facilities to assembly plants, i.e., inbound logistics (automotive industry), vehicles distribution, i.e., vehicles transport via port origin and port destination (maritime transport industry) and the final delivery to the importer. The performance of the Vehicle Transport SCS is accomplished through the provision of the Vehicle Transport Service (VTS) which is considered the CYRENE SCS-ToE. According to the above presentation of the three ToEs: -VTS-ToE I, includes the SCS processes of the VTS and the business partners interacting to meet these processes. 952690 CYRENE PROJECT PARTNERS 31/05/2021 74 D2.1 – Supply Chain Analysis and Requirements -VTS-ToE II consists of SCS processes of the VTS, the involved business partners along with the operating SC assets that are involved and participate in the operation and provision of the SCS. In the current deliverable only a high level infrastructure representation is provided. Asset models including technical specifications and security details will be analysed in D3.1, where the CY- RENE CA methodology will be thoroughly presented and described. -VTS-ToE II consists of SCS processes of the VTS, the involved business partners along with the operating SC assets that are involved and participate in the operation and provision of the SCS. In the current deliverable only a high level infrastructure representation is provided. Asset models including technical specifications and security details will be analysed in D3.1, where the CY- RENE CA methodology will be thoroughly presented and described. -VTS-ToE III reflects sector-specific processes of one business partner (in CYRENE the business partner is the Centro Ricerche FIAT ScpA and its World Class Manufacturing Research and In- novation (WCM R&I) department that has the goal to increase flexibility, quality, productivity, safety and ergonomics, energy and security of the logistics and manufacturing processes for FIAT Chrysler Automobiles (FCA)) and its SCS assets that hosts and uses in order to participate in the entire VTS. In the current deliverable only a high level infrastructure representation is provided. Asset models including technical specifications and security details will be analysed in D3.1, where the CYRENE CA methodology will be thoroughly presented and described. In the coming sections 5.1.1, 5.1.2 and 5.1.3, the business environment of the three ToEs is gradually displayed according to the following structure:  Identification and description of SCS business processes of the ToE. D2.1 – Supply Chain Analysis and Requirements p ( pp ) As ToE I is targeted into the business process evaluation, the current section is applicable only to VTS-ToE II and VTS-ToE III. Through this section, the ICT infrastructures of the cyber assets, the identified business partners utilize to perform their tasks within the un- derlined SC processes of the ToE are described and presented in a high-level overview. This is met according to the table format described in Table 8: ToE infrastructures of the SCS Process x (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description BP1 …… BP2 …… BP3 BPi …… Table 8 - ToE’s infrastructure description template. ToE infrastructures of the SCS Process x (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description BP1 …… BP2 …… BP3 BPi …… Table 8 - ToE’s infrastructure description template. Table 8 - ToE’s infrastructure description template.  Business process model generation To help the conformity assessor better comprehend the SCS processes, their workflows and the business partners and assets engaged across these flows, a process diagram is developed, visualizing each identified SCS process of the ToE.  Business process model generation To help the conformity assessor better comprehend the SCS processes, their workflows and the business partners and assets engaged across these flows, a process diagram is developed, visualizing each identified SCS process of the ToE. Example For the VTS process “Standard Cargo Manifest” the following business process diagram is developed utilizing the BPMN 2.0 business process modelling notation. Example For the VTS process “Standard Cargo Manifest” the following business process diagram is developed utilizing the BPMN 2.0 business process modelling notation. Figure 21 - “Standard Cargo Manifest”: a business process model example. Example For the VTS process “Standard Cargo Manifest” the following business process diagram is developed utilizing the BPMN 2.0 business process modelling notation. Figure 21 - “Standard Cargo Manifest”: a business process model example.  Identification and description of the business partners involved in the SCS pro- cesses of the ToE. Within this section the identified SCS processes of each ToE are further analysed into their embedded steps recognizing all the business partners participating together with their interactions and their business roles to fulfil these processes. The current description adopts the table format described in Table 7: 952690 CYRENE PROJECT PARTNERS 75 31/05/2021 ToE SC Process x analysis (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their inter- connections) Business Partners (BPi) Participating in the SC process x (Record business partners’ entities) BP1, BP2, BP3, … BPi Description (Process analysis describing how the business partners are involved in the SC process) ……. Table 7 - SCS Business Partners description template. 952690 CYRENE PROJECT PARTNERS 75 31/05/2021 ToE SC Process x analysis (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their inter- connections) Business Partners (BPi) Participating in the SC process x (Record business partners’ entities) BP1, BP2, BP3, … BPi Description (Process analysis describing how the business partners are involved in the SC process) ……. Table 7 - SCS Business Partners description template. Table 7 - SCS Business Partners description template. 952690 CYRENE PROJECT PARTNERS 75 31/05/2021 D2.1 – Supply Chain Analysis and Requirements The above figure shows the business process model of the “Standard Cargo Manifest” (SCM) process of the VTS. In particular, the process’s steps are visualized within the depicted workflow (Step1: Create SCM, Step2: SCM forwarded to Customs, Step3: Receive SCM) while the business partners involved in each step are represented into the three pools (swimlanes). Hence, in the current business process diagram the Ship Agent creates the SCM via the Port Community System (PCS) web application, the Port Authority forwards through its web service the SCM to the Customs to check and the Customs receives the document via the PCS web application and undertakes this responsibility 5.1.1 ToE I: Business view of the VTS-SCS The current ToE falls under the CYRENE business SCS perspective. The underlined SCS is the Vehicle Transport Service (VTS) as defined in the introduction of section 5.1. “ToE I” sets the scope to evaluate all process-related threat scenarios of the VTS. The current section encom- passes the business environment of ToE I: VTS processes and involved business partners. The processes under examination are dominant SCS processes that concern the overall VTS. Such SCS processes derive from an aggregation of industry sectors, such as automobile industry, mar- itime transport and logistics industry. The VTS-SCS is based upon the following four (4) business phases: A. Vehicles Purchase Phase; B. Shipment Phase; C. Pre-arrival Phase and Vessel Arrival, D. Vehicles Unloading and Delivery Phase. Vehicles Unloading and Delivery Phase The SCS phases encompass major SCS processes for the VTS provision as de A. Vehicles Purchase Phase Figure 21 - “Standard Cargo Manifest”: a business process model example. 952690 CYRENE PROJECT PARTNERS 76 D2.1 – Supply Chain Analysis and Requirements The Shipment Phase encapsulates all the shipping formalities that lead to the maritime transpor- tation of the vehicles from the port of origin to the destination port. It incorporates the Chartering Agreement Preparation & Negotiation, the Ship Formalities Arrangements and the Shipping Ar- rangements processes:  B1. Chartering Agreement Preparation & Negotiation process  B2. Ship Formalities Arrangements process  B3. Shipping Arrangements process A. Vehicles Purchase Phase The Vehicles Purchase Phase engages all the procedures an importer does for a vehicle pur- chase from an automobile Industry of the VTS. The SC processes of this phase are identi- fied in terms of the importer’s activities and interactions with the automobile industry to proceed with a purchase order of vehicles. The automobile manufacturer (e.g., FIAT Chrysler Automobiles (FCA)) realizes sectorial vehicles components supplying and manufacturing processes. For in- stance, they purchase and consume components from several auto parts suppliers in the vehicle assembly and auto parts (that can be several thousands of components of different variants and part numbers), which are assembled for the vehicle during the production of the automobiles. The current ToE considers an overview of the VTS. Therefore, such sector-specific processes are not identified and examined in the current ToE. The Vehicles Purchase Phase consists of the Vehi- cles Order Dispatch process and the Contract Agreement on the Vehicle Purchase process:  A1. Vehicles Order Dispatch process  A2. Contract Agreement on the Vehicle Purchase process B. Shipment Phase 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 77 C. Pre-arrival Phase The Pre-Arrival Phase possesses distribution chain procedures that have to be arranged by the importer through a corresponding representative (agent) and the port, engaging various formali- ties regarding the vessel docking and stevedoring of the vehicles at the designated port car ter- minal of the port and the agent’s interactions with other entities to accomplish these tasks, such as interactions with the Customs Authority. The phase encapsulates the Port Call Request, Standard Cargo Manifest, Entry Summary Declaration (ENS), Loading and Discharge List pro- cesses:  C.1 Port call request process  C.2 Standard cargo manifest process  C.3 Entry Summary Declaration (ENS) process  C.4 Loading and Discharge List process D. Vessel Arrival, Vehicles Unloading and Delivery Phase D. Vessel Arrival, Vehicles Unloading and Delivery Phase The Vessel Arrival, Vehicles Unloading and Delivery Phase includes a series of tasks among the key-players of the VTS that lead to a specific goal or produce a final result, referring to the delivery of the vehicles to the end customer (namely the importer in the current use case). This is instan- tiated by the completion of the vessel arrival procedures to the destination port and the vehi- cles unloading of the Car Carrier Vessel at the designated port car terminal. The current phase is supported by the Discharge vehicles, Customs declarations, Transportation order processes.  D1. Discharge vehicles process  D.2 Customs declarations process  D.3 Transportation order process Within the next two sections, the SCS processes are identified (section 5.1.1.1 Identification and description of SCS business processes of ToE I), the business partners are recognized and it is described how they are involved in each VTS process step (section 5.1.1.2 Identification and de- scription of SCS business partners of ToE I and corresponding SCS business process models) and eventually the business process models are developed for each corresponding SCS process. 5.1.1.1 Identification and description of SCS business processes of ToE I In this section, the recorded VTS processes for each SC phase, abovementioned, are specified. ToE “Contract Agreement on the Vehicle Purchase” process (Α general description of the business process and its business goal) The SC process “Contract Agreement on the Vehicle Purchase” of the VTS relates to the procedures undertaken, in order to issue and sign a contract agreement between the inter- ested parties towards a vehicles’ purchase request. 5.1.1.1 Identification and description of SCS business processes of ToE I In this section, the recorded VTS processes for each SC phase, abovemen 5.1.1.1 Identification and description of SCS business processes of ToE I In this section, the recorded VTS processes for each SC phase, abovementioned, are specified. n this section, the recorded VTS processes for each SC phase, abovementioned, A. Vehicles Purchase Phase A. Vehicles Purchase Phase 952690 CYRENE PROJECT PARTNERS  A1. Vehicles Order Dispatch process 31/05/2021 78 D2.1 – Supply Chain Analysis and Requirements ToE “Vehicles Order Dispatch” process (Α general description of the business process and its business goal) The process aims to satisfy the end customers’ requirements for automobile purchase and it refers to all the procedures undertaken for the preparation of the vehicles purchase order request. Table 9 - “Vehicle Order Dispatch” process description.  A2. Contract Agreement on the Vehicle Purchase process ToE “Contract Agreement on the Vehicle Purchase” process (Α general description of the business process and its business goal) The SC process “Contract Agreement on the Vehicle Purchase” of the VTS relates to the procedures undertaken, in order to issue and sign a contract agreement between the inter- ested parties towards a vehicles’ purchase request. Table 10 - “Contract Agreement on the Vehicle Purchase” process description. B. Shipment Phase  B1. Chartering Agreement Preparation & Negotiation process ToE “Chartering Agreement Preparation & Negotiation” process (Α general description of the business process and its business goal) The process aims to highlight all the procedures undertaken for ship chartering including the negotiation actions. Table 11 - “Chartering Agreement Preparation and Negotiation” process description.  B2. Ship Formalities Arrangements process ToE “Ship Formalities Arrangements” process (Α general description of the business process and its business goal) The current process describes some activities taken before the vessel reaches the local port to load or unload the vehicles. Critical activities in the current process are the Customs Clearance request, the docking permission and the control of the vessel’s course during the authorization process. Table 12 - “Ship Formalities Arrangements” process description.  B3. Shipping Arrangements process ToE “Shipping Arrangements” process D2.1 – Supply Chain Analysis and Requirements ToE “Vehicles Order Dispatch” process (Α general description of the business process and its business goal) The process aims to satisfy the end customers’ requirements for automobile purchase and it refers to all the procedures undertaken for the preparation of the vehicles purchase order request.  B1. Chartering Agreement Preparation & Negotiation process  B1. Chartering Agreement Preparation & Negotiation process D2.1 – Supply Chain Analysis and Requirements The process engages all the activities required to be implemented before the vehicles are distributed from the port of origin to the destination port via carrier vessel sea transport. The goal of the process is to fulfil all the prerequisites, in order to deliver the vehicles on time from one port to another, such as to prepare the respective documents and forms for the shipment, manage ship arrival procedures and control regional procedures. Table 13 - “Shipping Arrangements” process description. Table 13 - “Shipping Arrangements” process description. 952690 CYRENE PROJECT PARTNERS 80 31/05/2021 C. Pre-Arrival Phase  C.1 Port Call Request process ToE “Port Call Request” process (Α general description of the business process and its business goal) The SC process “Port Call Request” of the VTS can be described as the activities taken before the vessel reaches the local port for requesting port call including the nautical ser- vices (pilot, tugboats, and mooring). Table 14 - “Port Call Request” process description.  C.2 Standard Cargo Manifest process ToE “Standard Cargo Manifest process” (Α general description of the business process and its business goal) The current process describes some activities taken before the vessel reaches the local port for declaring all the goods in the vessel. Table 15 - “Standard Cargo Manifest” process description.  C.3 Entry Summary Declaration (ENS) process ToE “Entry Summary Declaration (ENS) process” (Α general description of the business process and its business goal) The current process describes some activities taken before the vessel reaches the local port for declaring all the goods on the vessel when accessing to the European customs area. Table 16 - “Entry Summary Declaration (ENS)” process description.  C.4 Loading and Discharge List process ToE “Loading and Discharge List process” (Α general description of the business process and its business goal) Table 12 - “Ship Formalities Arrangements” process description. Table 12 - “Ship Formalities Arrangements” process description.  B3. Shipping Arrangements process 952690 CYRENE PROJECT PARTNERS 79 31/05/2021  C.1 Port Call Request process The SC process “Port Call Request” of the VTS can be described as the activities taken before the vessel reaches the local port for requesting port call including the nautical ser- vices (pilot, tugboats, and mooring). The SC process “Port Call Request” of the VTS can be described as the activities taken before the vessel reaches the local port for requesting port call including the nautical ser- vices (pilot, tugboats, and mooring). Table 14 - “Port Call Request” process description.  C.2 Standard Cargo Manifest process The current process describes some activities taken before the vessel reaches the local port for declaring all the goods on the vessel when accessing to the European customs area. Table 16 - “Entry Summary Declaration (ENS)” process description. Table 16 - “Entry Summary Declaration (ENS)” process description.  C.4 Loading and Discharge List process ToE “Loading and Discharge List process” (Α general description of the business process and its business goal) ToE “Discharge Vehicles” (Α general description of the business process and its business goal) Once the car carrier vessel arrives at the destination port and the loading and the vehi- cles discharge list is sent to the Terminal Operator of the destination port on proper time and all preparation processes have been set, activities for discharging the vehicles from the vessel to the port take place. Table 18 - “Discharge Vehicles” process description. Table 18 - “Discharge Vehicles” process description.  C.4 Loading and Discharge List process  C.4 Loading and Discharge List process 952690 CYRENE PROJECT PARTNERS 31/05/2021 80 2.1 – Supply Chain Analysis and Requirements The current process describes some activities taken before the vessel reaches the local port for informing the good to be loaded and discharged. Table 17 - “Loading and Discharge List” process description. Vessel Arrival, Vehicles Unloading and Delivery Phase  D1. Discharge Vehicles process ToE “Discharge Vehicles” (Α general description of the business process and its business goal) Once the car carrier vessel arrives at the destination port and the loading and the vehi- cles discharge list is sent to the Terminal Operator of the destination port on proper time and all preparation processes have been set, activities for discharging the vehicles from the vessel to the port take place. Table 18 - “Discharge Vehicles” process description.  D.2 Customs Declarations process ToE “Customs Declarations” process (Α general description of the business process and its business goal) The Customs declarations is a mandatory process when importing goods with the Tax Agency's Custom. In this regard, the goods owner or its representative needs to submit the Single Administrative Document for goods from countries outside the EU or the correspond- ing document (e.g., Proof of Union Status). This is necessary before the goods can leave the customs compound, in this case the port. Table 19 - “Customs Declarations” process description.  D.3 Transportation Order process  D.3 Transportation Order process ToE “Transportation Order” process (Α general description of the business process and its business goal) Road transportation is an important part of the logistics in the container transport chain, including a variety of actors. The interested parties in order to control the transportation flow and obtain a common knowledge of the operations, there is a documentation flow in paral- lel running with the physical flow of goods, i.e., the ordered vehicles. The current process describes the documentation required for the road transportation of the vehicles to the de- livery to the end customer, namely the importer. Table 20 - “Transportation Order” process description. D2.1 – Supply Chain Analysis and Requirements The current process describes some activities taken before the vessel reaches the local port for informing the good to be loaded and discharged. The current process describes some activities taken before the vessel reaches the local port for informing the good to be loaded and discharged. D. Vessel Arrival, Vehicles Unloading and Delivery Phase D. Vessel Arrival, Vehicles Unloading and Delivery Phase  D1. Discharge Vehicles process Table 20 - “Transportation Order” process description. ToE “Customs Declarations” process (Α general description of the business process and its business goal) The Customs declarations is a mandatory process when importing goods with the Tax Agency's Custom. In this regard, the goods owner or its representative needs to submit the Single Administrative Document for goods from countries outside the EU or the correspond- ing document (e.g., Proof of Union Status). This is necessary before the goods can leave the customs compound, in this case the port. Table 19 - “Customs Declarations” process description. Table 21 - Business partners involved in the “Vehicles Order Dispatch” process. D2.1 – Supply Chain Analysis and Requirements 5.1.1.2 Identification and description of SCS business partners of ToE I and corresponding SCS business process models 5.1.1.2 Identification and description of SCS business partners of ToE I and corresponding SCS business process models In the current section, the SCS processes identified in section 5.1.1.1 Identification and descrip- tion of SCS business processes of ToE I are further analysed into consequent steps and the business partners involved in these processes for the provision of the VTS are specified along with their business roles. In the current section, the SCS processes identified in section 5.1.1.1 Identification and descrip- tion of SCS business processes of ToE I are further analysed into consequent steps and the business partners involved in these processes for the provision of the VTS are specified along with their business roles. After presenting the SCS processes of the VTS along with the business partners involved, busi- ness process diagrams are developed for each identified process. These business process model visualizations aim to help the reader better comprehend the process workflow and the involved parties. The business process models are represented using the BPMN 2.0 modelling specifica- tion and are depicted after each business partners identification table for the corresponding spec- ified SCS process. ToE “Transportation Order” process (Α general description of the business process and its business goal) Road transportation is an important part of the logistics in the container transport chain, including a variety of actors. The interested parties in order to control the transportation flow and obtain a common knowledge of the operations, there is a documentation flow in paral- lel running with the physical flow of goods, i.e., the ordered vehicles. The current process describes the documentation required for the road transportation of the vehicles to the de- livery to the end customer, namely the importer. Table 20 - “Transportation Order” process description. 952690 CYRENE PROJECT PARTNERS 31/05/2021 81  A1. Vehicles Order Dispatch process ToE “Vehicles Order Dispatch” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) ToE “Vehicles Order Dispatch” Business Partners (Process analysis, identifying and describing all the business partners participated in the current u SC process and their roles within the process (taking into account the cyber assets operating to sup interconnections) Business Partners (BPi) Participating in the process (R d b i ’ i i ) ( p ) Importer, Automobile Industry  A2. Contract Agreement on the Vehicle Purchase process  A2. Contract Agreement on the Vehicle Purchase process ToE “Contract Agreement on the Vehicle Purchase” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) Participating in the process (Record business partners’ entities) ( p ) Importer, Automobile Industry ( p ) Importer, Automobile Industry ( y g p p ) The “Vehicles Order Dispatch” order is a process executed between the Importer (and the Automobile Industry. The importer sets the order for the Automobile Industry according to the end customers’ demands. In particular, the following actions take place: Step 1. The Importer prepares a purchase order via his Ordering System that contains or- ders for a number of automobile Industries. Step 2. If the Automobile Industry does not approve it (e.g., lack of stock), the purchase order is returned to the customer (importer) for revision. If the Industry approves the task order, module generates a copy of the approval letter and e-mails it to the costumer (im- porter) together with the order file. The Manufacturer (Industry) deal with these procedures through an electronic commerce system (e.g.: a Part On line Catalogue and Ordering Sys- tem). ) Step 3. The Importer enters funding information and uploads appropriate supporting docu- ment attachments in the Part On line System (whether it is available to interact directly on line). 952690 CYRENE PROJECT PARTNERS 31/05/2021 82 D2.1 – Supply Chain Analysis and Requirements Figure 22 - Business process model for the “Vehicle Order Dispatch” process. Figure 22 - Business process model for the “Vehicle Order Dispatch” process.  A2. Contract Agreement on the Vehicle Purchase process ( p ) Automobile Industry, Importer D i i The process is performed between the Industry and the Importer and it is activated as soon as the Vehicles Order Dispatch process is fulfilled. The business partners involved follow the subsequent steps enlisted, below, to deal with the process: p y g Step 2. The Automobile Industry prepares a pre-contract agreement and sends it to the Importer. Step 3. The Importer delves into the terms of the pre-contract and with the cooperation of a lawyer, a financial consultant and an insurance consultant sends a report to the Industry for amendments. Step 4. Then the Automobile Industry arranges a meeting with the Importer in order to ne- gotiate for the contract. Step 4. Then the Automobile Industry arranges a meeting with the Importer in order to ne- gotiate for the contract. Step 5. During the meeting, they discuss with specialized officers about all the amendments required to get both parties satisfied. Step 5. During the meeting, they discuss with specialized officers about all the amendments required to get both parties satisfied. Step 6. Once both Importer and Industry have agreed upon the terms and conditions of the contract like pricing, documentation, freight charges, currency etc. and signed the contracts the latter proceeds to complete the order. Step 6. Once both Importer and Industry have agreed upon the terms and conditions of the contract like pricing, documentation, freight charges, currency etc. and signed the contracts the latter proceeds to complete the order. 952690 CYRENE PROJECT PARTNERS 31/05/2021 83 31/05/2021 D2.1 – Supply Chain Analysis and Requirements Lawyers and the Accounting Department of Industry issue all the appropriate formali- ties of the deal (invoicing, tax office declarations etc.) and send to the Importer the re- lated documents. The Automobile Industry involved parties issues the associated docu- mentation via enhanced technology platforms (i.e., ERPs) and database management sys- tems. Fiscal transactions are operated through e-government services provided by a Tax- ation Information System. Fund transfers are achieved through wire transfers, direct depos- its, ATM transactions and e-banking services via electronic, interactive communication channels. When the purchase procedures come to an end the Automobile Industry has to arrange the delivery of the vehicles to the Importer with Maritime Transportation at a spec- ified time. Table 22 - Business partners involved in the “Contract Agreement on the Vehicle Purchase” process. Figure 23 - Business process model for the “Contact Agreement on the Vehicle Purchase” process. Table 22 - Business partners involved in the “Contract Agreement on the Vehicle Purchase” process. - Business partners involved in the “Contract Agreement on the Vehicle Purchase” process. Figure 23 - Business process model for the “Contact Agreement on the Vehicle Purchase” process. B. Shipment Phase B. Shipment Phase 952690 CYRENE PROJECT PARTNERS 84 31/05/2021 31/05/2021 84 D2.1 – Supply Chain Analysis and Requirements  B1. Chartering Agreement Preparation & Negotiation process ToE “Chartering Agreement Preparation & Negotiation” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support thei interconnections) ( p ) Charterer (Automobile Industry), Ship Owner, Shipbroker The Automobile Industry is seeking to contract with the Ship Owner for delivering the vehicle to the destination port defined by the Importer. To accomplish this goal the following activi- ties take place: Step 1. Initiation of Chartering Agreement procedures. The Chartering Agreement (known as charter party) is a binding agreement between the Ship Owner and the Charterer (in the current scenario the Automobile Industry is the Charterer) indicating the certain conditions in which a vessel is rented regarding the vehicle transport. The most important clauses of a charter party are those defining the time-period allowed for loading and unloading the vessel and determining who undertakes the responsibility for the expenses involved. g p y p The Automobile Industry and the Ship Owner must come to a chartering agreement. More specifically, there are four principal methods of chartering a tramp ship summarized below: The Automobile Industry and the Ship Owner must come to a chartering agreeme specifically, there are four principal methods of chartering a tramp ship summarized  Voyage charter, is the most common type according to which chartering refers to a given price for the transport of a certain vehicle for a one-way voyage between specific ports p  Time charter, depends on hiring the vessel for a certain period of time  Bareboat or demise charter, is rarely used, described as an arrangement of hiring a vessel for a specified period without crew, insurance, stores or any other provision. As a result, the Charterer is entirely responsible for the vessel’s legal and financial support- ing  “Lump-sum” contracts are settled on a lump-sum basis, agreed upon a total and global price for simple and well-defined scope projects which are hardly possible to change. g Step 2. After continuing negotiations between the interested parties, the charter party agreement is set reflecting the following main aspects:  time offers  counter (may be one, two or several counters exchanged)  recap of terms (partially fixed on subjects)  clean recap (final revision)  execution of charter party Step 3.  B1. Chartering Agreement Preparation & Negotiation process The Automobile Industry informs the Shipbroker (an entity that acts as a negotiator between the Automobile Industry and the Ship Owner) about the specified shipment agree- g Step 2. After continuing negotiations between the interested parties, the charter party agreement is set reflecting the following main aspects:  time offers  counter (may be one, two or several counters exchanged)  counter (may be one, two or several counters exchanged) ( y  recap of terms (partially fixed on subjects) Step 3. The Automobile Industry informs the Shipbroker (an entity that acts as a negotiator between the Automobile Industry and the Ship Owner) about the specified shipment agree- ment conditions and terms dealt with the Importer. Step 3. The Automobile Industry informs the Shipbroker (an entity that acts as a negotiator between the Automobile Industry and the Ship Owner) about the specified shipment agree- ment conditions and terms dealt with the Importer. p Step 4. The Ship Owner reports the Shipbroker on cargo related details, declaring the actual vehicles carrying capacity of the vessel. p Step 4. The Ship Owner reports the Shipbroker on cargo related details, declaring the actual vehicles carrying capacity of the vessel. 952690 CYRENE PROJECT PARTNERS 31/05/2021 85 D2.1 – Supply Chain Analysis and Requirements Step 5. The Ship Owner and the Automobile Industry are under extended discussion through the Shipbroker, exchanging several counter offers until both parties lift the subjects on vessels. Step 5. The Ship Owner and the Automobile Industry are under extended discussion through the Shipbroker, exchanging several counter offers until both parties lift the subjects on essels Table 23 - Business partners involved in the “Chartering Agreement Preparation & Negotiation” process. Figure 24 - Business process model for the “Chartering Agreement Preparation & Negotiation” process. siness partners involved in the “Chartering Agreement Preparation & Negotiation” process. Figure 24 - Business process model for the “Chartering Agreement Preparation & Negotiation” process. Figure 24 - Business process model for the “Chartering Agreement Preparation & Negotiation” process.  B2. Ship Formalities Arrangements process ToE “Ship Formalities Arrangements” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Customs, Port Authority Description (Process analysis describing how the business partners are involved in the SC process) Vehicles import/export in Maritime transport is subject to local Customs’ audit. Only Local Customs can legitimate the vehicles shipment across the origin and destination port. Ac- cording to this, the Ship Agent undertakes the responsibility to deal with the appropriate formalities arrangements for gaining from the Local Customs the permission to proceed with the vehicles’ shipment. Customs Clearance is considered the documented permission  B2. Ship Formalities Arrangements process ToE “Ship Formalities Arrangements” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Customs, Port Authority Description (Process analysis describing how the business partners are involved in the SC process) Vehicles import/export in Maritime transport is subject to local Customs’ audit. Only Local Customs can legitimate the vehicles shipment across the origin and destination port. D2.1 – Supply Chain Analysis and Requirements given by the Customs to import or export vehicles. Customs Clearance proves that all Cus- toms duties have been paid and shipment procedures have been approved. To execute the process the following activities are undertaken: given by the Customs to import or export vehicles. Customs Clearance proves that all Cus- toms duties have been paid and shipment procedures have been approved. To execute the process the following activities are undertaken: p g Step 1. The Ship Agent submits the ship’s associated documentation to the Customs re- questing Customs Clearance. Typical formalities must have been fulfilled at the customs office for import/export activities including: Step 1. The Ship Agent submits the ship’s associated documentation to the Customs re- questing Customs Clearance. Typical formalities must have been fulfilled at the customs office for import/export activities including: office for import/export activities including:  the lodging and acceptance of the customs declaration;  the declaration verification n and the supporting documentation; the physical exam- ination of vehicles; measures for vehicles identification and controls on whether they are conformed to  measures for vehicles identification and controls on whether they are conformed to satisfy the appropriate conditions or restrictions; satisfy the appropriate conditions or restrictions;  payment for import/export procedures and other charges (e.g., VAT, excise dut  payment for import/export procedures and other charges (e.g., VAT, excise duties);  release of vehicles for the customs procedure concerned  release of vehicles for the customs procedure concerned. Step 2. Once the Ship Agent obtains the Custom Clearance approval, he submits a request to the Port Community System (PCS) of the Port Authority to grant permission for the vessel to dock at the port. The Ship Agent’s request is submitted via the corresponding online service of the PCS. Step 2. Once the Ship Agent obtains the Custom Clearance approval, he submits a request to the Port Community System (PCS) of the Port Authority to grant permission for the vessel to dock at the port. The Ship Agent’s request is submitted via the corresponding online service of the PCS. Step 3. The Port Community System (PCS) checks the Customs Clearance document au- thentication by data verification services requesting information from the Custom infor- mation system via an Electronic Data Interchange system (EDI) and reports the result. Dur- ing the authorization processes, Port Authority observes via the Automatic Identification System (AIS) the vessel’s course to ascertain that the marine transportation meets the pri- mary consignment’s specific requirements. Information on vessel position and an Arri- val/Departure timetable is requested from the Port Authority via the Vessel Traffic Service. Vessel Traffic Service is a Port’s Authority marine-traffic monitoring system, identifying ves- sel e-tracking and traffic services operations. The Port Authority uses the “MarineTraffic” intermodal web platform to retrieve information for the vessel’s course, its geo-coordinates location and the vessel traffic in the adjacent sea area and control remotely the vessel’s movement in case traffic on the water is high. Step 4. The Port Authority provides permission for the vessel to dock, only if the following prerequisites occur:  The vessel has not deviated from its route,  The vessel has not deviated from its route,  The vessel’s voyage has followed the regulatory compliance policies  Marine traffic does not prevent the vessel from approaching the Port.  Marine traffic does not prevent the vessel from approaching the Port. Step 5. As long as the above prerequisites are satisfied, the Local Agent must be informed from the Port Authority about the docking arrangements at a specific time before the vessel arrives at the local port to load (or unload) the vehicles. Step 6. Before the vessel reaches the local Port consults the marine traffic live map and informs the Ship’s Administration about the current sea traffic. Port Authority expert person- nel monitor the vessel’s movement and conducts it via AIS equipment to enter safely the port to dock. In particular, the internal communication between the Port Authority and the Local Agent is satisfied by utilizing AIS transponders broadcasting information via marine radar antennas VHF radio waves. In this manner, they can corporate in order to avoid a potential collision and to improve sea navigation. Step 6. Before the vessel reaches the local Port consults the marine traffic live map and informs the Ship’s Administration about the current sea traffic. Port Authority expert person- nel monitor the vessel’s movement and conducts it via AIS equipment to enter safely the port to dock. In particular, the internal communication between the Port Authority and the Local Agent is satisfied by utilizing AIS transponders broadcasting information via marine radar antennas VHF radio waves. Ac- di t thi th Shi A t d t k th ibilit t d l ith th i t ToE “Ship Formalities Arrangements” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Customs, Port Authority Description Ship Agent, Customs, Port Authority Ship Agent, Customs, Port Authority ( y g p p ) Vehicles import/export in Maritime transport is subject to local Customs’ audit. Only Local Customs can legitimate the vehicles shipment across the origin and destination port. Ac- cording to this, the Ship Agent undertakes the responsibility to deal with the appropriate formalities arrangements for gaining from the Local Customs the permission to proceed with the vehicles’ shipment. Customs Clearance is considered the documented permission 952690 CYRENE PROJECT PARTNERS 31/05/2021 86 In this manner, they can corporate in order to avoid a potential collision and to improve sea navigation. Step 7. The Ship Agent makes the arrangements with the Public Administration (manage- ment of the ship formalities) regarding the authorisation process, ranging from the entry of the ship into the local port to the vehicles loading onto the vessel for shipping them to the 31/05/2021 952690 CYRENE PROJECT PARTNERS 87 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements The Ship Agent (representing the Ship Owner) is obligated to deliver the vehicl The Ship Agent (representing the Ship Owner) is obligated to deliver the vehicles on time. A breach of contract can easily occur either for unreasonable delay or for unjustifiable de- parture from the usual and reasonable route. To achieve this, the following activities are implemented: The Ship Agent (representing the Ship Owner) is obligated to deliver the vehicles on time. A breach of contract can easily occur either for unreasonable delay or for unjustifiable de- parture from the usual and reasonable route. To achieve this, the following activities are implemented: Step 1. The Ship Agent undertakes the responsibility to send all the pertinent documents (e.g., the Manifest document of the vessel, the docking clearance document) to the Im- porter’s Local Agent. Step 1. The Ship Agent undertakes the responsibility to send all the pertinent documents (e.g., the Manifest document of the vessel, the docking clearance document) to the Im- porter’s Local Agent. Step 2. The Local Agent undertakes the responsibility for the ship arrival and controls the regional procedures:  he communicates the Insurance Company to make an assessment report of the ve- hicles physical status and their transportation’s service quality  he employs a Vehicle Transport Company and arranges to receive and load the vehicles from the vehicle terminal at the destination port at a specified time. he deals with the relevant shipping formalities (exchanging information through lo- cs infrastructure, multi-modal platforms and communicating via telematics)  he deals with the relevant shipping formalities (exchanging information through lo- gistics infrastructure, multi-modal platforms and communicating via telematics) Step 3. The Local Agent informs the Importer about the status of the aforementioned pro- cedures.  he deals with the relevant shipping formalities (exchanging information through lo- gistics infrastructure, multi-modal platforms and communicating via telematics) Step 3. The Local Agent informs the Importer about the status of the aforementioned pro- cedures. Table 25 - Business partners involved in the “Shipping Arrangements” process. Figure 26 - Business process model for the “Shipping Arrangements” process. Table 25 - Business partners involved in the “Shipping Arrangements” process. Figure 26 - Business process model for the “Shipping Arrangements” process. destination Port. The shipping arrangements are realized through data exchange using lo- gistics infrastructures and port integrated systems. During the authorization process, ship- ping operations are monitored and controlled through networking technology of SCADA and AIS systems. Table 24 - Business partners involved in the “Ship Formalities Arrangements” processs. Figure 25 - Business process model for the “Ship Formalities Arrangements” process. Table 24 - Business partners involved in the “Ship Formalities Arrangements” processs. Figure 25 - Business process model for the “Ship Formalities Arrangements” process. 952690 CYRENE PROJECT PARTNERS 88 31/05/2021  B3. Shipping Arrangements process ToE “Shipping Arrangements” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Local Agent, Insurance Company, Vehicles Transport Company, Importer Description (Process analysis describing how the business partners are involved in the SC process)  B3. Shipping Arrangements process ToE “Shipping Arrangements” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Local Agent, Insurance Company, Vehicles Transport Company, Importer Description (Process analysis describing how the business partners are involved in the SC process) 952690 CYRENE PROJECT PARTNERS 31/05/2021 88 C. Pre-Arrival Phase  C.1 Port Call Request process  C.1 Port Call Request process ToE “Port Call Request” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) 952690 CYRENE PROJECT PARTNERS 89 31/05/2021 D2.1 – Supply Chain Analysis and Requirements ( p ) Ship Agent, Port Authority, Customs, Terminal Operator Description The port calls process is a request from the Shipping Line or its Ship Agent to the Port Authority and the Harbourmaster’s office, requesting a berth, giving details of the call and the vessel authorized. To achieve this, the following activities are implemented: Step 1. The Ship Agent sends the Port Authority data including the port of arrival, name of vessel, the carrier, previous and following ports of call. Once the port call corresponding authorisations for these requests are received the Ship Agent provides more information about passengers and crew, waste, berth requirements, expected operations (pilot, tug- boats, and mooring), and other relevant data. The port calls process is a request from the Shipping Line or its Ship Agent to the Port Authority and the Harbourmaster’s office, requesting a berth, giving details of the call and the vessel authorized. To achieve this, the following activities are implemented: Step 1. The Ship Agent sends the Port Authority data including the port of arrival, name of vessel, the carrier, previous and following ports of call. Once the port call corresponding The port calls process is a request from the Shipping Line or its Ship Agent to the Port Authority and the Harbourmaster’s office, requesting a berth, giving details of the call and the vessel authorized. To achieve this, the following activities are implemented: Step 1. The Ship Agent sends the Port Authority data including the port of arrival, name of vessel, the carrier, previous and following ports of call. Once the port call corresponding authorisations for these requests are received the Ship Agent provides more information about passengers and crew, waste, berth requirements, expected operations (pilot, tug- boats, and mooring), and other relevant data. Step 2. Vehicles import/export in maritime transport is subject to local Customs’ audit. By sending the request of port call, automatically opens a Customs registry for the customs clearance of goods that must be loaded or unloaded from the vessel. Step 3. Table 27 - Business partners involved in the “Standard Cargo Manifest” process. ( p ) Ship Agent, Port Authority, Customs D i i ( p ) Ship Agent, Port Authority, Customs D i i ( y g p p ) The Standard Cargo Manifest is the document that includes all the information related to a ship and the cargo transported in a ship for a particular trip and which is intended to be discharged/loaded during its port call. In this way, the information contained in a Summary Declaration is mainly divided into two sections:  Information related to the ship, trip and port call: this section includes the data refer- ring to the name of the ship, flag, etc., indicating the ports of origin, destination that delimit the corresponding trip. In addition, it incorporates information for Customs, such as the concept of regular line, simplified transit procedures and the operations and actors involved in them.  Information related to the ship, trip and port call: this section includes the data refer- ring to the name of the ship, flag, etc., indicating the ports of origin, destination that delimit the corresponding trip. In addition, it incorporates information for Customs, such as the concept of regular line, simplified transit procedures and the operations and actors involved in them.  Information related to the Cargo transported: this section contains three blocks of data, those related to the level of Bill of Lading, (B/L), those related to the consignment (a consignment is characterized by having a single tariff code and be integrated into a single Bill of Lading), and those that refer to equipment. Also at the starting level, it contains data related to the customs situation and clearance.  Information related to the Cargo transported: this section contains three blocks of data, those related to the level of Bill of Lading, (B/L), those related to the consignment (a consignment is characterized by having a single tariff code and be integrated into a single Bill of Lading), and those that refer to equipment. Also at the starting level, it contains data related to the customs situation and clearance. Step 1. The Ship Agent, as the representative of the shipping Line, undertakes responsibil- ity for sending the Standard Cargo Manifest at least one day before the arrival of the ship. Step 2. The message is received by the Port Authority. p g y y Step 3. The Standard Cargo Manifest is forwarded to the Customs. D2.1 – Supply Chain Analysis and Requirements C. Pre-Arrival Phase The port calls information is used by Port Authority and the Terminal Operators to manage their resources accordingly preparing equipment, personnel, etc. All these communications are done using the Port Community System (PCS), which pro- vides the users a client application to launch the service rapidly and be able to fulfil the requirements to send documentation electronically to the involved entities. Table 26 - Business partners involved in the “Port Call Request” process. Figure 27 - Business process model for the “Port Call Request” process. Table 26 - Business partners involved in the “Port Call Request” process. Figure 27 - Business process model for the “Port Call Request” process.  C.2 Standard Cargo Manifest process  C.2 Standard Cargo Manifest process 52690 CYRENE PROJECT PARTNERS 90 31/05/2021  C.2 Standard Cargo Manifest process ToE “Standard Cargo Manifest” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) ToE “Standard Cargo Manifest” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) ToE “Standard Cargo Manifest” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) ToE “Standard Cargo Manifest” Business Partners s, identifying and describing all the business partners participated in the current underlined heir roles within the process (taking into account the cyber assets operating to support their interconnections) 952690 CYRENE PROJECT PARTNERS 90 31/05/2021 ( p ) Ship Agent, Port Authority, Customs D i i These communications are done using the Port Community System (PCS), which provides the users a client application to launch the service rapidly and be able to fulfil the require- ments to send documentation electronically to the involved entities. 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 91 D2.1 – Supply Chain Analysis and Requirements Figure 28 - Business model for the “Standard Cargo Manifest” process. Figure 28 - Business model for the “Standard Cargo Manifest” process.  C.3 Entry Summary Declaration (ENS) process ToE “Entry Summary Declaration (ENS)” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Customs, Port Authority Description ToE “Entry Summary Declaration (ENS)” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Customs, Port Authority Description ( p ) Ship Agent, Customs, Port Authority The Entry Summary Declaration (ENS) is a mandatory document which includes all the information related to the cargo transported in a ship. It is needed only when arriving to the first European port. The information in the document is quite similar to the Standard Cargo Manifest, although in this case contains all the cargo in the vessel and not only the cargo to be discharged to a specific port. Step 1. The Ship Agent, as the representative of the shipping Line, is engaged to send the ENS at least one day before the arrival of the ship together with the Standard Cargo Manifest. Step 2. The message is received by the Port Authority. Step 2. The message is received by the Port Authority. Step 2. The message is received by the Port Authority. Step 3 The message is forwarded to the Customs Step 2. The message is received by the Port Authority. S 3 Th i f d d h C Step 3. The message is forwarded to the Customs. Step 3. The message is forwarded to the Customs. Table 28 - Business partners involved in the “Entry Summary Declaration (ENS)” process. ( p ) Ship Agent, Port Authority, Customs D i i These communications are done using the Port Community System (PCS), which provides the users a client application to launch the service rapidly and be able to fulfil the require- ments to send documentation electronically to the involved entities. 952690 CYRENE PROJECT PARTNERS 31/05/2021 92 D2.1 – Supply Chain Analysis and Requirements Figure 29 - Business process model for the “Entry Summary Declaration (ENS)” process. Figure 29 - Business process model for the “Entry Summary Declaration (ENS)” process. Table 29 - Business partners involved in the “Loading and Discharge List” process.  C.4 Loading and Discharge List process ToE “Loading and Discharge List” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Ship Agent, Terminal Operator, Port Authority Description ( p ) Ship Agent, Terminal Operator, Port Authority The loading and discharge list is a document which contains all the vessel loading and discharge containers, cars, or goods. The main information included in the document is the container type, dimensions, weight, temperature, seal, dangerous goods information, etc. Step 1. The Ship Agent sends the loading and discharge list to the Terminal Operator at least 12 hours before the arrival. g , , g container type, dimensions, weight, temperature, seal, dangerous goods information, etc. Step 1. The Ship Agent sends the loading and discharge list to the Terminal Operator at least 12 hours before the arrival. Step 2. The Terminal Operator needs this information to manage all the resources needed for the process. Step 3. The Ship Agent receives a confirmation from the Terminal Operator of the loading and discharge of these containers when it is accomplished. These communications are done using the Port Community System (PCS), which provides the users a client application to launch the service rapidly and be able to fulfil the require- ments to send documentation electronically to the involved entities. Table 29 - Business partners involved in the “Loading and Discharge List” process. 952690 CYRENE PROJECT PARTNERS 31/05/2021 93 D2.1 – Supply Chain Analysis and Requirements Figure 30 - Business process model for the “Loading and Discharge List” process. Figure 30 - Business process model for the “Loading and Discharge List” process. D. Vessel Arrival, Vehicles Unloading and Delivery Phase  D1. Discharge Vehicles process Terminal Operator (Docker) y Step 2. Dockers have a mobile device connected to the TOS (Terminal Operating system) for receiving indications of the vehicles to be discharged and the area in the terminal yard to be parked. 952690 CYRENE PROJECT PARTNERS 94 31/05/2021 31/05/2021 952690 CYRENE PROJECT PARTNERS 95 31/05/2021 Step 3. With this information terminal operators managers are aware of the situation of each vehicle and are able to manage the resources and the space. Table 30 - Business partners involved in the “Discharge Vehicles” process. Figure 31 - Business process model for the “Discharge Vehicles” process.  D.2 Customs Declarations process ToE “Customs Declarations” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Freight Forwarder, Customs, Port Authority Description (Process analysis describing how the business partners are involved in the SC process) The Customs declarations is a mandatory process when importing goods with the Tax Agency's Custom. For that the goods owner or its representative needs to submit the Single Administrative Document for goods from countries outside the European union or the cor- responding document (e.g. Proof of Union Status). This is necessary before the goods can leave the customs compound, in this case the port. The Single Administrative Document Table 30 - Business partners involved in the “Discharge Vehicles” process. Table 30 - Business partners involved in the “Discharge Vehicles” process. Figure 31 - Business process model for the “Discharge Vehicles” process. Figure 31 - Business process model for the “Discharge Vehicles” process. Figure 31 - Business process model for the “Discharge Vehicles” process. Figure 31 - Business process model for the “Discharge Vehicles” process. Figure 31 - Business process model for the “Discharge Vehicles” process.  D.2 Customs Declarations process ToE “Customs Declarations” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Freight Forwarder, Customs, Port Authority Description (Process analysis describing how the business partners are involved in the SC process) The Customs declarations is a mandatory process when importing goods with the Tax Agency's Custom. For that the goods owner or its representative needs to submit the Single Administrative Document for goods from countries outside the European union or the cor- responding document (e.g. Proof of Union Status). This is necessary before the goods can leave the customs compound, in this case the port. The Single Administrative Document D2.1 – Supply Chain Analysis and Requirements Step 3. With this information terminal operators managers are aware of the situation of each vehicle and are able to manage the resources and the space. Step 3. With this information terminal operators managers are aware of the situation of each vehicle and are able to manage the resources and the space.  D.2 Customs Declarations process  D.2 Customs Declarations process Freight Forwarder, Customs, Port Authority ( y g p p ) The Customs declarations is a mandatory process when importing goods with the Tax Agency's Custom. For that the goods owner or its representative needs to submit the Single Administrative Document for goods from countries outside the European union or the cor- responding document (e.g. Proof of Union Status). This is necessary before the goods can leave the customs compound, in this case the port. The Single Administrative Document 952690 CYRENE PROJECT PARTNERS 31/05/2021 95 D2.1 – Supply Chain Analysis and Requirements includes the type of import, content, type of goods, value, currency, invoice, transport doc- uments, origin, destination, weight, etc. includes the type of import, content, type of goods, value, currency, invoice, transport doc- uments, origin, destination, weight, etc. includes the type of import, content, type of goods, value, currency, invoice, transport doc- uments, origin, destination, weight, etc. Step 1. Before the truck arrives at the port to pick up the vehicles, the Freight Forwarder has to submit the Single Administrative Document to Customs. S C includes the type of import, content, type of goods, value, currency, invoice, transport doc- uments, origin, destination, weight, etc. Step 1. Before the truck arrives at the port to pick up the vehicles, the Freight Forwarder has to submit the Single Administrative Document to Customs. Step 2. Whether predefined conditions are met, the Customs authorises the re- quest and the response is sent to the PCS in order to be available for involved stakeholders, including the automatic gate system in the port allows the trucks to leave the port. This process is done directly through the Customs system. yp p yp g y p uments, origin, destination, weight, etc. Step 1. Before the truck arrives at the port to pick up the vehicles, the Freight Forwarder has to submit the Single Administrative Document to Customs. Step 2 Whether predefined conditions are met the Customs authorises the re- Step 1. Before the truck arrives at the port to pick up the vehicles, the Freight Forwarder has to submit the Single Administrative Document to Customs. Step 2. Whether predefined conditions are met, the Customs authorises the re- quest and the response is sent to the PCS in order to be available for involved stakeholders, including the automatic gate system in the port allows the trucks to leave the port. 52690 CYRENE PROJECT PARTNERS 96 31/05/2021 c ud g t e auto at c gate syste t e po t a o s t e t uc s to ea e t e po t This process is done directly through the Customs system. Table 31 - Business partners involved in the “Customs Declarations” process. Figure 32 - Business process model for the “Customs Declarations” process.  D.3 Transportation Order process ToE “Transportation Order” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Freight Forwarder, Ship Agent, Terminal Operator, Haulier Company Description (Process analysis describing how the business partners are involved in the SC process) Road transportation is an important part of the logistics in the container transport chain, and several actors are involved such as Freight Forwarders, Haulier Companies, Terminals, or Ship Agents. In order to have a common understanding of the operations, there is a docu- mentation flow parallel to the physical flow of goods. For road transportation it is needed a Table 31 - Business partners involved in the “Customs Declarations” process. Figure 32 - Business process model for the “Customs Declarations” process. Table 31 - Business partners involved in the “Customs Declarations” process. Table 31 - Business partners involved in the “Customs Declarations” process. Figure 32 - Business process model for the “Customs Declarations” process. D2.1 – Supply Chain Analysis and Requirements transport order, a document which determines the obligations between carrier and shipper. This document is local and therefore is regulated by national law. The main information included in the document is the container type, dimensions, weight, temperature, seal, dan- gerous goods information, etc. transport order, a document which determines the obligations between carrier and shipper. This document is local and therefore is regulated by national law. The main information included in the document is the container type, dimensions, weight, temperature, seal, dan- gerous goods information, etc. Step 1. The process starts when the importer or its Freight Forwarder request a transport. Step 2. The Ship Agent provides the transport order documentation to the Freight For- warder and the Terminal Operator. Step 3. The Haulier Company completes the document assigning a truck. Step 3. The Haulier Company completes the document assigning a truck. Step 4. Finally, when the prerequisites are met, the Terminal Operator confirms the deliv- ery. Step 4. Finally, when the prerequisites are met, the Terminal Operator confirms the deliv- ery Although this process is done thought the Port Community System (PCS) in the container transport, the vehicles transport process is still on paper and email. During the road transport operation, the driver needs the documentation is case is required by the traffic police or other security forces. Table 32 - Business partners involved in the “Transportation Order” process. Figure 33 - Business process model for the “Transportation Order” process. Table 32 - Business partners involved in the “Transportation Order” process. Figure 33 - Business process model for the “Transportation Order” process.  D.3 Transportation Order process  D.3 Transportation Order process ToE “Transportation Order” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Freight Forwarder, Ship Agent, Terminal Operator, Haulier Company Description (Process analysis describing how the business partners are involved in the SC process) Road transportation is an important part of the logistics in the container transport chain, and several actors are involved such as Freight Forwarders, Haulier Companies, Terminals, or Ship Agents. In order to have a common understanding of the operations, there is a docu- mentation flow parallel to the physical flow of goods. For road transportation it is needed a  D.3 Transportation Order process ToE “Transportation Order” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) participating in the process (Record business partners’ entities) Freight Forwarder, Ship Agent, Terminal Operator, Haulier Company Description (Process analysis describing how the business partners are involved in the SC process) Road transportation is an important part of the logistics in the container transport chain, and several actors are involved such as Freight Forwarders, Haulier Companies, Terminals, or Ship Agents. In order to have a common understanding of the operations, there is a docu- t ti fl ll l t th h i l fl f d F d t t ti it i d d Road transportation is an important part of the logistics in the container transport chain, and several actors are involved such as Freight Forwarders, Haulier Companies, Terminals, or Ship Agents. In order to have a common understanding of the operations, there is a docu- mentation flow parallel to the physical flow of goods. For road transportation it is needed a 952690 CYRENE PROJECT PARTNERS 31/05/2021 96 D2.1 – Supply Chain Analysis and Requirements 5.1.2.1 Identification and description of SCS business processes of ToE II Within this section, the recorded VTS processes for each identified SCs phase are The VTS-ToE II SCS processes abovementioned, have already been described and ana- lysed (section 5.1.1.1 Identification and description of SCS business processes of ToE I) and as they remain the same, no other actions required to be fulfilled in this section. 5.1.2 ToE II: Technical view of the VTS-SCS ToE II reflects the CYRENE technical SCS perspective. The underlined SCS is the Vehicle Transport Service (VTS) as defined in the introduction of section 5.1. “ToE II” sets the scope to evaluate SC processes of the VTS under a technical view that goes to a further analysis of the ICT infrastructure operating within these processes. The current section encompasses the busi- ness environment of ToE II: VTS processes, involved business partners and SC stakehold- ers and assets utilized for the completion of these processes. As mentioned previously in the beginning of section 5.1, in the current deliverable under examination assets will be presented only with a high-level ICT infrastructure view, as it is referred at the beginning of section 5.1. VTS-SCS processes derive from an aggregation of industry sectors, such as the automobile in- dustry, maritime transport and logistics industry. The VTS-SCS, as presented in the previous sec- tion, are: Vehicles Purchase Phase; Shipment Phase; Pre-arrival Phase and Vessel Arrival, Ve- hicles Unloading and Delivery Phase. Each of the VTS-SCS phase encompasses major SCS pro- cesses for the VTS provision, which have been described in detail in ToE I (the business view of the VTS-SCS). Regarding the asset view description of the VTS-SCS, for the purpose of the current project to illustrate the CYRENE CA methodology, ToE II will reflect assets of the SCS processes concerning the Pre-Arrival phase and Vessel Arrival, Vehicles Unloading and Deliv- ery phases, which mostly engage port-related processes and distribution chain processes (the port authority business partner of the current VTS use case will be the Valenciaport Foundation which is a pilot partner of the CYRENE project). 5.1.1.3 ToE’s infrastructure description Since ToE I adopts the business view, the assets and infrastructures do not belong to the current ToE’s environment and they will not be subject to evaluation. The current section is N/A for ToE I (only the business processes together with their workflows are applied here). 952690 CYRENE PROJECT PARTNERS 31/05/2021 97 5.1.2.2 Identification and description of SCS business partners of ToE II Process analysis and business partners identification of VTS-ToE II have already been described (section 5.1.1.2 Identification and description of SCS business partners of ToE I and correspond- ing SCS business process models) and as they remain the same, no other actions required to be fulfilled in this section. 5.1.2.3 ToE’s II infrastructure description 5.1.2.3 ToE’s II infrastructure description p has the last Microsoft operating system Windows 10 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements C. Pre-arrival Phase  C.1 Port Call Request process VTS-SCS Assets involved in the “Port Call Request” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners (BPs) of the underlined SCS process; data exchange services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Port Authority The Port Authority manages the PCS, an open and neutral elec- tronic platform, that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware: (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has a recovery site with all the systems replicated, to en- sure the availability. (b) A router Extreme Networks SLX 9640 that allows the intercon- nection of LAN networks and provide access to the system. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct communica- tion with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data interchange via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Ship Agent The Ship Agent is one of the main actors in the port call process. It needs access to the PCS to manage the operations. 5.1.2.3 ToE’s II infrastructure description The systems used for that activity are the following hardware and software.; (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U pro- cessor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 5.1.2.3 ToE’s II infrastructure description The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. p Agent The Ship Agent is one of the main actors in the port call process. It needs access to the PCS to manage the operations. The systems used for that activity are the following hardware and software.; (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U pro- cessor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system Windows 10 s ved in the “Port Call Request” Process assets and the overall technical equipment required to cover com- aged business partners (BPs) of the underlined SCS process; data systems and interoperable functionalities are also mentioned wher- Description hority manages the PCS, an open and neutral elec- m, that allows a safe and smart information exchange ic and private agents in order to improve the competi- n the port. The is composed of the following elements nd hardware: ystems are hosted on a server HPE ProLiant DL580 a recovery site with all the systems replicated, to en- ability. Extreme Networks SLX 9640 that allows the intercon- N networks and provide access to the system. ting system on the server is a Windows Server 2019 Microsoft with extended security. he data about all the actors involved in the activity and d, it has a relational database Microsoft SQL server has a web application deployed on a web server IIS 10. ccess to all information and documents available and ecessary documents. as its own mail domain that allows direct communica- users. It uses the SMTP server of Microsoft, Exchange n FTP Server for file transferring and data interchange FTP Server supports the following three web services; ocessor Service, Movement Retrieval Service, Data etrieval Service. the security and integrity of the entire system has been he server an antivirus Symantec. nt is one of the main actors in the port call process. It s to the PCS to manage the operations. The systems activity are the following hardware and software.; the PCS, the Ship Agent has a laptop LENOVO Yoga following characteristics: Intel® Core™ i5- 8250U pro- RAM DDR4, and 512 GB SSD storage. C. Pre-arrival Phase C. Pre arrival Phase  C.1 Port Call Request process VTS-SCS Assets involved in the “Port Call Request” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to munications and transactions among the engaged business partners (BPs) of the underlined SCS p exchange services between heterogeneous systems and interoperable functionalities are also men ever exist) Business Partner (BPi) Description Port Authority The Port Authority manages the PCS, an open and neu tronic platform, that allows a safe and smart information between public and private agents in order to improve the tive position in the port. The is composed of the following of software and hardware: (a) All PCS systems are hosted on a server HPE ProLia Gen10. It has a recovery site with all the systems replicat sure the availability. (b) A router Extreme Networks SLX 9640 that allows the nection of LAN networks and provide access to the system (c) The operating system on the server is a Windows Se developed by Microsoft with extended security. (d) To store the data about all the actors involved in the a data produced, it has a relational database Microsoft SQ 2019. (e) The PCS has a web application deployed on a web serv This allows access to all information and documents ava provide the necessary documents. (f) The PCS has its own mail domain that allows direct co tion with the users. It uses the SMTP server of Microsoft, Server 2019. (g) There is an FTP Server for file transferring and data in via PCS. The FTP Server supports the following three web Message Processor Service, Movement Retrieval Serv Movement Retrieval Service. (h) To ensure the security and integrity of the entire system installed on the server an antivirus Symantec. Ship Agent The Ship Agent is one of the main actors in the port call p needs access to the PCS to manage the operations. The used for that activity are the following hardware and softw (a) To access the PCS, the Ship Agent has a laptop LENO C930 with the following characteristics: Intel® Core™ i5- 8 cessor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, W x64. 5.1.2.3 ToE’s II infrastructure description 5.1.2.3 ToE s II infrastructure description Since ToE II adopts a technical view, assets used for the VTS-SCS processes execution will be presented. The VTS-business partners (BPs) that participate in the SCS processes of the Pre- Arrival phase and Vessel Arrival, Vehicles Unloading and Delivery phases, were asked to report all assets used in the VTS processes. As mentioned previously, in the current document, only high-level infrastructure representations of the BPs that encompass these assets are displayed. The under examination SCS processes according to which the infrastructure representations are depicted are enlisted below adopting the template structure that is stressed in the beginning of section 5.1: 952690 CYRENE PROJECT PARTNERS 31/05/2021 98 Supply Chain Analysis and Requirements -arrival Phase C.1 Port Call Request process VTS-SCS Assets involved in the “Port Call Request” Process astructure representations of the cyber assets and the overall technical equipment required to cover com- cations and transactions among the engaged business partners (BPs) of the underlined SCS process; data ange services between heterogeneous systems and interoperable functionalities are also mentioned wher- exist) Business Partner (BPi) Description t Authority The Port Authority manages the PCS, an open and neutral elec- tronic platform, that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware: (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has a recovery site with all the systems replicated, to en- sure the availability. (b) A router Extreme Networks SLX 9640 that allows the intercon- nection of LAN networks and provide access to the system. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct communica- tion with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data interchange via PCS. C. Pre-arrival Phase  C.1 Port Call Request process VTS-SCS Assets involved in the “Port Call Request” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners (BPs) of the underlined SCS process; data exchange services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) ( ) Port Authority Port Authority The Port Authority manages the PCS, an open and neutral elec- tronic platform, that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware: (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has a recovery site with all the systems replicated, to en- sure the availability. (b) A router Extreme Networks SLX 9640 that allows the intercon- nection of LAN networks and provide access to the system. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct communica- tion with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data interchange via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (g) There is an FTP Server for file transferring and data interchange via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. 952690 CYRENE PROJECT PARTNERS 31/05/2021 99 D2.1 – Supply Chain Analysis and Requirements (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mi- crosoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivirus against viruses and malware software. Terminal Operator The systems needed by the Terminal Operator in order to access the PCS and manage the documental activity are the following hard- ware and software. (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. Customs Customs needs to receive the request to start the customs clearance of goods process. The systems used for that activity are the follow- ing hardware and software. (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. Table 33 - Identified infrastructures of the “Port Call Request” process. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mi- crosoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivirus against viruses and malware software. The systems needed by the Terminal Operator in order to access the PCS and manage the documental activity are the following hard- ware and software. (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. Customs needs to receive the request to start the customs clearance of goods process. The systems used for that activity are the follow- ing hardware and software. (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. Table 33 - Identified infrastructures of the “Port Call Request” process.  C.2 Standard Cargo Manifest process VTS-SCS Assets involved in the of the “Standard Cargo Manifest” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged BPs of the underlined SCS process; data exchange services between heterogeneous systems and interoperable functionalities are also mentioned wherever exist) Business Partner (BPi) Description Port Authority The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware:  C.2 Standard Cargo Manifest process VTS-SCS Assets involved in the of the “Standard Cargo Manifest” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged BPs of the underlined SCS process; data exchange services between heterogeneous systems and interoperable functionalities are also mentioned wherever exist) Business Partner (BPi) Description Port Authority The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. Table 33 - Identified infrastructures of the “Port Call Request” process. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Ship Agent The Ship Agent is one of the main actors in the Standard cargo man- ifest process. It needs access to the PCS to manage the operations. The systems used for that activity are the following hardware and software: (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Customs Customs needs to receive the request to manage the customs clear- ance of goods process. The systems used for that activity are the following hardware and software: (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. The is composed of the following elements of software and hardware: The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware: 952690 CYRENE PROJECT PARTNERS 31/05/2021 100 D2.1 – Supply Chain Analysis and Requirements It has recovery cated, to ensure the availabilit (b) A router Extreme Networks S connection of LAN networks a tem. (c) The operating system on the 2019 developed by Microsoft (d) To store the data about all the and data produced, it has a SQL server 2019. (e) The PCS has a web applicat IIS 10. This allows access to available and provide the nec (f) The PCS has its own mail dom nication with the users. It uses Exchange Server 2019. (g) There is an FTP Server for f change via PCS. The FTP three web services; Message Retrieval Service, Data Movem (h) To ensure the security and int been installed on the server a Ship Agent The Ship Agent is one of the main act ifest process. It needs access to the P The systems used for that activity a software: (a) To access the PCS, the Ship Yoga C930 with the following i5- 8250U processor, 8GB R storage. (b) The laptop has the last Micros 10 x64. (c) The Ship Agent uses Microsof to manage and create their do (d) The browser chosen to acces Microsoft Edge. (e) The shipping agent’s laptop is rus against viruses and malwa Customs Customs needs to receive the reques ance of goods process. The systems following hardware and software: (a) To receive the information f server Dell PE R740. (b) The operating system on the Server 2019. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Ship Agent The Ship Agent is one of the main actors in the Standard cargo man- ifest process. It needs access to the PCS to manage the operations. The systems used for that activity are the following hardware and software: (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Customs Customs needs to receive the request to manage the customs clear- ance of goods process. The systems used for that activity are the following hardware and software: (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (a) All PCS systems are hosted DL580 Gen10. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. The Ship Agent is one of the main actors in the Standard cargo man- ifest process. It needs access to the PCS to manage the operations. The systems used for that activity are the following hardware and software: (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Customs needs to receive the request to manage the customs clear- ance of goods process. The systems used for that activity are the following hardware and software: 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 101 (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. D2.1 – Supply Chain Analysis and Requirements (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. Table 34 - Identified infrastructures of the “Standard Cargo Manifest” process. (c) Customs uses Microsoft Office 365 and Adobe Reader manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud a tivirus against viruses and malware software. Table 34 - Identified infrastructures of the “Standard Cargo Manifest” process.  C.3 Entry Summary Declaration (ENS) process VTS-SCS Assets involved in the “Entry Summary Declaration (ENS)” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover co munications and transactions among the engaged business partners of the underlined SC process; data change services between heterogeneous systems and interoperable functionalities are also mentioned wh ever exist) Business Partner (BPi) Description Port Authority The Port Authority is managing the PCS, an open and neutral ele tronic platform that allows a safe and smart information exchan between public and private agents in order to improve the compe tive position in the port. The is composed of the following elemen of software and hardware. (a) All PCS systems are hosted on a server HPE ProLia DL580 Gen10. It has recovery site with all the systems rep cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the int connection of LAN networks and provide access to the sy tem. (c) The operating system on the server is a Windows Serv 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activ and data produced, it has a relational database Micros SQL server 2019. (e) The PCS has a web application deployed on a web serv IIS 10. This allows access to all information and documen available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct comm nication with the users. It uses the SMTP server of Microso Exchange Server 2019. (g) There is an FTP Server for file transferring and data int change via PCS. The FTP Server supports the followi three web services; Message Processor Service, Moveme Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system h been installed on the server an antivirus Symantec. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. Table 34 - Identified infrastructures of the “St  C.3 Entry Summary Declaration (ENS) proces VTS-SCS Assets involved in the “Entry Sum (Infrastructure representations of the cyber assets and the ov munications and transactions among the engaged business change services between heterogeneous systems and intero ever exist) Business Partner (BPi) Port Authority The Port Authority is manag tronic platform that allows a between public and private tive position in the port. The of software and hardware. (a) All PCS systems a DL580 Gen10. It has cated, to ensure the (b) A router Extreme Ne connection of LAN n tem. (c) The operating syste 2019 developed by M (d) To store the data ab and data produced, SQL server 2019. (e) The PCS has a web IIS 10. This allows a available and provid (f) The PCS has its own nication with the use Exchange Server 20 (g) There is an FTP Se change via PCS. T three web services; Retrieval Service, Da (h) To ensure the secur been installed on the Table 34 - Identified infrastructures of the “Standard Cargo Manifest” process.  C.3 Entry Summary Declaration (ENS) process VTS-SCS Assets involved in the “Entry Summary Declaration (ENS)” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Port Authority The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security.  C.3 Entry Summary Declaration (ENS) process  C.3 Entry Summary Declaration (ENS) process Business Partner The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. 952690 CYRENE PROJECT PARTNERS 31/05/2021 102 (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. able 35 - Identified infrastructures of the “Entry Summary Declaration (ENS)” process. Table 35 Identified infrastructures of the Entry Summary Declaration (ENS) process.  C.4 Loading and Discharge List process VTS-SCS Assets involved in the “Loading and Discharge List” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Port Authority The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability.  C.4 Loading and Discharge List process  C.4 Loading and Discharge List process Table 35 - Identified infrastructures of the “Entry Summary Declaration (ENS)” process. D2.1 – Supply Chain Analysis and Requirements Ship Agent The Ship Agent is one of the main actors in the Entry Summary Dec- laration process. It needs access to the PCS to manage the opera- tions. The systems used for that activity are the following hardware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Customs Customs needs to receive the request to manage the customs clear- ance of goods process. The systems used for that activity are the following hardware and software. (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. Table 35 - Identified infrastructures of the “Entry Summary Declaration (ENS)” process. Ship Agent The Ship Agent is one of the main actors in the Entry Summary Dec- laration process. It needs access to the PCS to manage the opera- tions. The systems used for that activity are the following hardware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Customs needs to receive the request to manage the customs clear- ance of goods process. The systems used for that activity are the following hardware and software. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Ship Agent The Ship Agent is one of the main actors in the Loading and dis- charge list process. It needs access to the PCS to manage the op- erations. The systems used for that activity are the following hard- ware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Terminal Operator The systems needed by the Terminal Operator in order to access the PCS and manage the documental activity are the following hard- ware and software. (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents.  C.4 Loading and Discharge List process The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. The is composed of the following elements of software and hardware. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. 952690 CYRENE PROJECT PARTNERS 103 31/05/2021 D2.1 – Supply Chain Analysis and Requirements (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. The Ship Agent is one of the main actors in the Loading and dis- charge list process. It needs access to the PCS to manage the op- erations. The systems used for that activity are the following hard- ware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. The systems needed by the Terminal Operator in order to access the PCS and manage the documental activity are the following hard- ware and software. (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. Table 36 - Identified infrastructures of the “Loading and Discharge List” process. D. Vessel Arrival, Vehicles Unloading and Delivery Phase  D1. Discharge Vehicles process VTS-SCS Assets involved in the “Discharge Vehicles” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Terminal Operator The systems needed by the Terminal Operator in order to manage the Discharge vehicles process are the following hardware and soft- ware. (a) To manage the operations, the Terminal Operator has a lap- top HP x360 1040 G6 with the following characteristics: In- tel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. (f) Dckers exchange information with a MUNBYN PDA Android 8.1 Honeywell with barcode scanner. (g) TOS systems is hosted on a server Lenovo ThinkSystem SR665. Table 37 - Identified infrastructures of the “Discharge Vehicles” process Table 37 - Identified infrastructures of the “Discharge Vehicles” process. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. 952690 CYRENE PROJECT PARTNERS 31/05/2021 104  D1. Discharge Vehicles process VTS-SCS Assets involved in the “Discharge Vehicles” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) The systems needed by the Terminal Operator in order to manage the Discharge vehicles process are the following hardware and soft- ware. (a) To manage the operations, the Terminal Operator has a lap- top HP x360 1040 G6 with the following characteristics: In- tel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. (f) Dckers exchange information with a MUNBYN PDA Android 8.1 Honeywell with barcode scanner. (g) TOS systems is hosted on a server Lenovo ThinkSystem SR665. D2.1 – Supply Chain Analysis and Requirements (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. (d) The browser chosen to access the PCS web application is Google Chrome. Table 37 - Identified infrastructures of the “Discharge Vehicles” process. Table 37 - Identified infrastructures of the “Discharge Vehicles” process. D2.1 – Supply Chain Analysis and Requirements  D.2 Customs Declarations process  D.2 Customs Declarations process 952690 CYRENE PROJECT PARTNERS 105 31/05/2021  D.2 Customs Declarations process VTS-SCS Assets involved in the “Customs Declarations” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description VTS-SCS Assets involved in the “Customs Declarations” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 105 Port Authority (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. Freight Forwarder The systems needed by the Freigt Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware. (a) To send the information the Freight Fowarder has a laptop Asus ZenBook UX325EA-EG016T with the following charac- teristics: Intel® Core™ i5- 1135G7 processor, 8GB RAM and 512 GB SSD storage. (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Customs systems are managing the Customs declarations requests. The systems used for that activity are the following hardware and software: (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. The systems needed by the Freigt Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware. The systems needed by the Freigt Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware. Port Authority (a) To send the information the Freight Fowarder has a laptop Asus ZenBook UX325EA-EG016T with the following charac- teristics: Intel® Core™ i5- 1135G7 processor, 8GB RAM and 512 GB SSD storage. The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. It receives Single Administrative Document request response from Customs. The is composed of the following elements of software and hardware: tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. It receives Single Administrative Document request response from Customs. The is composed of the following elements of software and hardware: (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Customs Customs systems are managing the Customs declarations requests. The systems used for that activity are the following hardware and software: (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. Port Authority 52690 CYRENE PROJECT PARTNERS 106 31/05/2021 Port Authority The Port Authority is managing the PCS, an open and neutral elec- tronic platform that allows a safe and smart information exchange between public and private agents in order to improve the competi- tive position in the port. It receives Single Administrative Document request response from Customs. The is composed of the following elements of software and hardware: (a) All PCS systems are hosted on a server HPE ProLiant DL580 Gen10. It has recovery site with all the systems repli- cated, to ensure the availability. (b) A router Extreme Networks SLX 9640 that allows the inter- connection of LAN networks and provide access to the sys- tem. (c) The operating system on the server is a Windows Server 2019 developed by Microsoft with extended security. (d) To store the data about all the actors involved in the activity and data produced, it has a relational database Microsoft SQL server 2019. (e) The PCS has a web application deployed on a web server IIS 10. This allows access to all information and documents available and provide the necessary documents. (f) The PCS has its own mail domain that allows direct commu- nication with the users. It uses the SMTP server of Microsoft, Exchange Server 2019. (g) There is an FTP Server for file transferring and data inter- change via PCS. The FTP Server supports the following three web services; Message Processor Service, Movement Retrieval Service, Data Movement Retrieval Service. (h) To ensure the security and integrity of the entire system has been installed on the server an antivirus Symantec. Customs Customs systems are managing the Customs declarations requests. The systems used for that activity are the following hardware and software: (a) To receive the information from the PCS Customs has a server Dell PE R740. (b) The operating system on the server is a Microsoft Windows Server 2019. (c) Customs uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The server is protected with a Kaspersky Security Cloud an- tivirus against viruses and malware software. Freight Forwarder The systems needed by the Freigt Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware. Port Authority (a) To send the information the Freight Fowarder has a laptop Asus ZenBook UX325EA-EG016T with the following charac- teristics: Intel® Core™ i5- 1135G7 processor, 8GB RAM and 512 GB SSD storage. 952690 CYRENE PROJECT PARTNERS 31/05/2021 106 D2.1 – Supply Chain Analysis and Requirements (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Freight Fowarder uses Microsoft Office 365 and and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mozilla Firefox. (e) The Freight Fowarder’s laptop is protected with a McAfee Total Protection antivirus against viruses and malware soft- ware. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Freight Fowarder uses Microsoft Office 365 and and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mozilla Firefox. (e) The Freight Fowarder’s laptop is protected with a McAfee Total Protection antivirus against viruses and malware soft- ware. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Freight Fowarder uses Microsoft Office 365 and and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mozilla Firefox. (e) The Freight Fowarder’s laptop is protected with a McAfee Total Protection antivirus against viruses and malware soft- ware.  D.3 Transportation Order process VTS-SCS Assets involved in the “Transportation Order” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Ship Agent The Ship Agent is one of the actors in the Transportation order pro- cess. It needs to exchange documents with other stakeholders in- volved to manage the road operations. The systems used for that activity are the following hardware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Terminal Operator The systems needed by the Terminal Operator in order to manage the documental activity are the following hardware and software: (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. Business Partner (BPi) Description Ship Agent The Ship Agent is one of the actors in the Transportation order pro- cess. It needs to exchange documents with other stakeholders in- volved to manage the road operations. The systems used for that activity are the following hardware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. Table 38 - Identified infrastructures of the “Customs Declarations” processs. (e) The Terminal Op 360 antivirus aga Freight Forwarder The systems needed by t age the documental act ware: (a) To send the infor Asus ZenBook UX teristics: Intel® Co 512 GB SSD stor (b) The laptop has th 10 x64. (c) The Freight Fowa Reader to manag (d) The browser cho Mozilla Firefox. (e) The Freight Fow Total Protection a ware. Haulier Company The Haulier Company is needs access to the orde this document is the nece enter the port premises. following hardware and s (a) To access the PC A515-54-72TH w Core™ i7- 10510 storage. (b) The laptop has th 10 x64. (c) The Haulier uses Reader to manag (d) The browser cho Google Chrome. (e) The haulier’s lap tyZone antivirus a (f) The haulier need has to have the Brother HL- L237 Table 39 - Identified infrastructures of th The business process models of the current VTS- already developed in section 5.1.1.2 and as they re fulfilled in this section. (d) The browser chosen to access the PCS web application is Google Chrome. (d) The browser chosen to access the PCS web application is Google Chrome. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software The systems needed by the Freight Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware: The systems needed by the Freight Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware: (a) To send the information the Freight Forwarder has a laptop Asus ZenBook UX325EA-EG016T with the following charac- teristics: Intel® Core™ i5- 1135G7 processor, 8GB RAM and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Freight Fowarder uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mozilla Firefox. (e) The Freight Fowarder’s laptop is protected with a McAfee Total Protection antivirus against viruses and malware soft- ware. The Haulier Company is involved in the transport operations, so it needs access to the order of transportation document.  D.3 Transportation Order process (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. Terminal Operator The systems needed by the Terminal Operator in order to manage the documental activity are the following hardware and software: (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. The Ship Agent is one of the actors in the Transportation order pro- cess. It needs to exchange documents with other stakeholders in- volved to manage the road operations. The systems used for that activity are the following hardware and software activity are the following hardware and software. (a) To access the PCS, the Ship Agent has a laptop LENOVO Yoga C930 with the following characteristics: Intel® Core™ i5- 8250U processor, 8GB RAM DDR4, and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Ship Agent uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Microsoft Edge. (e) The shipping agent’s laptop is protected with an AVG antivi- rus against viruses and malware software. The systems needed by the Terminal Operator in order to manage the documental activity are the following hardware and software: (a) To access the PCS, the Terminal Operator has a laptop HP x360 1040 G6 with the following characteristics: Intel® Core™ i5-8265U processor, 8GB RAM DDR4, and 256 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Terminal Operator uses Microsoft Office 365 and Adobe Reader to manage and create their documents. 952690 CYRENE PROJECT PARTNERS 31/05/2021 107 D2.1 – Supply Chain Analysis and Requirements (d) The browser chosen to access the PCS web application is Google Chrome. (e) The Terminal Operator’s laptop is protected with a Norton 360 antivirus against viruses and malware software. Freight Forwarder The systems needed by the Freight Fowarder to exchange and man- age the documental activity are the following hardware and soft- ware: (a) To send the information the Freight Forwarder has a laptop Asus ZenBook UX325EA-EG016T with the following charac- teristics: Intel® Core™ i5- 1135G7 processor, 8GB RAM and 512 GB SSD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Freight Fowarder uses Microsoft Office 365 and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Mozilla Firefox. (e) The Freight Fowarder’s laptop is protected with a McAfee Total Protection antivirus against viruses and malware soft- ware. Haulier Company The Haulier Company is involved in the transport operations, so it needs access to the order of transportation document. Furthermore this document is the necessary justification to pick up the goods and enter the port premises. The systems used for that activity are the following hardware and software. (a) To access the PCS the haulier has a laptop Acer Aspire 3 A515-54-72TH with the following characteristics: Intel® Core™ i7- 10510U processor, 8GB RAM and 256GB HDD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Haulier uses Microsoft Office 2016 and and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The haulier’s laptop is protected with a Bitdefender Gravi- tyZone antivirus against viruses and malware software. (f) The haulier needs a printer because the driver of the truck has to have the order of transportation printed. It has a Brother HL- L2370DN. Table 39 - Identified infrastructures of the “Transportation Order” process. The business process models of the current VTS-ToE II SCS respective processes have bee already developed in section 5.1.1.2 and as they remain the same no other actions required to b ulfilled in this section. (d) The browser cho Google Chrome. Table 39 - Identified infrastructures of the “Transportation Order” process. D2.1 – Supply Chain Analysis and Requirements 5.1.3 ToE III: Sectorial view of the VTS-SCS Furthermore this document is the necessary justification to pick up the goods and enter the port premises. The systems used for that activity are the following hardware and software. (a) To access the PCS the haulier has a laptop Acer Aspire 3 A515-54-72TH with the following characteristics: Intel® Core™ i7- 10510U processor, 8GB RAM and 256GB HDD storage. (b) The laptop has the last Microsoft operating system, Windows 10 x64. (c) The Haulier uses Microsoft Office 2016 and and Adobe Reader to manage and create their documents. (d) The browser chosen to access the PCS web application is Google Chrome. (e) The haulier’s laptop is protected with a Bitdefender Gravi- tyZone antivirus against viruses and malware software. (f) The haulier needs a printer because the driver of the truck has to have the order of transportation printed. It has a Brother HL- L2370DN. The business process models of the current VTS-ToE II SCS respective processes have been already developed in section 5.1.1.2 and as they remain the same no other actions required to be fulfilled in this section. 108 31/05/2021 952690 CYRENE PROJECT PARTNERS 31/05/2021 108 Table 43 - “Vehicle Assembly” process description. Table 43 - “Vehicle Assembly” process description. This process is related to the transportation of finished components from the warehouse where they have been stored after intermediate assembly to the car manufacturing plant, where they are assembled with the rest of vehicle parts. This process involves the following operations: transportation documents arrangement, loading of components on containers, truck formalities arrangement, loading of containers on the truck. The shipment is executed by an external supply service provider, while the management of the whole process is by FCA Supply Chain Management Area. Table 41 - “Supply of finished components” process description. 5.1.3 ToE III: Sectorial view of the VTS-SCS “ToE III” reflects sector-specific processes of automotive manufacturer, and its SC assets that hosts and uses in order to participate in the VTS entire SCS. “ToE III” sets the scope to evaluate SC processes under a sectorial perspective that goes to a further analysis of the ICT infrastructure operating within these processes. As for the previous ToE, the current section encompasses the business environment of ToE III: processes, involved business partners and SC stakeholders and the ICT infrastructure they utilize for the completion of these processes. The processes under examination that will be analysed are enlisted below adopting the template structure that is stressed in the beginning of the section 5.1. The SC processes described here are related to the Inbound Logistics phase of components shipment, from the assembly plant to the production plant, and they are listed below: 1. Supply of partial-assembled components 2. Supply of the finished components 3. Monitoring of components during transportation 4. Vehicle Assembly 1. Supply of partial-assembled components 2. Supply of the finished components 3. Monitoring of components during transportation 4. Vehicle Assembly ToE III reflects the CYRENE sectorial SC perspective. It is linked to the Vehicle Transport SC and to the related Vehicle Transport Service (VTS) defined in the introduction of section 5.1. 5.1.3.1 Identification and description of SCS business processes of ToE III Within this section, the business processes for the Inbound Logistics phase are specified.  1 Supply of partial-assembled components The process involves the steps for the supply and execution of the final assembly of partial- assembled parts provided by external suppliers. The current process consists in all the op- erations needed to supply the pre-assembled parts and perform the final assembly, check their quality and store them before shipping them to the car manufacturing plant. Not all parts need an intermediate assembly and in that case they are directly supplied from the components supplier plant to the vehicle production plant. 952690 CYRENE PROJECT PARTNERS 109 31/05/2021 Table 40 - Supply of partial-assembled components process description.  2 Supply of finished components ToE “Supply of finished components” process Table 40 - Supply of partial-assembled components process description. Table 40 - Supply of partial-assembled components process description. Table 40 - Supply of partial-assembled components process description.  2 Supply of finished components 952690 CYRENE PROJECT PARTNERS 109 31/05/2021 D2.1 – Supply Chain Analysis and Requirements  4 Vehicle Assembly The components are received, assembled and the final quality is checked in the produc- tion plant. Main steps in this process regards the unloading of components, the registra- tion in the plant internal systems and the vehicle manufacturing. ToE “Monitoring of components during transportation” process (Α general description of the business process and its business goal) During the Inbound Logistics, IoT are used to track, trace, and monitor the quality of components transported on containers from the supplier plant to the vehicle production plant. The main objective for the use of these devices is the monitoring of containers parameters during transportation from the assembly plant to the FCA production plant. The current process happens in parallel with the previous one. Table 42 - “Monitoring of components” process description.  4 Vehicle Assembly ToE “Vehicle Assembly” process (Α general description of the business process and its business goal) The components are received, assembled and the final quality is checked in the produc- tion plant. Main steps in this process regards the unloading of components, the registra- tion in the plant internal systems and the vehicle manufacturing. Table 42 - “Monitoring of components” process description. Table 42 - “Monitoring of components” process description. Table 42 - “Monitoring of components” process description. 5.1.3.2 Identification and description of SCS business partners of ToE III In the current section, the SC processes identified in section 5.1.3.1 are further analyzed into consequent steps and the business partners involved in these processes for the provision of the Inbound Logistics are specified along with their business roles.  1 Supply of partial-assembled components 952690 CYRENE PROJECT PARTNERS 110 D2.1 – Supply Chain Analysis and Requirements (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Vehicle plant SC Manager, Warehouse, Supply service provider This process starts with the request of components supply by the SC manager. The request is received and then the seal needed for the transportation of the material is produced. The information on the warehouse stock is updated and then the Supply service provider pro- ceed with the loading of containers on the trucks. After all documentation is ready and the truck has been loaded, the transportation towards the final assembly plant starts. 952690 CYRENE PROJECT PARTNERS 111 31/05/2021 p y pp y p p  3 Monitoring of components during transportation ToE “Monitoring of components during transportation” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) Participating in the process (Record business partners’ entities) 952690 CYRENE PROJECT PARTNERS 111 31/05/2021  3 Monitoring of components during transportation ToE “Monitoring of components during transportation” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) Participating in the process (Record business partners’ entities) ( p ) Components Supplier, Intermediate assembly plant, Vehicle Plant SC Manager, Warehouse Components Supplier, Intermediate assembly plant, Vehicle Plant SC Manager, Warehouse This process is executed in the Intermediate Assembly plant. Here, the Supply Chain Man- ager makes order of partial assembled components, according to the production plant de- mand. Then, the external supplier responds to the request, sending the material to the plant where the parts assembly is executed. Finally, after the components are finished, they are stored in a warehouse, before they are shipped to the manufacturing plant, where they are assembled with the other vehicle parts. Table 44 - Business partners identification and analysis of the “Supply of partial-assembled components” process.  2 Supply of finished components ToE “Supply of finished components” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) Participating in the process (Record business partners’ entities) Vehicle plant SC Manager, Warehouse, Supply service provider Description (Process analysis describing how the business partners are involved in the SC process) This process starts with the request of components supply by the SC manager. The request is received and then the seal needed for the transportation of the material is produced. The information on the warehouse stock is updated and then the Supply service provider pro- ceed with the loading of containers on the trucks. After all documentation is ready and the truck has been loaded, the transportation towards the final assembly plant starts. Table 45 - Business partners identification and analysis of the “Supply of finished components” process.  3 Monitoring of components during transportation ToE “Monitoring of components during transportation” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Table 44 - Business partners identification and analysis of the “Supply of partial-assembled components” process. ( ) Vehicle assembly plant, Vehicle plant SC Management As soon as the components arrive at the final assembly plant, the SC manager updates information about the stock available. The components are registered in the internal sys- tems of the plant. Then their quality is checked and if the control is passed, they proceed to the production, where they will be assembled with the other parts of the vehicle. Table 47 - Business partners identification and analysis of the “Vehicle Assembly” process. D2.1 – Supply Chain Analysis and Requirements Supply Chain Manager, Vehicle Assembly plant D i ti The FCA Supply Chain Manager is the only actor in the monitoring process. He needs to access a web application to monitor the behavior of containers’ parameters and localization. By using this system, he/she can have knowledge about events that could endanger the quality of the transported material, i.e.: high vibrations or temperature shocks, and also he/she can be informed about the position of the truck, and then update the Estimated Time of Arrival (ETA). Thanks to the near-real time data about quality conditions and localization of the material during the shipment, the final assembly plant is able to schedule and/or reschedule, if needed, the production. Table 46 - Business partners identification and analysis of the “Monitoring of components during transportation” process. Table 46 - Business partners identification and analysis of the “Monitoring of components during transportation” process.  4 Vehicle Assembly ToE “Vehicle Assembly” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) Participating in the process (Record business partners’ entities) Vehicle assembly plant, Vehicle plant SC Management Description (Process analysis describing how the business partners are involved in the SC process) As soon as the components arrive at the final assembly plant, the SC manager updates information about the stock available. The components are registered in the internal sys- tems of the plant. Then their quality is checked and if the control is passed, they proceed to the production, where they will be assembled with the other parts of the vehicle. Table 47 - Business partners identification and analysis of the “Vehicle Assembly” process.  3 Monitoring of components during transportation  3 Monitoring of components during transportation ToE “Monitoring of components during transportation” Business Partne (Process analysis, identifying and describing all the business partners participated in the current u SC process and their roles within the process (taking into account the cyber assets operating to sup interconnections) 952690 CYRENE PROJECT PARTNERS 111 5.1.3.3 ToE’s infrastructure description Through this section, high level ICT infrastructure representations that are used by business part- ners who are involved in the identified SC processes are provided.  4 Vehicle Assembly  4 Vehicle Assembly ToE “Vehicle Assembly” Business Partners (Process analysis, identifying and describing all the business partners participated in the current underlined SC process and their roles within the process (taking into account the cyber assets operating to support their interconnections) Business Partners (BPi) Participating in the process (Record business partners’ entities) Vehicle assembly plant, Vehicle plant SC Management ToE “Vehicle Assembly” Business Partners (Process analysis, identifying and describing all the business partners participated in the current u SC process and their roles within the process (taking into account the cyber assets operating to sup interconnections)  1 Supply of partial-assembled components Warehouse Here the main actors are operators who provide to organize and ex- ecute procedures and manual operations for the material handling. The system utilized for managing data about warehouse material is the Warehouse Management System. (a) This system is hosted on a server. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. Table 48 - Identified infrastructures of the “Supply of partial-assembled components” process Table 48 - Identified infrastructures of the “Supply of partial-assembled components” proces D2.1 – Supply Chain Analysis and Requirements up-The asset utilized by the components supply in this process is the so called “eSupplierConnect”. It is a portal providing useful features to improve performance, collaboration and communication between FCA and supplier partners. It allows this BP to receive orders by the SC Manager requiring the material. (a) This system is hosted on a database. (a) This system is hosted on a database. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) It is reachable through a laptop. (d) To ensure the security and integrity of the entire system, FCA internal regulations in terms of ICT security are followed. Intermediate assem- bly plant In the Intermediate Assembly plant, the main assets utilized are the Manufacturing Execution System (MES) and the Material Require- ments Planning (MRP). The first one is used to track and document all the steps of the transformation of raw materials to finished com- ponents, while the second one is used for process planning, sched- uling and inventory control. The hardware required for these sys- tems are servers with high computational capacity. In fact, they lev- erage on optimization algorithms that uses a lot of input parameters in order to organize, plan, schedule, reschedule, register all the as- sembly materials and operations, maximizing efficiency and improv- ing the assembly output. ing the assembly output. Vehicle Plant SC Manager This BP is the actor who starts the whole process. He utilizes the eSupplierConnect portal, that has been already described above, to make the demand for material supply. Warehouse Here the main actors are operators who provide to organize and ex- ecute procedures and manual operations for the material handling. The system utilized for managing data about warehouse material is the Warehouse Management System. (a) This system is hosted on a server. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. Vehicle Plant SC Manager This BP is the actor who starts the whole process. He utilizes the eSupplierConnect portal, that has been already described above, to make the demand for material supply.  1 Supply of partial-assembled components  1 Supply of partial-assembled components ToE infrastructures of the “Supply of partial-assembled components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description ToE infrastructures of the “Supply of partial-assembled components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description 952690 CYRENE PROJECT PARTNERS 112 2.1 – Supply Chain Analysis and Requirements Components Sup- plier The asset utilized by the components supply in this process is the so called “eSupplierConnect”. It is a portal providing useful features to improve performance, collaboration and communication between FCA and supplier partners. It allows this BP to receive orders by the SC Manager requiring the material. (a) This system is hosted on a database. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) It is reachable through a laptop. (d) To ensure the security and integrity of the entire system, FCA internal regulations in terms of ICT security are followed. Intermediate assem- bly plant In the Intermediate Assembly plant, the main assets utilized are the Manufacturing Execution System (MES) and the Material Require- ments Planning (MRP). The first one is used to track and document all the steps of the transformation of raw materials to finished com- ponents, while the second one is used for process planning, sched- uling and inventory control. The hardware required for these sys- tems are servers with high computational capacity. In fact, they lev- erage on optimization algorithms that uses a lot of input parameters in order to organize, plan, schedule, reschedule, register all the as- sembly materials and operations, maximizing efficiency and improv- ing the assembly output. Vehicle Plant SC Manager This BP is the actor who starts the whole process. He utilizes the eSupplierConnect portal, that has been already described above, to make the demand for material supply. Warehouse H th i t t h id t i d Here the main actors are operators who provide to organize and ex- ecute procedures and manual operations for the material handling. The system utilized for managing data about warehouse material is the Warehouse Management System. (a) This system is hosted on a server. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. 952690 CYRENE PROJECT PARTNERS 31/05/2021 113 31/05/2021 D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 114 31/05/2021 Figure 34 - Business process model for the”Supply of partial-assembled components” process.  2 Supply of finished components ToE infrastructures of the “Supply of finished components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Vehicle Plant SC Manager This BP is the actor who starts this process. He utilizes the eSup- plierConnect portal, that has been already described above, to notify to the SC service provider that the material is ready for pick-up. Warehouse The main tool utilized in the Warehouse in this process is the Trans- portation Management System (TMS), which is a FCA internal sys- tem. Here all the data related to the shipment operations are man- aged and collected and the transport documentation is issued. (a) This system is hosted on a server. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) It is reachable through a laptop. D2.1 – Supply Chain Analysis and Requirements Figure 34 - Business process model for the”Supply of partial-assembled components” process. Figure 34 - Business process model for the”Supply of partial-assembled components” process.  2 Supply of finished components ToE infrastructures of the “Supply of finished components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Vehicle Plant SC Manager This BP is the actor who starts this process. He utilizes the eSup- plierConnect portal, that has been already described above, to notify to the SC service provider that the material is ready for pick-up. Warehouse The main tool utilized in the Warehouse in this process is the Trans- portation Management System (TMS), which is a FCA internal sys- tem. Here all the data related to the shipment operations are man- aged and collected and the transport documentation is issued. (a) This system is hosted on a server. (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) It is reachable through a laptop.  2 Supply of finished components (b) Data about all the actors involved in the activity and data pro- duced are stored in a relational database Microsoft SQL server. (c) It is reachable through a laptop. 952690 CYRENE PROJECT PARTNERS 31/05/2021 114 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements Table 49 - Identified infrastructures of the "Supply of finished components" process. (d) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. Moreover, the WMS is used also in this process, in order to update information on material available in the warehouse. Supply Service pro- vider The main system used by this BP is the eSupplierConnect already described above. In fact, through it, he receives the demand for providing supply service and then, after he sends the information Table 49 - Identified infrastructures of the "Supply of finished components" process. (d) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. Moreover, the WMS is used also in this process, in order to update information on material available in the warehouse. Supply Service pro- vider The main system used by this BP is the eSupplierConnect already described above. In fact, through it, he receives the demand for providing supply service and then, after he sends the information Table 49 - Identified infrastructures of the "Supply of finished components" process. (d) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. Moreover, the WMS is used also in this process, in order to update information on material available in the warehouse. Supply Service pro- vider The main system used by this BP is the eSupplierConnect already described above. In fact, through it, he receives the demand for providing supply service and then, after he sends the information (d) To ensure the security and integrity of the entire system, company internal regulations in terms of ICT security are fol- lowed. Moreover, the WMS is used also in this process, in order to update information on material available in the warehouse. pply Service pro- er The main system used by this BP is the eSupplierConnect already described above. In fact, through it, he receives the demand for providing supply service and then, after he sends the information Table 49 - Identified infrastructures of the "Supply of finished components" process. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 115 D2.1 – Supply Chain Analysis and Requirements Figure 35 - Business process model for the "Supply of finished components" process. Figure 35 - Business process model for the "Supply of finished components" process. Vehicle Assembly Plant The main tool utilized in this process are agement System (TMS), the Warehous (WMS) and the Material Requirements P tures of the hardware needed for using the of the ones listed in the tables above. Table 50 - Identified infrastructures of the "Monitoring of comp Table 50 - Identified infrastructures of the "Monitoring of components" process. He needs access to the i4.0 web application to monitor the behaviour of process parameters. The systems used for that activity are the following hardware and software. (a) He uses the Transport Management System, for the docu- ments and all information related to the material supply (i.e. information on the orders, on the transportation documenta- tion, on the trucks, and so on) (b) Data from the loggers are visualized on the web application (c) To access the web application he needs a laptop and cre- dentials (d) Data can be visualized in real time or downloaded for analy- sis. Downloaded data can be visualized by using Microsoft Excel (e) Data are stored on an external SQL server Loggers with 3G/4G connection are installed on the containers. The wireless connection (3G/4G) has no protection measures (e.g. en- cryption) and the data are sent as clear text. There is only a basic authentication mechanism for local access, and the suppliers have permission to access/modify. Vehicle Assembly Plant The main tool utilized in this process are the Transportation Man- agement System (TMS), the Warehouse Management System (WMS) and the Material Requirements Planning (MRP). The fea- tures of the hardware needed for using these systems are the same of the ones listed in the tables above. Table 50 - Identified infrastructures of the "Monitoring of components" process.  3 Monitoring of components during transportation ToE infrastructures of the “Monitoring of compone (Infrastructure representations of the cyber assets and the overall technical equ munications and transactions among the engaged business partners of the un change services between heterogeneous systems and interoperable functionali ever exist) Business Partner (BPi) Description Supply Chain Man- ager The FCA Supply Chain Manager the m process. He needs access to the i4.0 web a behaviour of process parameters. The syst are the following hardware and software. (a) He uses the Transport Manageme ments and all information related to information on the orders, on the tra tion, on the trucks, and so on) (b) Data from the loggers are visualize (c) To access the web application he dentials (d) Data can be visualized in real time sis. Downloaded data can be visua Excel (e) Data are stored on an external SQL Loggers with 3G/4G connection are installe wireless connection (3G/4G) has no prote cryption) and the data are sent as clear te authentication mechanism for local access permission to access/modify. 952690 CYRENE PROJECT PARTNERS 31/05/2021 116 D2.1 – Supply Chain Analysis and Requirements  3 Monitoring of components during transportation ToE infrastructures of the “Monitoring of components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Supply Chain Man- ager The FCA Supply Chain Manager the main actor in monitoring process. He needs access to the i4.0 web application to monitor the behaviour of process parameters. The systems used for that activity are the following hardware and software. (a) He uses the Transport Management System, for the docu- ments and all information related to the material supply (i.e. information on the orders, on the transportation documenta- tion, on the trucks, and so on) (b) Data from the loggers are visualized on the web application (c) To access the web application he needs a laptop and cre- dentials (d) Data can be visualized in real time or downloaded for analy- sis. Downloaded data can be visualized by using Microsoft Excel (e) Data are stored on an external SQL server Loggers with 3G/4G connection are installed on the containers. The wireless connection (3G/4G) has no protection measures (e.g. en- cryption) and the data are sent as clear text. There is only a basic authentication mechanism for local access, and the suppliers have permission to access/modify. Vehicle Assembly Plant The main tool utilized in this process are the Transportation Man- agement System (TMS), the Warehouse Management System (WMS) and the Material Requirements Planning (MRP). The fea- tures of the hardware needed for using these systems are the same of the ones listed in the tables above. Table 50 - Identified infrastructures of the "Monitoring of components" process.  3 Monitoring of components during transportation ToE infrastructures of the “Monitoring of components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Supply Chain Man- ager The FCA Supply Chain Manager the main actor in monitoring process.  3 Monitoring of components during transportation  Vehicle Assembly ToE infrastructures of the “Vehicle Assembly” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Vehicle Plant SC Manager The systems used here by the BP are the Material Requirements Planning (MRP) and the Transportation Management System (TMS) Vehicle Assembly Plant The tools used in this process are the Transportation Management System (TMS), the Warehouse Management System (WMS) and the Manufacturing Execution System (MES). Table 51 - Identified infrastructures of the "Vehicle Assembly" process. Figure 36 - Business process model for the "Monitoring of components" process. Figure 36 - Business process model for the "Monitoring of components" proces Figure 36 - Business process model for the "Monitoring of components" process. g p g p p  Vehicle Assembly ToE infrastructures of the “Vehicle Assembly” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) Business Partner (BPi) Description Vehicle Plant SC Manager The systems used here by the BP are the Material Requirements Planning (MRP) and the Transportation Management System (TMS) Vehicle Assembly Plant The tools used in this process are the Transportation Management System (TMS), the Warehouse Management System (WMS) and the Manufacturing Execution System (MES). Table 51 - Identified infrastructures of the "Vehicle Assembly" process.  3 Monitoring of components during transportation ToE infrastructures of the “Monitoring of components” Process (Infrastructure representations of the cyber assets and the overall technical equipment required to cover com- munications and transactions among the engaged business partners of the underlined SC process; data ex- change services between heterogeneous systems and interoperable functionalities are also mentioned wher- ever exist) n-The FCA Supply Chain Manager the main actor in monitoring process. He needs access to the i4.0 web application to monitor the behaviour of process parameters. The systems used for that activity are the following hardware and software. (a) He uses the Transport Management System, for the docu- ments and all information related to the material supply (i.e. information on the orders, on the transportation documenta- tion, on the trucks, and so on) (b) Data from the loggers are visualized on the web application (c) To access the web application he needs a laptop and cre- dentials (d) Data can be visualized in real time or downloaded for analy- sis. Downloaded data can be visualized by using Microsoft Excel (e) Data are stored on an external SQL server Loggers with 3G/4G connection are installed on the containers. The wireless connection (3G/4G) has no protection measures (e.g. en- cryption) and the data are sent as clear text. There is only a basic authentication mechanism for local access, and the suppliers have permission to access/modify. Vehicle Assembly Plant The main tool utilized in this process are the Transportation Man- agement System (TMS), the Warehouse Management System (WMS) and the Material Requirements Planning (MRP). The fea- tures of the hardware needed for using these systems are the same of the ones listed in the tables above. 952690 CYRENE PROJECT PARTNERS 31/05/2021 117 D2.1 – Supply Chain Analysis and Requirements D2.1 Supply Chain Analysis and Requirements Figure 36 - Business process model for the "Monitoring of components" process.  Vehicle Assembly 952690 CYRENE PROJECT PARTNERS 31/05/2021 118 D2.1 – Supply Chain Analysis and Requirements Figure 37 - Business process model for the “Vehicle Assembly” process. Figure 37 - Business process model for the “Vehicle Assembly” process. 5.2.1 Requirements for CYRENE ToE I ToE Ι requirements for self-conformity assessment corresponds to the assurance level “basic”, which is EAL 1 level of assurance, as defined in the CC (CC-PART3, 2017). The assurance class for the evaluation will be the Class AVA: Vulnerability Assessment dealing with the first compo- nent of the class, which is AVA_VAN.1 (CC-PART3, 2017). The purpose of the vulnerability as- sessment activity is to identify the exploitability of flaws or weaknesses in the ToE’s environment (CC-PART3, 2017). In this section, the ToE I requirements are presented according to the ToE I business environment description. As defined in section 5.1, ToE I is the “Vehicle Transport Service” (VTS) and refers to the CY- RENE business SC aspect. The Vehicle Transport Service is a complex SCS, including several 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 119 D2.1 – Supply Chain Analysis and Requirements ToE_I_CA_15 ToE I environment identification must be compliant with the Common Criteria Evaluation ToE_I_CA_16 Paperwork of the identified SC processes are in line with the description of ToE I business environment ToE_I_CA_17 The business flow of the SC processes meets the description of ToE I business environment ToE_I_CA_18 If privacy considerations and additional conventions described in ToE I busi- ness environment are fulfilled ToE_I_CA_19 Identification of the security issues of each SC process of the VTS ToE_I_CA_20 Identification of possible threat scenarios that address the defined security is- sue ToE_I_CA_21 Identification of the protection profile according to the ToE I environment Table 52 - Requirements for CYRENE ToE I. 5.2.2 Requirements for CYRENE ToE II D2.1 – Supply Chain Analysis and Requirements collaborating actors coming from different industries and it has been selected regarding its high impact to the EU economy (see section 5.1). According to the scenario described in section 5.1, ToE I business environment refers to the identified SC processes. These processes were decom- posed to a thorough analysis identifying the business partners involved and the business roles they have in each process for the provision of the VTS. On this grounds, CYRENE ToE I concerns self-conformity assessment requirements, which are as follows: Identifier Description ToE_I_CA_1 Set the boundaries of the ToE’s environment (VTS as defined in section 5.1) ToE_I_CA_2 Clarify what will be evaluated (i.e., the identified VTS processes, exploring the actors involved, their interactions, information exchange, business logic) ToE_I_CA_3 Assign a Business Agreement between the involved SC parties with an em- bedded Security Declaration statement ToE_I_CA_4 ToE I must be compliant with the content of the Business Agreement and the embedded Security Declaration statement ToE_I_CA_5 Identify accepted certifications of the business partners involved in the ToE, related to the SC processes they execute for the provision of the VTS ToE_I_CA_6 In case the business partners SCS processes are certified, it must be exam- ined if the flow and the execution of these processes are compliant with the corresponding certification requirements ToE_I_CA_7 The SCS processes must address what has been declared in ToE I business environment ToE_I_CA_8 Clear identification and concrete analysis of the SCS processes content ToE_I_CA_9 Business partners of the VTS participate in the identified SCS processes as defined in ToE I business environment ToE_I_CA_10 Business roles of the participants in each VTS process must be in line with what has been described in ToE I business environment ToE_I_CA_11 Information exchanges in the identified VTS processes must be carried out as it has been described in ToE I business environment ToE_I_CA_12 Business partners interactions undertaken in each process must be in line with what has been described in ToE I business environment ToE_I_CA_13 Each VTS process shall be fulfilled according to the business goal identified for this process in ToE I business environment ToE_I_CA_14 If the organizational business policies (i.e., invoice policy), security policies, security plans and security measures are enforced, the business partners should adopt these processes (declared in the Security Declaration statement) and take care to be implemented properly 952690 CYRENE PROJECT PARTNERS 120 D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 121 31/05/2021 ToE_I_CA_15 ToE I environment identification must be compliant with the Common Criteria Evaluation ToE_I_CA_16 Paperwork of the identified SC processes are in line with the description of ToE I business environment ToE_I_CA_17 The business flow of the SC processes meets the description of ToE I business environment ToE_I_CA_18 If privacy considerations and additional conventions described in ToE I busi- ness environment are fulfilled ToE_I_CA_19 Identification of the security issues of each SC process of the VTS ToE_I_CA_20 Identification of possible threat scenarios that address the defined security is- sue ToE_I_CA_21 Identification of the protection profile according to the ToE I environment Table 52 - Requirements for CYRENE ToE I. 5.2.2 Requirements for CYRENE ToE II TOE II requirements include the technical requirements related to the ICT systems and assets involved in the VTS and are as follows: Identifier Description ToE_II_CA_1 Identification of the compromised assets among all the VTS systems ToE_II_CA_2 All the related activity in the VTS should be auditable ToE_II_CA_3 Ensure data privacy in all the communication with external systems following the European and National legislation ToE_II_CA_4 Ensure confidentiality of the communications through the different channels ToE_II_CA_5 Ensure integrity of the data, so that anybody can not change anything from the message ToE_II_CA_6 Ensure the authenticity of the involved actors avoiding identity fraud ToE_II_CA_7 Set the boundaries of the ToE’s environment (VTS as defined in section 5.1) ToE_II_CA_8 Clarify what will be evaluated (i.e. the identified VTS processes, exploring the actors involved, their interactions, the infrastructure they utilize and the assets they operate for the provision of the VTS) ToE_II_CA_9 Assign a Business Agreement between the involved SC parties with an em- bedded Security Declaration statement 5.2.2 Requirements for CYRENE ToE II involved in the VTS and are as follows: Identifier Description ToE_II_CA_1 Identification of the compromised assets among all the VTS systems ToE_II_CA_2 All the related activity in the VTS should be auditable ToE_II_CA_3 Ensure data privacy in all the communication with external systems following the European and National legislation ToE_II_CA_4 Ensure confidentiality of the communications through the different channels ToE_II_CA_5 Ensure integrity of the data, so that anybody can not change anything from the message ToE_II_CA_6 Ensure the authenticity of the involved actors avoiding identity fraud ToE_II_CA_7 Set the boundaries of the ToE’s environment (VTS as defined in section 5.1) ToE_II_CA_8 Clarify what will be evaluated (i.e. the identified VTS processes, exploring the actors involved, their interactions, the infrastructure they utilize and the assets they operate for the provision of the VTS) ToE_II_CA_9 Assign a Business Agreement between the involved SC parties with an em- bedded Security Declaration statement 952690 CYRENE PROJECT PARTNERS 121 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements ToE_II_CA_10 ToE II must be compliant with the content of the Business Agreement and the embedded Security Declaration statement ToE_II_CA_11 Identify accepted certifications of the business partners involved in VTS-ToE II, related to the SC processes they execute for the provision of the VTS ToE_II_CA_12 Identify accepted certifications of the business partners involved in VTS-ToE II, related to the SC assets they operate for the provision of the VTS ToE_II_CA_13 Identify implemented security controls of the SCS assets of the business part- ners they operate for the provision of the VTS and they are included in the current ToE (and to the signed Security Declaration statement as a conse- quence) ToE_II_CA_14 Identify accepted certifications of the business partners involved in VTS-ToE II, related to the security controls of the SCS assets they operate for the provi- sion of the VTS ToE_II_CA_15 Ensure that after performing the risk assessment in order to implement an op- timal mitigation strategy, all business partners involved will undertake appro- priate security controls (whether and wherever required) in order to reach the VTS desired security level which they have agreed upon through the Security Declaration signed commitment ToE_II_CA_16 In case business partners VTS processes are certified, it must be examined if the flow and the execution of these processes are compliant with the relevant certification requirements ToE_II_CA_17 The VTS processes must address what has been declared in VTS-ToE II en- vironment ToE_II_CA_18 Clear identification and concrete analysis of the VTS processes content ToE_II_CA_19 Business partners of the VTS participate in the identified SCS processes as defined in ToE II business environment ToE_II_CA_20 Business roles of the participants in each VTS process must be in line with what has been described in VTS-ToE II business environment ToE_II_CA_21 Information exchanges in the identified VTS processes must be carried out as it has been described in VTS-ToE II business environment ToE_II_CA_22 Business partners interactions undertaken in each process must be in line with what has been described in VTS-ToE II business environment ToE_II_CA_23 Identify the existing asset interdependencies for the provision of the VTS (within a participated organization and between VTS interacted business part- ners) and check whether they are illustrated in the corresponding VTS pro- cesses according to the description of VTS-ToE II environment ToE_II_CA_24 ICT infrastructure (or parts of the infrastructure) that operate for the provision of the VTS must be in consensus with VTS-ToE II description 952690 CYRENE PROJECT PARTNERS 122 D2.1 – Supply Chain Analysis and Requirements ToE_II_CA_25 Each VTS process shall be fulfilled according to the business goal identified for this process in ToE II business environment ToE_II_CA_26 If the organizational business policies (i.e., invoice policy), security policies, security plans and security measures are enforced, the business partners should adopt these processes (declared in the Security Declaration statement) and take care to be implemented properly ToE_II_CA_27 VTS-ToE II environment identification must be compliant with the Common Cri- teria Evaluation ToE_II_CA_28 Check whether the security assumptions (intended use of the implementation of the ToE) of VTS-ToE II that will be thoroughly analyzed in D2.2 are fulfilled ToE_II_CA_29 VTS-ToE II must be assessed taking into account the key concepts of protec- tion profiles (PP), packages of security requirements, the specified topic of conformance and the consequences of evaluation according to the CC ToE_II_CA_30 VTS-ToE II must address the identified security issue and the described Secu- rity Target (ST) and through the implementation of the CA process examine if the described security objectives are satisfied ToE_II_CA_31 VTS-ToE II meets the privacy considerations determined in the Security Dec- laration statement ToE_II_CA_32 CYRENE CA process for the VTS-ToE II is in line with the content of Class AVA: Vulnerability Assessment dealing with the description of the first compo- nent of the class, which is AVA_VAN.1 (CC-PART3, 2017) Table 53 - Requirements for CYRENE ToE II. for this process in ToE II business environment ToE_II_CA_26 If the organizational business policies (i.e., invoice policy), security policies, security plans and security measures are enforced, the business partners should adopt these processes (declared in the Security Declaration statement) and take care to be implemented properly ToE_II_CA_27 VTS-ToE II environment identification must be compliant with the Common Cri- teria Evaluation ToE_II_CA_28 Check whether the security assumptions (intended use of the implementation of the ToE) of VTS-ToE II that will be thoroughly analyzed in D2.2 are fulfilled ToE_II_CA_29 VTS-ToE II must be assessed taking into account the key concepts of protec- tion profiles (PP), packages of security requirements, the specified topic of conformance and the consequences of evaluation according to the CC ToE_II_CA_30 VTS-ToE II must address the identified security issue and the described Secu- rity Target (ST) and through the implementation of the CA process examine if the described security objectives are satisfied ToE_II_CA_31 VTS-ToE II meets the privacy considerations determined in the Security Dec- laration statement ToE_II_CA_32 CYRENE CA process for the VTS-ToE II is in line with the content of Class AVA: Vulnerability Assessment dealing with the description of the first compo- nent of the class, which is AVA_VAN.1 (CC-PART3, 2017) Table 53 - Requirements for CYRENE ToE II. 5.2.3 Requirements for CYRENE ToE III TOE III requirements include the sectorial requirements related to the ICT systems and assets involved in the Automotive SC and are as follows: Identifier Description ToE_III_CA_1 Identification of the compromised assets among all the Automotive SC sys- tems ToE_III_CA_2 All the related activities in the Automotive SC should be auditable ToE_III_CA_3 Ensure the identification of the vulnerable assets ToE_III_CA_4 Perform impact assessment on top of verified risks ToE_III_CA_5 Ensure data privacy in all the communication with external systems following the European and National legislation ToE_III_CA_6 Ensure confidentiality of the communications through the different channels 5.2.3 Requirements for CYRENE ToE III 123 D2.1 – Supply Chain Analysis and Requirements ToE_III_CA_7 Ensure integrity of the data, so that anybody can not change anything from the message ToE_III_CA_8 Ensure the authenticity of the involved actors avoiding identity fraud ToE_III_CA_9 All occurred risks should be reported ToE_III_CA_10 Perform forecasting of possible threats/attacks ToE_III_CA_11 Dashboards should be available to final users (i.e. for visual inspection and management) ToE_III_CA_12 Mitigation actions should be proposed by the system ToE_III_CA_13 Support to the end-user should be provided in the decision-making process ToE_III_CA_14 Set the boundaries of the ToE’s environment (VTS as defined in section 5.1) ToE_III_CA_15 Clarify what will be evaluated (i.e. the identified Automotive SC processes, ex- ploring the actors involved, their interactions, the infrastructure they utilize and the assets they operate) ToE_III_CA_16 Assign a Business Agreement between the involved SC parties with an em- bedded Security Declaration statement ToE_III_CA_17 ToE III must be compliant with the content of the Business Agreement and the embedded Security Declaration statement ToE_III_CA_18 Identify accepted certifications of the business partners involved in ToE III, re- lated to the SC processes they execute ToE_III_CA_19 Identify accepted certifications of the business partners involved in ToE III, re- lated to the SC assets they operate ToE_III_CA_20 Identify implemented security controls of the SCS assets of the business part- ners they operate for the provision of the Automotive SCS and they are in- cluded in the current ToE III (and to the signed Security Declaration as a con- sequence) ToE_III_CA_21 Identify accepted certifications of the business partners involved in ToE III, re- lated to the security controls of the SCS assets they operate for the provision of the Automotive SC ToE_III_CA_22 Ensure that after performing the risk assessment in order to implement an op- timal mitigation strategy, all business partners involved will undertake appro- priate security controls (whether and wherever required) in order to reach the desired security level which they have agreed upon through the Security Dec- laration signed commitment ToE_III_CA_23 In case business partners Automotive SC processes are certified, it must be examined if the flow and the execution of these processes are compliant with the corresponding certification requirements 952690 CYRENE PROJECT PARTNERS 124 31/05/2021 D2.1 – Supply Chain Analysis and Requirements ToE_III_CA_24 The Automotive SC processes must address what has been declared in ToE III environment ToE_III_CA_25 Clear identification and concrete analysis of the Automotive SC processes con- tent should be performed ToE_III_CA_26 Business partners of the Automotive SC participate in the identified SCS pro- cesses as defined in ToE III business environment ToE_III_CA_27 Business roles of the participants in each Automotive SC process must be in line with what has been described in ToE III business environment ToE_III_CA_28 Information exchanges in the identified Automotive SC processes must be car- ried out as it has been described in ToE III business environment ToE_III_CA_29 Business partners interactions undertaken in each process must be in line with what has been described in ToE III business environment ToE_III_CA_30 Identify the existing asset interdependencies for the provision of the Automo- tive SC and check whether they are illustrated in the corresponding processes according to the description of ToE III environment ToE_III_CA_31 ICT infrastructure (or parts of the infrastructure) that operate for the provision of the Automotive SC must be in consensus with ToE III description ToE_III_CA_32 Each Automotive SC process shall be fulfilled according to the business goal identified for this process in ToE III business environment ToE_III_CA_33 ToE III environment identification must be compliant with the Common Criteria Evaluation Table 54 - Requirements for CYRENE ToE III. 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 125 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 7. 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Online available: https://www.iso.org/obp/ui/#iso:std:iso:28000:ed-1:v1:en 952690 CYRENE PROJECT PARTNERS 31/05/2021 131 Appendix A – Glossary and Examples The glossary is available and continuously updated in the project website59. It is an aggregation of terms and definitions based on different sources, such as Common Criteria and other ISO standards, NIS Directive, EU Cybersecurity Act and other Regulations, ENISA reports, EU Horizon2020 projects, NIST, CVSS, etc. It also contains examples, where necessary, to help the reader obtain a better understanding of these terms and the thin lines that may exist among them. Another objective of the glossary is to integrate all the definitions possible and state their differences, if any, when they refer to the same term. For this purpose, there is a Notes/Remarks column, which the reader can also use for additional reading. This glossary is divided into three main parts: the Security and Certification Concepts, the Supply Chain and Business Concepts and the Maritime Transport Concepts. Table 55, Table 56, and Table 57, are extracts from the three different parts are presented as an example of the glossary contents included in the project website59. 952690 CYRENE PROJECT PARTNERS 132 31/05/2021 Term Abbrevi ation Definition(s) Reference(s) Example(s) Notes/ Remarks Security Concepts Information - Any communication or representation of knowledge such as facts, data, or opinions in any medium or form, including textual, numerical, graphic, cartographic, narrative, or audiovisual. [CNSSI No. 4009] Information system set of applications, services, information technology assets, or other information- handling components [ISO/IEC 27000:2018] An Enterprise Resource Planning (ERP) system Information Security Managemen t System ISMS Set of interrelated or interacting elements of an organization to establish policies and [ISO/IEC 27000:2018] ISO 27001 59 https://www.cyrene.eu/glossary/ 132 D2.1 – Supply Chain Analysis and Requirements objectives and processes to achieve those objectives Confidentiali ty - Property that information is not made available or disclosed to unauthorized individuals, entities, or processes [ISO/IEC 27000:2018] Integrity - Property of accuracy and completeness [ISO/IEC 27000:2018] Availability - Property of being accessible and usable on demand by an authorized entity. [ISO/IEC 27000:2018] Accountabili ty - The state of being answerable (in response) for assigned actions and decisions. [ISO/IEC 27000:2018] Authenticity - Property that an entity is what it claims to be. [ISO/IEC 27000:2018] Reliability - Property of consistent intended behaviour and results [ISO/IEC 27000:2018] Non- repudiation - Ability to prove the occurrence of a claimed event or action and its originating entities [ISO/IEC 27000:2018] Table 55 - Extract from Security and Certification concept glossary. Appendix A – Glossary and Examples D2.1 – Supply Chain Analysis and Requirements Table 55 - Extract from Security and Certification concept glossary. 952690 CYRENE PROJECT PARTNERS 133 31/05/2021 Term Abbrevi ation Definition(s) Reference(s) Example(s) Notes/ Remarks Supply Chain and Business Concepts Application - IT solution, including application software, ISO/IEC 27032:2012 133 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 134 31/05/2021 application data and procedures, designed to help an organization’s users perform particular tasks or handle particular types of IT problems by automating a business process or function Application service - Software with functionality delivered on-demand to subscribers through an online model which includes web based or client-server applications. ISO/IEC 27032:2012 Online storage, Customs online service Application service provider: operator who provides a hosted software solution that provides application services which includes web based or client- server delivery models [ISO/IEC 27032:2012]. Application software - Software designed to help users perform particular tasks or handle particular types of problems, as distinct from software that controls the computer itself ISO/IEC 26514:2008 Asset - Something (item, thing or entity) that has value (potential or actual value) to the organization. An asset extends beyond physical goods or hardware, and includes software, information, people, and reputation. [ISO/IEC 27001: 2013; ISO/IEC 20000- ISO/IEC 27001: 2013; ISO/IEC 20000-1: 2018 An asset can be for example an application server, a presence sensor, a mobile or a municipal building. The only difference of the two terms is that the second makes provision for individuals and the separation of governments from organizations. 952690 CYRENE PROJECT PARTNERS 134 D2.1 – Supply Chain Analysis and Requirements D2.1 Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 135 31/05/2021 1: 2018] Information asset: Anything that has value to an individual, an organization or a government. [ISO/IEC 27032: 2012] Business objective - result to be achieved. An objective can be strategic, tactical, or operational. Objectives can relate to different disciplines (e.g. financial, health and safety, and environmental goals) and can apply at different levels (e.g. strategic, organization-wide, project, product and process. An objective can be expressed in other ways, e.g. as an intended outcome, a purpose, an operational criterion, as an information security objective or by the use of other words with similar meaning (e.g. aim, goal, or target). ISO/IEC 27000: 2018 Cosignee / Shipper / Cosignor - A person or company that consigns or receives goods for transportation. Appendix A – Glossary and Examples EU H2020- DS-2014-01 project “MITIGATE” Critical Infrastructur e CI An asset, system or part thereof located in Member States which is essential for the maintenance of vital societal functions, health, safety, security, economic or Council Directive 2008/114/EC SCADA, port, Port Community System (PCS) European critical infrastructure (ECI) is a critical infrastructure located in Member States the disruption or destruction of 135 D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements social well-being of people, and the disruption or destruction of which would have a significant impact in a Member State as a result of the failure to maintain those functions which would have a significant impact on at least two Member States. The significance of the impact shall be assessed in terms of cross- cutting criteria. This includes effects resulting from cross-sector dependencies on other types of infrastructure [Council Directive 2008/114/EC]. Critical Information Infrastructur e CII ICT systems that are Critical Infrastructures for themselves or that are essential for the operation of Critical Infrastructures (telecommunications, computers/software, Internet, satellites, etc.) Council Directive 2008/114/EC Human Machine Interface (HMI) Critical services - A critical service is a service that is essential for the maintenance of critical societal or economic activities. NIS Directive, 2016 Customer Person or organization that could or does receive a product or a service that is intended for or required by this person or organization ISO 9000:2015 Consumer, client, end- user, retailer, receiver of product or service from an internal process, beneficiary 952690 CYRENE PROJECT PARTNERS 136 D2.1 – Supply Chain Analysis and Requirements and purchaser. Table 56 - Extract from Supply Chain and Business concept glossary. Term Abbreviati on Definition(s) Reference(s) Example(s) Notes/Remarks Maritime Transport Concepts Baltic and Internatio nal Maritime Council BIMCO BIMCO is one of the greatest international shipping associations representing ship owners. It undertakes the control of around 65 percent of the world's tonnage and it has a strong membership, engaging more than 120 countries, involving managers, brokers and agents. BIMCO’s main objective is to protect its global membership via the provision of information and consulting that forwards fair business practices and invests on the harmonisation and standardization of commercial shipping practices and contracts. Baltic and International Maritime Council, Den Store Danske Encyklopædi. Denstoredan ske.dk. Online available: https://densto redanske.lex. dk/Baltic_and _International _Maritime_C ouncil?utm_s ource=densto redanske.dk &utm_mediu m=redirect&u tm_campaign =DSDredirect https://www.bimc o.org/ Barge operator - A company that provides barge capacity and barge transport. EU H2020- DS-2014-01 project "MITIGATE" Berth - Those systems are "Port D2.1 – Supply Chain Analysis and Requirements and purchaser. Table 56 - Extract from Supply Chain and Business concept glossary. D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 137 Table 56 - Extract from Supply Chain and Term Abbreviati on Definition(s) Maritime Transp Baltic and Internatio nal Maritime Council BIMCO BIMCO is one of the greatest international shipping associations representing ship owners. It undertakes the control of around 65 percent of the world's tonnage and it has a strong membership, engaging more than 120 countries, involving managers, brokers and agents. BIMCO’s main objective is to protect its global membership via the provision of information and consulting that forwards fair business practices and invests on the harmonisation and standardization of commercial shipping practices and contracts. Barge operator - A company that provides barge capacity and barge transport. Berth Managem ent Systems - Those systems are used by Port Authorities to manage and ensure safety in D2.1 – Supply Chain Analysis and Requirements Table 56 - Extract from Supply Chain and Business concept glossary. 137 D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 138 31/05/2021 mooring processes: warnings and alerts, meteorological data, video cameras streams, berth allocation management, etc. cybersecurity in the maritime sector", ENISA, 2019 Border Control - The border control authorities are responsible of taking measures to monitor the state borders and to regulate the movement of people, animals and goods. In the EU, with Schengen agreement, the crews and passengers are controlled only once when they come from a non-EU country. "Port Cybersecurit y - Good practices for cybersecurity in the maritime sector", ENISA, 2019 Bunkerin g - The provision of solid, liquid or gaseous fuel or of any other energy source used for the propulsion of the waterborne vessel as well as for general and specific energy provision on board of the waterborne vessel whilst at berth. Regulation (EU) 2017/352, Article 2 Cargo - Items that are placed on the ship to be transported to another port, such as boxes, pallets, cargo transport units, and bulk liquid and non- liquid matter. ISO 20858:2007 Cargo Communi ty System CCS Usually owned and managed by port stakeholders that are "Port Cybersecurit y - Good 138 Table 57 - Extract from Maritime Transport concept glossary. Appendix B – Validation of CYRENE ToEs This Appendix presents the three CYRENE Advisory Boards that have been formed in the first six months of the project’s lifetime. It also presents the organization of the first project workshop, in which members of the Advisory Boards participated, and the feedback received by them. D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 139 31/05/2021 usually private companies in charge of the terminal port operations. This system is used to share information on port operations related to the cargo and containers between all involved stakeholders (content of the cargo, localisation of a container, hour of its transfer, customs declarations, etc.) practices for cybersecurity in the maritime sector", ENISA, 2019 Cargo handling - The organisation and handling of cargo between the carrying waterborne vessel and the shore, whether it be for import, export or transit of the cargo, including the processing, lashing, unlashing, stowing, transporting and temporary storage of the cargo on the relevant cargo- handling terminal and directly related to the transporting of the cargo, but excluding, unless the Member State determines otherwise, warehousing, stripping, repackaging or any other value added services related to the cargo. Regulation (EU) 2017/352, Article 2 Carrier - Freight transporting company EU H2020- DS-2014-01 139 D2.1 – Supply Chain Analysis and Requirements project "MITIGATE" Centre for Internatio nal Maritime Security CIMSEC A 501(c)3 non- partisan think tank incorporated as a non-profit in the state of Maryland. CIMSEC was formed in 2012 and as of 2020 has 20 international chapters and over 2,000 members and subscribers in 60 countries. CIMSEC does not take organizational positions and encourages a diversity of views in the belief that a broad range of perspectives strengthens our understanding of the challenges and opportunities in the maritime domain. http://cimsec. org/about Table 57 - Extract from Maritime Transport concept glossary. D2.1 – Supply Chain Analysis and Requirements 952690 CYRENE PROJECT PARTNERS 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 140 B.1 CYRENE Advisory Boards The mem- bers of the Advisory Boards are the following: The three Advisory Boards were formed in the bers of the Advisory Boards are the following: User Advisory Board: B.1 CYRENE Advisory Boards CYRENE has defined three Advisory Boards, which are expected to provide their expert advice on key topics the project addresses, related to the certification of security aspects of supply chains. The role and expectations of the three boards are as follows: User Advisory Board (UAB): it will support the project with its requirements collection and evaluation tasks, including User Advisory Board (UAB): it will support the project with its requirements collection and evaluation tasks, including (i) providing sound and concrete advice on requirements related to security aspects of modern supply chains, as well as the design of tools, scenarios and user stories that will be devel- oped in the context of the project, (ii) participating in the evaluation phases of the developed tools and prototypes, and (iii) acting as channels and multipliers for the exploitation and market uptake of the project’s results. External Advisory Board (EAB): it will support the project with its requirements and dissemination objectives, including: (i) Ensuring that the project’s outcomes address not only the requirements of the specific do- mains via the project’s pilots, namely the domain of automotive industry and the one of vehicle transportation, but from other industrial sectors as well. (ii) Bringing in touch the project with potential users (citizens, local authorities, ministries) of the solutions to be developed, other similar projects and research initiatives, as well as policy makers and standardization bodies. (iii) Advising on the compliance of project’s results with legal, ethics, standardization and certi- fication regulations and relevant legislation. (iii) Advising on the compliance of project’s results with legal, ethics, standardization and certi- fication regulations and relevant legislation. Ethical Advisory Board (EthAB): it will support the project for implementing its ethical management strategy including: (i) adherence to privacy laws and regulations, (ii) handling of anonymization processes, (iii) handling of informed consent processes for participants of the pilot operations, (iv) processes associated with secure storage of incident handling datasets (iii) handling of informed consent processes for participants of the pilot operations, (iv) processes associated with secure storage of incident handling datasets. The three Advisory Boards were formed in the first six months of the project’s lifetime. The mem- bers of the Advisory Boards are the following: The three Advisory Boards were formed in the first six months of the project’s lifetime. User Advisory Board: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952690. D2.1 – Supply Chain Analysis and Requirements (i) Notis Iliopoulos, Director Cyber Security Operations, Neurosoft, S.A. (ii) Leandros Maglaras, Associate Professor, De Montfort University, Leicester, U.K. (iii) Stefan Schauer, Scientist, Austrian Institute of Technology (AIT) (iv) Antonio Skarmeta, Professor, University of Murcia (v) Valentin Carlan, Researcher, University of Antwerp (vi) Andrea Lanzi, Associate Professor at Università degli Studi di Milano External Advisory Board: (i) Loredana Mancini, Cybersecurity senior consultant (ii) Vangelis Gazis, Chief Architect Iot Security, HUAWEI (iii) Michel Bosco, Consultant, Strategic Advisor Ethical Advisory Board. (i) Dusko Martic, Cybercrime and Privacy lawyer, Head of Content Protection and the GDPR Compliance Manager for the United Media Group (ii) Diorela Prisacaru, Security Consultant, Capgemini (iii) George Exarchou, Legal Advisor, DFDL (Ho Chi Minh City, Vietnam) (iv) Milos Mijomanovic, Cybercrime, Interpol (i) Notis Iliopoulos, Director Cyber Security Operations, Neurosoft, S.A. (ii) Leandros Maglaras, Associate Professor, De Montfort University, Leicester, U.K. (iii) Stefan Schauer, Scientist, Austrian Institute of Technology (AIT) (iv) Antonio Skarmeta, Professor, University of Murcia (v) Valentin Carlan, Researcher, University of Antwerp (vi) Andrea Lanzi, Associate Professor at Università degli Studi di Milano Ethical Advisory Board. (i) Dusko Martic, Cybercrime and Privacy lawyer, Head of Content Protection and the GDPR Compliance Manager for the United Media Group (ii) Diorela Prisacaru, Security Consultant, Capgemini (iii) George Exarchou, Legal Advisor, DFDL (Ho Chi Minh City, Vietnam) (iv) Milos Mijomanovic, Cybercrime, Interpol (i) Dusko Martic, Cybercrime and Privacy lawyer, Head of Content Protection and the GDPR Compliance Manager for the United Media Group (ii) Diorela Prisacaru, Security Consultant, Capgemini (iii) George Exarchou, Legal Advisor, DFDL (Ho Chi Minh City, Vietnam) (iv) Milos Mijomanovic, Cybercrime, Interpol B.2 First Project Workshop CYRENE organized its first (web) workshop on March 31, 2021. The objective of the workshop was the elicitation of feedback from the three Advisory Boards on requirements relating to the certification of security aspects of supply chains that had been collected by the project in the first six months. The workshop agenda is shown in Table 58. Time (CET) Topic Speaker 10:00-10:05 Welcome Farhan Sahito, PN 10:05-10:20 CYRENE Overview Sofoklis Efremidis, MAG Sophia Karageorgou, UBI 10:20-10:30 Security Aspects of Supply Chains Haris Mouratidis, SU 10:30-10:50 Supply Chains (SC) as Targets of Evaluation (ToE), CYRENE ToEs Alexandra Michota, FP Elma Kalogeraki, MAG 10:50-11:10 End user requirements Danijela Boberic, UNSPMF 11:10-11:40 CYRENE Questionnaire Feedback, discussion, Q/A by AB members (collection of end user requirements) Danijela Boberic, UNSPMF 952690 CYRENE PROJECT PARTNERS 142 D2.1 – Supply Chain Analysis and Requirements 11:40-11:45 Conclusions and closing remarks 11:40-11:45 Conclusions and closing remarks Farhan Sahito, PN Table 58 - Workshop agenda. D2.1 – Supply Chain Analysis and Requirements D2.1 – Supply Chain Analysis and Requirements Table 58 - Workshop agenda. The workshop agenda, a copy of the presentations, and a tailored version of D2.1 were communicated to the Advisory Board members before the workshop. During the workshop the presentations that were given by the project focused on: 1. The objectives of the project and its technical approach. 2. The State of the Art on security aspects of supply chains and related risks. 3. The European cybersecurity certification initiatives and approaches to certification of sup- ply chains. 3. The European cybersecurity certification initiatives and approaches to certification of sup- ply chains. 4. The compiled by the project Supply Chain requirements, expressed as Targets of Evalu- ation. 5. Details of business process, assets and infrastructure, and sector specific requirements. o o g t e p oject p ese tat o s, a Q& sess o too p ace o ac tat g te act o s t t e Advisory Boards and receiving their feedback and comments on the presented Supply Chain security certification requirements. The Q&A session started by summarizing the outcome of the project, which is a methodology and a set of tools for the Conformity Assessment of security aspects of Supply Chains. The main issues raised by Advisory Board members, are summarized in Table 59, along with the approach the project takes to address them. The main issues raised by Advisory Board members, are summarized in Table 59, along with the approach the project takes to address them. Suggestion CYRENE approach IoT devices be included as SC assets CYRENE plans to address IoT as assets of supply chains Cyber-physical systems be addressed by the project CYRENE plans to address cybersecurity aspects of CPS Security aspects of cloud-based services supporting supply chains to be considered CYRENE will consider developments in the security of cloud-based services. The supply chain certification scheme to be designed and proposed by CYRENE is based on a similar scheme on cloud services certification proposed by ENISA Enlarge the base of supply chains addressed and include Hydrogen-LNG SC CYRENE takes a general approach to security certification of SCs but it will focus it pilots on the two supply chain scenarios that have been prescribed. Consider certification of software products CYRENE addresses SCs and infrastructures that support them, but not certification of software products per se, which falls into the realm of other certification initiatives 143 31/05/2021 Table 59 - Suggestions from the Advisory Boards. Further input was sought by Advisory Board members who were asked to provide offline their feedback to a questionnaire that had been prepared and setup by the project at https://ec.europa.eu/eusurvey/runner/cyrene-questionnaire. The replies to the questionnaire received were analyzed, and the results are presented in Chapter 4. The workshop completed with a slight delay with respect to its original schedule. 952690 CYRENE PROJECT PARTNERS 31/05/2021 31/05/2021 144
https://openalex.org/W2616838737	https://journalsojs3.fe.up.pt/index.php/jim/article/download/2183-0606_005.001_0005/270	English	null	Efforts to build a culture of innovation in the Brazilian energy sector	Journal of innovation management	2,017	cc-by	7,228	Journal of Innovation Management Cândido, Magro, Roczanski, Jamil JIM 5, 1 (2017) 40-57 HANDLE: http://hdl.handle.net/10216/103566 SM: Dec/2015 AM: Dec/ 2016 Efforts to build a culture of innovation in the Brazilian energy sector Ana Clara Cândido Professor at Federal University of Santa Catarina; Associated Researcher INESC P&D Brazil; Campus Universitário Reitor João David F. Lima Trindade - Florianópolis - SC – Brasil, CEP: 88040-900 ana.candido@ufsc.br Dalva Magro Collaborator at State University of Santa Catarina; Associated Researcher INESC P&D Brazil dalva.magro@inescbrasil.org.br Carla Regina Magagnin Roczanski Collaborator at State University of Santa Catarina, Brazil carla.roczanski@udesc.br George Leal Jamil Associated Researcher InescTec, Porto gljamil@gmail.com Journal of Innovation Management JIM 5, 1 (2017) 40-57 HANDLE: http://hdl.handle.net/10216/103566 SM: Dec/2015 AM: Dec/ 2016 Journal of Innovation Management JIM 5, 1 (2017) 40-57 HANDLE: http://hdl.handle.net/10216/103566 SM: Dec/2015 AM: Dec/ 2016 Keywords: Culture of Innovation, Energy Sector, Brazil, ANEEL. ISSN 2183-0606 http://www.open-jim.org http://creativecommons.org/licenses/by/3.0 Cândido, Magro, Roczanski, Jamil Cândido, Magro, Roczanski, Jamil 1 Introduction The importance of the knowledge and technology is undeniable for the balanced and sustainable development of the nations. This effect can be evidenced on product innovations, the more knowledge and technology are inserted to the new products and services, the higher your market value, consequently more benefits to society, through boosting the economy and improving the quality of life. The investment in Research and Development (R&D) became a crucial factor for most sectors of the economy to prosper and exceed the challenges that concern them. In the eletrical power sector, this situation is strongly identified, considering its stong influence to the economy, environmental, politics and others key sectors for the development of nations. The culture of innovation in Brazil was started recently, this theme gained more attention since the 90’s, through the creation of support mechanisms to the productive sector in the context of policies in science, technology and innovation. This study intends to contribute to an analysis and evaluation of the innovation policies in Brazilian eletric power sector. Therefore, the purpose is to analyze and evaluate regulatory framework in Brazil in relation with innovation efforts to create a culture of innovation at national level. The methodological procedures were based on descriptive statistics through secondary data collected from ANEEL database (submitted proposals by companies to "R&D Program ANEEL", available on the agency's website). Although can be listed numerous benefits of the R&D Program of ANEEL, the results released until now draw attention to some aspects: Of the 2257 projects between 2008 and march 2015, only two are market production insertion. This reflects the need for projects with more applicability and greater technical and economic impact for the Brazilian electricity sector. The need for more practical application of the projects, originated new products to market, was encouraged by ANEEL when proposed changes to the program. On that occasion were created mechanisms which encouraged the development of projects with proposals for potential new products insertion into the market. Figures relating to the projects of R&D ANEEL between 2008 and early 2015 show that program structure had no effect so far after changes. The creation of mechanisms to encourage the development of Market production insertion, by itself, was not sufficient. It is necessary to step back and see how these projects are being perceived by the companies. Efforts to build a culture of innovation in the Brazilian energy sector Professor at Federal University of Santa Catarina; Associated Researcher INESC P&D Brazil; Campus Universitário Reitor João David F. Lima Trindade - Florianópolis - SC – Brasil, CEP: 88040-900 ana.candido@ufsc.br Dalva Magro Collaborator at State University of Santa Catarina; Associated Researcher INESC P&D Brazil dalva.magro@inescbrasil.org.br Carla Regina Magagnin Roczanski Collaborator at State University of Santa Catarina, Brazil carla.roczanski@udesc.br George Leal Jamil Associated Researcher InescTec, Porto gljamil@gmail.com Abstract. Innovation and innovation management are essential topics for any organizational system nowadays. Public services, commercial and industrial corporations are pressured to study, plan and promote innovations as strategic pillars for their activities and reputation in their competitive scenario. This paper analyzes the efforts on building and improving cultural factors that can foster innovation and innovation management in a critical, competitive and technology- driven sector of electrical power production in Brazil, examining records from ANEEL, its regulatory agency. From these records, values and facts concerning Brazilian program for investment in innovation were evaluated, together with related projects data, showing results that indicated: (a) Investments were conducted in mandatory fashion, not following strategic policies; (b) Expressive amount of investments were also done in the basic and applied research, not offering fair perspectives on more qualified or value-aggregated innovations and; (c) This investment program, executed by the regulatory agency, is opportune to sponsor innovation in this important economic sector. Methodological aspects, such as indexes choice, comparisons and analysis applied in this paper can also build a basis for other studies around the same context, allowing further comparisons to other sectors – such as those in this value-aggregated chain or even with other countries, with perspectives of richer results that can provide another level of innovation investments programs comprehension. Keywords: Culture of Innovation, Energy Sector, Brazil, ANEEL. 40 40 Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 http://www.open-jim.org 1 Introduction The mistaken view of investing in R & D to fulfill obligations imposed by the regulatory mechanism of ANEEL, could compromise the performance of this sector. The creation and consolidation of the program proposed by ANEEL is an important step forward for the technological development of the Brazilian electricity sector, however, the role of innovation culture needs to be strengthened further in this context. Finally, some important concepts should be incorporated in this environment to ensure the successful operations of such mechanisms, for example: collaborative networks, institutional partnerships, innovation management, innovation indicators, commitment to results, innovation strategy, among others. 41 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 2 Concepts and Premisses of Innovation Conceptualize innovation and its associated management principles are constant reviews against the common-sense understanding. These problems are caused mainly by the remarkable market events produced by some product innovations, as those emerged from information technology, automotive, aerospace and mobile communication sectors. Although supported by the background theoretical reviews, they can be considered as some ways on how innovations can modify people´s lives, including when considering us in the organizational context as members of several organizations, for example, employees of market companies. http://www.open-jim.org 2.1 Although referring like new ways of production or for entrepreneurial activities, the citations by Joseph Schumpeter are considered as the first landmark for this conceptual work. In its fundamental work – Schumpeter (1934) – he announces six types of “new combinations” of entrepreneurship, in order to promote social changes. These definitions are stated in Table 1 below: Table 1. Innovation concepts Innovation as… From Schumpeter (1934) Product “a new good” (or a new quality or functionality). Process “a new method of production…” (with a reference to fundamentals on a scientific definition or new ways to handle products for commercial offering). New market “a country…” where a manufacturer have not entered yet. New source of supply “of raw materials…” or semi-manufactured goods. New organization As the creation of a “monopolist” positioning by a competitive strategy on a new market. Source: Authors (October, 2015). “a new method of production…” (with a reference to fundamentals on a scientific definition or new ways to handle products for commercial offering). These concepts are discussed, debated, validated and supported by many authors and organizational sources since their original edition. For example, the Oslo Manual (2005) refers to TPP – technologically-supported products and processes – or new actions or approachs to propose, define, plan and implement new product lines and related processes in organizational environments which are based on technology support and scientific applications. From Drucker (1985), it is possible to understand a wider conception for an innovative process, reinforcing “as a process”, “a new market” and “new organization” definitions, when this remarkable author studied and proposed several analyses on businesses process. In his view, these processes were oriented to support business negotiations with final customers (new market, process) and advantageous strategic positioning 42 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 (new organization), corroborating Schumpeter definitions. (new organization), corroborating Schumpeter definitions Interestingly, from Utterbach (1994), it can be seen a thorough discussion around the innovation concept, in which this important author validates some of Schumpeter findings, promoting a proposition of one new view. In this work, Utterbach compares, immediately, innovation products and process lifecycles, observing how they appear in national innovation systems and can be implemented in industrial and services organizations. 2.1 His work focuses on designing a model for innovation lifecycle, approaching several real cases, adjusting these to a generic context that, at the end, will serve to understand his propositions to comprehend how one innovation is firstly introduced in a market, then develops its acceptance, technological adjustments and, finally, ends its commercial, technologic and social impact, receiving progressively less changes, becoming a “commoditized” version of a product or process. He also depicts, as a result of these observations around innovation as products and processes, the understanding about new ways of organization, as a response to changes in processes and new ways of products supply, as an integration of processes to provide new products to final customers. This market-oriented view results in an opportune conceptualization on how competitiveness provokes innovation, which eventually leads to understand how a new type of innovation – that produced by market competition – is also conceptualized. In a practical consolidation of this first view, the works by Schumpeter and Utterbach result in six different conceptions for innovation (as we combine four of them, considering them as equal, observing the two remaining as additions), which are: (Innovation as…) (1) Product; (2) Process; (3) New organization; (4) New market; (5) New source of supply and (6) Result of competitive forces. It is also interesting to observe that Strategy is a collateral concept which can be intensively observed in each of these definitions, and also when they are taken combined. These concepts also cannot be faced as exclusive, or, as supported by many authors as those already cited in this section, strictly demanding that one innovation is “only” from one type. It is reasonable to think that the introduction of a new product may demand a new idea to offer, or also a new way to position, promote and sell it to a new market, combining two or three conceptualizations (Drucker, 1985; Davila, Epstein & Shelton, 2006; Bés & Kotler, 2011). There are numberless definitions for innovation, usually as contributions from academic and scientific areas such as Strategy, Marketing, Human resources management, Information technology, Computing Science, Engineering (several different fields), among many others. For the purpose of this review, those first six definitions, added by the qualifications discussed below, are sufficient to announce the following study about a system to manage innovation in organizations. 2.2 Qualifying an innovation Additionally, many views for innovation initiatives, processes and planning, result in complementary approaches, that are affirmed as “qualifying” for the innovation concept. First, it is opportune to approach, as announced by Engen & Holen (2014), citing Tushman & Romanelli (1985), the discussion around radical and incremental innovations. Radical innovations, as it happens with the common-sense perception of 43 43 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 considering product as the only type of innovation, is regarded as “the innovation” whatsoever. It is a major change, a disruptive action to propose something completely new, in which will mandatorily replace the older offer – product or service – with a complete new applicative scenario. But, carefully analyzing markets, strategic positioning and marketing stories, it is easy to perceive, as studied by these authors, several cases of small changes, applied to already existing components, parts or even complete commercial solutions, that were really successful. These small but identifiable changes, which resulted eventually in new ways to use products and services, are regarded as incremental innovations. Davila, Epstein & Shelton (2006) analyzed, in a very detailed and analytical way, how technological and business model drivers should be observed to define if an innovation can be defined as radical, semi-radical or incremental. In their model, three drivers for each dimension – technology and business model – must be evaluated to check if there was, on one hand small, on the other hand, expressive sustainable changes from the former offer, resulting in those three qualifications. Another approach, focusing more on the process innovation itself, was previously announced by Henry Chesbrough, He defined a context of intense, interactive, cyclic and perennial cooperation of economic agents to innovate, called “Open Innovation” (Chesbrough, 2003). In this proposition, innovation planning, design and implementation is an intensive cooperative context, where signals, information, communication and knowledge flow around the value-aggregated organizational chain, integrating customers and other economic agents, as participative elements to produce the original concept for any proposed innovation. This context diverges from the “closed innovation” former view, where an organization tries to develop its new types of innovation completely by itself, generally working to offer the final conception to the market, eventually dictating how customers will receive it. 2.2 Qualifying an innovation In general, approaches to Chesbrough works, this “closed” fashion is related to older, strict and classical markets-oriented corporations, progressively becoming extinct by new competitive models and competitive scenarios. http://www.open-jim.org 2.3 Innovation Management Taking into account those different conceptualizations for innovation, its related management is also complex and broad, becoming challenging to focus for a conceptual base detail. First, it is recommendable to understand what can be regarded as innovation management, how it can occur in real organizational arrays and, after these steps, understand how it can be defined. Observing in the former subsection, it is possible to define innovation from six different points of view. As a product (the most usual and perceived), as a process, as the relationships with new markets and customers, as new ways to organize the final customer service (new organizational models), as how to apply new basic and modified supplies, basic materials and, finally, as results of competitive reactions. It is provocative to think, analyzing from the literature discussed before and from other sources, presented in the following, some possible contributions to manage innovation. The principles to build such way of thinking is to merely observe how each type of those six definitions of innovation demands specific management principles, The principles to build such way of thinking is to merely observe how each type of those six definitions of innovation demands specific management principles, 44 http://www.open-jim.org 44 Cândido, Magro, Roczanski, Jamil Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 fundamentals and actions and perceive, in an initial view, some management areas and tasks which are demanded by all those six types, in general. The following text discusses by this way, presenting its references and, in the final, a consolidation is produced. Innovation as product requires principles of Project Management – PMBoK (2012) – specific Engineering, Design, Production Engineering – Trott (2011) -, among several other themes that define and build logic and organizational fundamentals, proposed to structure the production of “something” tangible, eventually never tested before and also functionally acceptable by customers (Christensen, 2015). It is interesting to notice that, as said before, this type of innovation is immediately perceived by customers, in general, then any other type mention previously, also showing the largest base of references discussed by researchers and practitioners for the first decades of 20th Century, just because the other forms to innovate weren´t so much noticed as strategic resources by organizations. 2.3 Innovation Management Innovation as process is more understood from other points of view, like those related with business process management, being better exemplified with the approach of processes that namely deal with the “flow” of strategic, production materials or even critical resources, as information or money (BPM CBoK, 2013; Hill, 2015). Modifying a process is not a completely transparent innovation for final users, frequently aiming to improve, optimize or at least change some internal organizational aspects. Although this lack of image for external agents, processes innovation in areas like Finance, Supply chain and Production management eventually produce quantitative results of impact in overall organizational performance. New markets – in the sense of creating it or even exploiting an old one in a new approach – are usually discussed by Marketing disciplines (Kotler and Keller, 2015). Several evidences from new markets observed occurred in the last years. Emerging markets, such as those from instantaneous, impulsive and sometimes unsustainable national Economies (like from the countries of the BRICS block), demanded new forms of supply, businesses models, competitive regulations, among other actions and agreements. Marketing, Production, Human Resources, Commercial and some other professional areas were dynamically adapted to deal with these new competitive fronts. This resulted in a practical productive scenario where scientific and academic knowledge have to be produced and applied in a sudden, eventual way (Johnson, Christensen & Kagermann, 2008). As a result from the two former types of innovation – process and market – or even as new, stand-alone innovative approach, one organization can also define a new structure to answer changing competitive external signals, as fast customer change of preferences, invasion by an external competitor or even a risk of technological replacement (Weldeken, et. al., 2014). New design for business models, an event that is still being studied and not correctly comprehended by entrepreneurs and other economic agents, is remarkably being proposed by new competitive organizations, as social media providers, sharing resource partnership promoters (as AirBnB or Uber), industrial dynamic outsourcers (as micro-factories that are now producing from beer to car parts) or by integrative platforms of services, as entertainment tickets sellers or food delivery firms (Dijk, 2015). 2.3 Innovation Management New ideas of treating old materials, or even integrating or exploiting these old basic 45 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 supplies in another way and also exploiting new materials, are also interesting fronts of innovation, as approached by Dangelico & Pujari (2010) and Gerstlberger, Knudsen & Stampe (2014). It is opportune both to affirm about the technological front, where technical and engineering approach are increasingly successful, and observe how the sustainability issue also provokes researchers and practitioners to understand new materials and new ways for handling of the old ones impact the environment, demanding by this way new degrees of comprehension on how innovations from this kind are valuable for humanity. Finally, competitive forces, as those presented by Strategy authors who studied Innovation –Porter (2008), Mintzberg, Ahlstrand & Lampel (2009) and Barney (2011) – are recognized as a drive for organizational innovation. Several demands from markets, as new ways to optimize human resources management, finance, supply chain, productive arrays and other aggregated-chain components are examples of these competitive requests for innovation. Organizations competing in these new markets face the demand to change promptly their conditions to understand and propose a productive rivalry, integrating their efforts to occupy market spaces and improve the final customer perceived value. 2.4 The Integrative Context of Innovation Management The process itself, as it is possible to observe in new business models, can differentiate from a traditional, up-down, scaling process (Porter, 1998) to new interactive methods, although with the same overall guidance main objectives (Sniukas, 2015). Another way to think is that strategy regards innovation, as conceptualized, for organizational future positioning. As strategy is unfolded in strategic-tactical plans, defining potential relationships of management areas of one organization to achieve predicted goals, it is important to understand these innovation management components. A brief observation about those areas / subjects that support the organizational strategy focus themes such as Human Resources, Marketing, Commercial, Logistics, Information systems design, Communication, Financial, Operations, Project Management, Production among others, where its specific plans must consider innovation culture and management to produce suitable scenarios that allow to propose innovations as a result of strategic thinking and planning. Specific approaches to these areas are beyond this first-level theoretical review, although it can be oriented by some of the titles referenced until this part of the study. 3 The Energy Sector in Brazil An important movement in the Brazilian energy sector occurred through Law 9.991/2000, established by Brazilian Electricity Regulatory Agency (ANEEL). This law provides investments in R&D by concessionary enterprises, permissionaires and authorized companies of the electric power sector. Thus, this law requires obligatory investment, of at least 1%, net revenue from companies in the sector. The initiative is known as "R&D Program of ANEEL”. It is important to note that from 2008 there were some changes in the structure of the R&D Program ANEEL, including the new classification modalit: • Basic Research (BR) • Applied Research (AR) • Experimental Development (ED) • Head Production Series (HD) • Piooner Production Lot (HS) • Market Production Insertion (MI) These classifications of projects identify the stage of maturity of the businesses proposals, in addition to greater dissemination of results. In this sense, considering that it's been more than 10 years of this investment effort in innovative projects in this sector, some studies have already realized to assess the performance of this regulatory framework as a study conducted by Institute for Applied Economic Research (Ipea) in collaboration with ANEEL. The results obtained showed strengths and points which needed to be strengthened that the programme could be more efficient. 2.4 The Integrative Context of Innovation Management As stated in the last subsection, innovation management is a complex and unlimited context, where several management techniques and methods play a decisive role. Concluding this objective view of innovation management, is opportune to mention its integrative context referring both to the fact that it integrates these relevant disciplines of technological and managerial contributions and is, mainly, open to additional thoughts that aim to allow one organization to produce and interact with innovations. Among the topics that can be easily identified in the literature, it is important to define Strategy as the base for innovation management composition. Strategy can be regarded as one coordinated view for one organization´s future (Porter, 2008; Mintzberg, Ahlstrand & Lampel, 2009). This coordinated view must consider innovation, in the proposed broader view conformed conceptualized before, as a strategic formulation component for one organization. Innovation can be proposed as, for example, a new process to answer customer demands for more flexible services or even to correctly point out a new focus for launching a specific product, as a result for the availability of new technology (Ma, Jill & Ziang, 2015). Thinking this way, it is possible to affirm that even when it is considered that innovations must disrupt a company´s strategy, it is perceivable that this rupture occurs with meaning to former objectives, supported by new approaches to goal definitions processes and specifications for tactical and operational plans, i. e., it considers the rupture from a conventional, traditional strategic view (Christensen & Raynor, 2003). Another considerable observation is that one can propose innovation as a part of the organizational strategy, but the strategic proposition, itself, can become an innovation (Ettlie & Reza, 1992). For a development of this affirmation, it is possible to understand the characteristic of strategic planning – it is one organizational process, defined by several authors as “the” organizational process” (Hammer & Stanton, 1999), as the 46 http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 main integration flow for corporative decisions and implementations. The process itself, as it is possible to observe in new business models, can differentiate from a traditional, up-down, scaling process (Porter, 1998) to new interactive methods, although with the same overall guidance main objectives (Sniukas, 2015). main integration flow for corporative decisions and implementations. 3.1 The role and intervention of Brazilian Electricity Regulatory Agency (ANEEL) According to Powder and Abrucio (2004), one of the results of the state reform process 47 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 Cândido, Magro, Roczanski, Jamil in Brazil was the creation of regulatory agencies. During the first generation of reforms have created regulatory agencies related to the privatization and break of the state monopoly in the sectors of infrastructure, cases of the National Electric Energy Agency (Aneel), the National Telecommunications Agency (Anatel) and the National Petroleum Agency (ANP). in Brazil was the creation of regulatory agencies. During the first generation of reforms have created regulatory agencies related to the privatization and break of the state monopoly in the sectors of infrastructure, cases of the National Electric Energy Agency (Aneel), the National Telecommunications Agency (Anatel) and the National Petroleum Agency (ANP). This first generation of regulatory agencies, created since 1996 in the context of privatization, breaking the state monopoly and inspired by international experience, was set up as public entities endowed with independence from the executive branch. In its creation the work of the Congress was important, as well as the recommendations of the Council of State Reform, an advisory body attached to the President, though less participation of the Ministry of Federal Administration and State Reform (Mare) (Pacheco, 2004). The creation of ANEEL project was sent by the Federal Executive to the National Congress in late 1995, later to the first privatization in the sector, causing some problems of legitimacy, especially regarding the arbitration of disputes (Salgado, 2003). ANEEL was created by the Law 9427, on December 26, 1996 and regulated by Decree No. 2,335, on October 6, 1997, which approved its regimental structure. The Aneel management contract had its first version adopted in 1998 and the Target Plan approved in 1999.2 The agency, set up as an independent regulatory and linked to the Mines and Energy Ministry (MME), is to regulate and inspecting the production, transmission and sale of electricity in accordance with the policies and guidelines of the federal government. Aneel has managerial and financial autonomy and competence to regulate technical issues as well as decision-making autonomy, guaranteed by fixed terms of its board, whose conformation is designed to ensure technical quality and neutrality in their decisions. The Law No. 9991 on July 24, 2000, changed by Law No.10,438, on April 26, 2002, No. http://www.open-jim.org 3.1 The role and intervention of Brazilian Electricity Regulatory Agency (ANEEL) 10,848, on March 15, 2004, No.11 465, on March 28, 2007, 12.111 on December 09, 2009 and No.12.212 on January 20, 2010, concessionaires of public distribution services, transmission and generation of electricity, the licensees of public services of electricity distribution and authorized for the independent production of electricity, excluding those that generate energy exclusively from wind installations, solar, biomass, qualified cogeneration and small power plants hydropower, should apply annually a minimum percentage of their net operating income - ROL in Research and Technological Development of the Electricity Sector - R&D, according to regulations established by ANEEL. According to this law (Article 1), concessionaires and licensees of electricity distribution are required to apply annually a minimum of 0.75% (Seventy-five hundredths percent) of their ROL in research and development of the electricity sector and 0.25% (twenty five percent) on energy efficiency - EE in the final use, and should be subject to the transition period these percentage. As for the generation companies, authorized the independent production of electricity and transmission concessionaires were required to apply annually at least 1% (one percent) of ROL in research and development of the electricity sector. By Exemption, were excluded from the obligation companies that generate power exclusively from wind installations, solar, biomass, small hydro and qualified cogeneration, observing, for the latter, the provisions of 48 Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 Resolution No. 652 of 9 December 2003. Resolution No. 652 of 9 December 2003. That change occurred to modify the previous rules that force generation concessionaires to invest in research and development of electric power industry annually a minimum of 0.25% (twenty-five hundredths percent) of its ROL. To the distribution concessionaires that percentage was 0.1% (one tenth percent). The projects should be guided by innovation, for the purpose of the market and technological challenges in the electrical sector. Thus, the R&D project in this sector needs to be original and innovator. 4 Methodological Procedures and Analysis of Data This study was conducted through the submitted proposals by companies to "R&D Program ANEEL". As such, data were collected from ANEEL database during the period 2008 to 2014. The data considered in this study was “annual expenditures on R&D Projects” reported by energy companies for the aproval by the ANEEL in the period from 2008 to 2014, i.e. after the establishment of the priority research themes, namely: Alternative Sources of Electric Power; Thermoelectric Generation; Basin and Reservoir Management; Environment; Security; Energy Efficiency; Electrical Power Systems Planning; Operation of Power Systems; Supervision; Control and Protection Systems for Electric Energy; Quality and Reliability of Electric Energy Services; Metering, billing and control of commercial losses, and others. The table 2 shows the number of submitted and currently projects as well as the financial amounts to be expended. Table 2. Investments by year and projects submitted Table 2. Investments by year and projects submitted Table 2. Investments by year and projects submitted Year Research and Development R$ Submission Ongoing projects 2008 44.265.986,36 33 30 2009 483.321.604,26 301 102 2010 839.291.149,32 568 293 2011 1.110.007.426,81 483 301 2012 1.772.905.013,27 505 256 2013 586.246.812,17 168 98 2014 584.301.518,42 198 155 Total 5.420.339.510,61 2.256 1.235 Source: ANEEL (march, 2015). Source: ANEEL (march, 2015). 49 http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 The table above shows an increase in R&D by companies from 2008 to 2012. There is a sharp drop in investment due to the loss of revenue resulting from the companies’ renewal of concession agréments, during the period 2013-2014, made by the federal government. Concerning the number of submissions and the number of ongoing projects, the power company may at any time inform the ANEEL lack of interest in carrying out projects that have already been subjected to evaluation. This lack of interest may be of any type ranging from financial problems to the technology to be developed in the project be obsolete or have been exceeded. The project number is running 54% of the submitted projects, but when analyzing the total project investment financial expenditure decreased only 20%. The Figure 1 contains information about the percentage of R & D projects from the project scope. Fig. 1. Percentage by Bussiness Scope of Project (ANEEL, march 2015). http://www.open-jim.org 4 Methodological Procedures and Analysis of Data The graph shows that 53% of the Submitted projects are related to generation, 27% in distribution, 18% transmission and only two percent in energy trading area. The Figure 2 present expenditures (R$) for projects research themes evaluated by ANEEL. Fig. 1. Percentage by Bussiness Scope of Project (ANEEL, march 2015). The graph shows that 53% of the Submitted projects are related to generation, 27% in distribution, 18% transmission and only two percent in energy trading area. The graph shows that 53% of the Submitted projects are related to generation, 27% in distribution, 18% transmission and only two percent in energy trading area. The Figure 2 present expenditures (R$) for projects research themes evaluated by ANEEL. The Figure 2 present expenditures (R$) for projects research themes evaluated by ANEEL. 50 http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 Journal of Innovation Management JIM 5, 1 (2017) 40-57 Fig. 2. Distribution of project research theme (ANEEL, march 2015). 1 870 852 095,1 1 115 384 100,34 212 293 036,90 355 939 291,89 170 150 686,69 187 058 558,32 490 191 819,23 352 111 243,94 580 217 747,65 229 498 336,93 235 990 896,93 620 545 536,69 Project Research Theme Alternative power generation sources Thermal Power generation Basin and Reservoir Management Environment Safety Energy efficiency Electrical Power Systems Planning Systems operation Electricity Supervision, Control and Protection Systems for Electric Energy Quality and Reliability of Electricity Services Metering, billing and control of commercial losses Other Fig. 2. Distribution of project research theme (ANEEL, march 2015). Fig. 2. Distribution of project research theme (ANEEL, march 2015). It´s observed in the graph that the issue has the highest demand is the Sources of electricity generation alternatives with 35% of expenditure on research, followed by Supervision, Control and Protection of Electrical Power Systems with 11%, Systems Planning electricity with 9% and Environment 7%. The other theme accumulates 11%. In phase of the Innovation Chain seen in the Figure 3, investments in research focus on applied research and experimental development. In the analysis of expenditures by modality in the innovation chain is observed that 62% of this was allocated in Applied Research and Experimental Development 29%. These two items account for over 90% of investment in R & D fitting Basic Research 5%, Head Production Series 3%. The phases Market Production Insertion and Pioneer Production Lot account for less than 1% of the investment. 51 51 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 Fig. 3. Phase of Innovation Chain (ANEEL, march 2015). Fig. 4. Main products of projects (ANEEL, march 2015). 248 845 568,1 3 3 409 846 415, 23 1 547 391 974, 67 156 801 166,0 6 54 951 048,84 2 503 337,68 Phase of Innovation Chain Basic Research (BR) Applied Research (AR) Experimental Development (ED) Head Production Series (HD) Piooner Production Lot (HS) Market Production Insertion (MI) 1 994 608 516,22 445 878 750,87 951 099 776,99 134 092 744,50 345 430 177,79 1 549 229 544,25 Main products of projects Concept or methodology Software System Material or substance Component or device Machine or equipment Fig. 3. Phase of Innovation Chain (ANEEL, march 2015). 248 845 568,1 3 3 409 846 415, 23 1 547 391 974, 67 156 801 166,0 6 54 951 048,84 2 503 337,68 Phase of Innovation Chain Basic Research (BR) Applied Research (AR) Experimental Development (ED) Head Production Series (HD) Piooner Production Lot (HS) Market Production Insertion (MI) Fig. 3. Phase of Innovation Chain (ANEEL, march 2015). 248 845 568,1 3 3 409 846 415, 23 1 547 391 974, 67 156 801 166,0 6 54 951 048,84 2 503 337,68 Phase of Innovation Chain Basic Research (BR) Applied Research (AR) Experimental Development (ED) Head Production Series (HD) Piooner Production Lot (HS) Market Production Insertion (MI) 2 2 503 337,68 Phase of Innovation Chain Fig. 4. Main products of projects (ANEEL, march 2015). http://www.open-jim.org Fig. 2. Distribution of project research theme (ANEEL, march 2015). 1 994 608 516,22 445 878 750,87 951 099 776,99 134 092 744,50 345 430 177,79 1 549 229 544,25 Main products of projects Concept or methodology Software System Material or substance Component or device Machine or equipment 52 http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 The expenditure related to the project's main product is closely linked results to academia, so 36% concerns to concept or methodology, followed by 28% Machinery and Equipment and 17% System. The remaining items as software component or device and material or substance amount to 19%. The Intellectual property is divided between the power companies and the performers of the project, as conditions envisaged by Brazilian Innovation Law. Fig. 5. Intellectual Property (ANEEL, march 2015) 188 882 382,90 700 481 979,85 51 613 028,04 2 324 912 946,6 9 1 076 813 613,2 5 Project Results (Intellectual Property) Public domain Exclusive of companies electricity Exclusive (s) organization (s) executor (s) Shared between the company (s) electric power and authority (s) executor (s) Uninformed 188 882 382,90 6 813 613 2 Project Results (Intellectual Property) Fig. 5. Intellectual Property (ANEEL, march 2015) In the Figure 5 it can be observed that the allocation of intellectual property of the results of projects being split between electric power companies and implementing agencies (54%), only 16% of the property is unique to electric company while in the public domain are only 4%. Also that 25% of the ownership of intellectual property was not informed. Regarding investments can be seen in the graph below. The project submissions and the ones going to final are quite different. 53 53 http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 Fig. 6. Submission x ongoing projects (ANEEL, march 2015). 0 100 200 300 400 500 600 2008 2009 2010 2011 2012 2013 2014 Submission x Run Submission Ongoing projects Fig. 6. Submission x ongoing projects (ANEEL, march 2015). Analyzing the graphic (Figure 6) the rising line in R&D investments from 2008 to 2012, it can be observed that the population increased on average 0.9% per year, while per capita consumption showed an average growth of 3.8% per year in the period (EEAA, 2014). This growth was due to the inclusion of low-income consumers under the Light for All Program (LpT). Fig. 2. Distribution of project research theme (ANEEL, march 2015). The program, over its 10 years of existence, accumulated more than 3 million power connections, representing 5% of all residential consumers in the country, and totaling about 15 million people benefited from access to electricity (EEAA, 2014). The decline in the number of projects submitted and executed in 2013 and 2014 is due to the renewal of contracts for large dealers with the federal government, some claim loss of revenue that can be seen in the graph, since there is a requirement for investment in R & D, it can not be accumulated for more than two years. 5 Conclusion In general, the program proposed by ANEEL brought to Brazilian eletric power sector a thousand of projects that were attended by hundreds of research institutions and qualified professionals in their developments. It is therefore important to mention that training and technological capabilities were direct benefits of R&D projects. In addition, new materials and processes have been incorporated to reduce costs, improve the quality of services, and improve the productive capacity. The first conclusion is that 89% increase in the number of companies, now have the obligation of investment of ROL - Net operating revenue in R&D, from 49 companies in 2008 to 91 companies in 2014. 54 54 http://www.open-jim.org http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 However, the billings and investments in R&D projects do not achieved the same growth rate as can be seen in the graph below, which shows the number of projects submitted and the number of projects effectively achieved. The second conclusion is that Brazil is spending too much on basic and applied research, and the results are not progressing in the innovation chain, as the graph shows, the phases Market Production Insertion and Pioneer Production Lot, they account for less than 1% of the investment, i.e., only two and a half million have been invested in these final stages of innovation, a total of more than five billion real (R$) invested in these seven years. Considering the relevance of the subject in an intensely strategic sector for the Brazilian economy, we encourage new future studies to examine the projects that followed the trajectory to market. Finally, it´s worth mentioning that these efforts by ANEEL Program are essential for building a culture of innovation in the Brazilian electric sector. After the analysis performed in this study, we conclude that this challenge is associated with the global challenge in this sector, such as: strengthening the local industry competitiveness, supply chain development and development of new technologies. 6 References AEEE (2014). Anuário Estatístico de Energia Elétrica 2013 - Empresa de Pesquisa Energética - Ministério das Minas de Energia. Barney, J. (2011). Strategic management and competitive advantage. 4th Edition. New Jersey: Prentice Hall. Bés, F. T. & Kotler, P. (2011). Winning at innovation. London: Palgrave McMillan. BPM CBoK (2013). Business process management Common Body of Knowledge. Association of Business process management professionals (ABPMP), Version 3.0, 1st. Ed. Chesbrough, H.W. (2003). Open Innovation: the new imperative for creating and profiting from technology. Boston: Harvard Business School Press. Christensen, C. Disruptive innovation. Clayton Christensen blog. URL: http://www.claytonchristensen.com/key-concepts/. Accessed November, 2015. Christensen, C. & Raynor, M. E. (2003). The innovator solution: creating and sustaining successful growth. Boston: Harvard School Press. Dangelico, M. R. & Pujari, D. (2010). Mainstreaming Green Product Innovation: Why and How Companies Integrate Environmental Sustainability. Journal of Business Ethics 95, 471–486. Davila, T.; Epstein, M. J. & Shelton, R. (2012). Making Innovation Work. New Jersey: Pearson. Dijk, M. Business model commitment and experimentation. InnovationManagment.SE, available at http://www.innovationmanagement.se/2015/11/18/business-model- commitment-and-experimentation/. Accessed on November, 2015. 55 55 http://www.open-jim.org Cândido, Magro, Roczanski, Jamil Journal of Innovation Management JIM 5, 1 (2017) 40-57 Drucker, P. (1985). Innovation and Entrepreneurship. New York: Collins Business. Engen, M. & Holen, I. E. (2014). Radical Versus Incremental Innovation: The Importance of Key Competences in Service Firms. Technology Innovation Management Review 4(4): 15–25. Ettlie, J. E. & Reza, E. M. Organizational integration and process innovation. Academy of Management Journal 4 (35), 795-827. Ferreira, C.K.L. (2000). Privatização no setor elétrico no Brasil. In: Fukasaru, K; Pinheiro, A.C. (orgs.). A privatização no Brasil. Rio de Janeiro: BNDES. Gerstlberger,W.; Knudsen, M. P. & Stampe, I. (2014). Sustainable Development Strategies for Product Innovation and Energy Efficiency. Business Strategy and the Environment Business Strategies 23, 131–144. Hammer, M. & Stanton, S. (1999). How process enterprises really work. Harvard Business Review 6 (77), 108-118. Johsnon, M. W.; Christensen, C. M. & Kagermann, H. (2008). Reiventing your Business Model. Harvard Business Review. Ma, Z.; Gill, T. & Ziang, Y. Core versus peripheral innovations: The effect of innovation locus on consumer adoption of new products. Journal of Marketing Research: American Marketing Association 52, 309-324. Mintzberg, H.; Alhstrand, B. & Lampel, J. (2009). Strategy Safari: the complete guide through the wilds of strategic management, 2nd edition. New Jersey: Pearson Education Limited. OECD – Organisation for Economic Co-operation and Development (2005). OSLO MANUAL.European Comission. Eurostat. Pacheco, R. S. (2004). http://www.open-jim.org 6 References Agências reguladoras na infraestrutura e na área social no Brasil: gênese e indistinções. In: Encontro Annual da ANPOCS 28, Caxambu. PMBoK (2012). A Guide to the Project Management Body of Knowledge, 5th Ed. Pó, M. V.; Abrucio, F. L. (2004). Gênese e indefinições da accountability nas agências reguladoras brasileiras: o caso da Anatel e da ANS. In: Encontro Anual da ANPAD 28, Curitiba. Porter, M. (1998). Competitive Advantage. Free Press. Porter, M. (2008). On Competition. Harvard Business School Press. Salgado, L. H. (2003). Agências reguladoras na experiência brasileira: um panorama do atual desenho institucional. Brasília: Ipea. Sniukas, M. (2015). Design Thinking + Business models innovation. Acessed at http://www.innovationmanagement.se/2015/08/25/design-thinking-business- model-innovtion/. Acessed on September 2015. Trott, P. (2011). Innovation management and new product development. New Jersey: Prentice Hall. Tushman, M. L. & Romanelli, E. (1985). Organizational Evolution: A Metamorphosis Model of Convergence and Reorientation. In L. L. Cummings & M. B. Staw (Eds.). Research in Organizational Behavior 7: 171-222. Greenwich: JAI press. 56 http://www.open-jim.org http://www.open-jim.org Journal of Innovation Management JIM 5, 1 (2017) 40-57 Cândido, Magro, Roczanski, Jamil Wendelken, A., Danzinger, F., Rau, C., & Moeslein, K. M. (2014). Innovation without me: why employees do (not) participate in organizational innovation communities. R&D Management 44(2), 217-236. Utterbach, J. M. (1994). 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https://openalex.org/W4246420939	https://www.researchsquare.com/article/rs-83731/v1.pdf?c=1601603988000	English	null	Squamous cell carcinoma antigens (SCCAs) are sensitive biomarkers for atopic dermatitis in children and adolescents: a cross-sectional study	Research Square (Research Square)	2,020	cc-by	5,278	Squamous cell carcinoma antigens (SCCAs) are sensitive biomarkers for atopic dermatitis in children and adolescents: a cross-sectional study Junya Hirayama Mie University School of Medicine Takao Fujisawa (  eosinophilosophy@gmail.com ) National Hospital Organization Mie National Hospital https://orcid.org/0000-0002-9196-9436 Mizuho Nagao National Hospital Organization Mie National Hospital Yu Kuwabara National Hospital Organization Mie National Hospital Keigo Kainuma National Hospital Organization Mie National Hospital Yoshinori Azuma Sino-Test Corperation Junya Ono Sino-Test Corperation Shoichiro Ohta Saga University School of Medicine Masahiro Hirayama Mie University School of Medicine Kenji Izuhara Saga University School of Medicine Research Research Page 1/15 Page 1/15 Version of Record: A version of this preprint was published at Asia Pacific Allergy on January 1st, 2021. See the published version at https://doi.org/10.5415/apallergy.2021.11.e42. Version of Record: A version of this preprint was published at Asia Pacific Allergy on January 1st, 2021. See the published version at https://doi.org/10.5415/apallergy.2021.11.e42. Version of Record: A version of this preprint was published at Asia Pacific Allergy on January 1st, 2021. See the published version at https://doi.org/10.5415/apallergy.2021.11.e42. Page 2/15 Abstract Background We recently reported that squamous cell carcinoma antigen 2 (SCCA2) is a reliable biomarker for atopic dermatitis (AD) in children. To further clarify its utility, we investigated for possible effects of comorbid allergies and AD treatment on serum SCCA levels in children and adolescents. Methods Volunteers aged less than 18 years were recruited through our website. Their allergic status was elucidated using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. We also recruited pediatric patients who needed to be hospitalized because of severe AD. The serum levels of SCCA1 and SCCA2 were measured by ELISAs. In the severe AD patients, the levels of thymus and activation-regulated chemokine (TARC), SCCA1 and SCCA2 were measured before and after hospitalization. The severity of AD was assessed using the severity scoring of atopic dermatitis (SCORAD) index. Results A total of 576 participants (547 volunteers and 29 patients) were enrolled in the study. The levels of SCCA1 and SCCA2 were significantly higher in volunteers with mild AD and patients with severe AD than in healthy volunteers without allergic diseases. In contrast, the levels were not elevated in those who had mild bronchial asthma or mild allergic rhinitis without AD. TARC, SCCA1 and SCCA2 were decreased in patients with severe AD, reflecting clinical improvement in response to treatment. Linear regression analysis for predicting a decrease in the SCORAD index showed R2 values of 0.16, 0.38 and 0.48 for TARC, SCCA1 and SCCA2, respectively.. Conclusions SCCAs, especially SCCA2, are sensitive biomarkers for detecting AD in children and adolescents, even in the mild stage, and for assessing the severity and response to treatment of severe AD. Background Atopic dermatitis (AD) is a chronic inflammatory skin disorder, and its increasing prevalence is a major public health concern. The goal of treatment of AD is to control skin eruptions and bothersome symptoms, such as itching, so that patients can lead an uninterrupted social life with the best possible quality of life [1]. To achieve that goal, full control of skin inflammation is essential. However, accurate assessment of skin inflammation is sometimes difficult since a physician’s visual examination may not always identify subclinical inflammation [2]. To date, many biomarkers for monitoring skin inflammation in AD have been proposed [3]. However, there are few reliable biomarkers at present, and the American Academy of Dermatology even states that there are no specific biomarkers that can be recommended for diagnosis and/or assessment of disease severity [4]. Squamous cell carcinoma antigens (SCCAs) are serine proteinase inhibitors that belong to the serpin superfamily of proteins [5]. They are expressed in various epithelial tissues [6] and may be involved in epidermal barrier homeostasis [7, 8]. They were originally purified from squamous cell carcinoma of the uterine cervix [9] and have been utilized as markers in various cancers [10–14]. Later, SCCA serum levels Page 3/15 were found to be elevated in various inflammatory skin diseases, including AD [15–18]. Since SCCAs were found to be induced by Th2 cytokines, such as interleukin-4 (IL-4) and IL-13 [16], they may be potential biomarkers for AD as downstream markers of Th2-type immune responses. Several studies suggested that measurement of SCCA1 and SCCA2 in the serum may be promising for evaluating/monitoring the clinical severity of AD [16, 19–23]. Recently, we and others in a multi-center study [23] reported the clinical utility of SCCA2 in a relatively large number of pediatric AD patients. However, the marker was not measured in patients with comorbid allergic diseases such as asthma and allergic rhinitis, which may confound interpretation of the data in AD since most children with AD have multiple allergic diseases. Here, we conducted a cross-sectional study to assess the utility of SCCA1 and SCCA2 in the pediatric population, focusing especially on comorbid allergies. We also assessed the relationship between their serum levels and the disease severity/treatment responses in patients with severe AD. Study participants and outcomes Volunteers aged less than 18 years were recruited through the Mie National Hospital website. Subjects were excluded from the study if they had a non-allergic chronic disease or an acute infectious disease. All volunteers completed the questionnaires of the International Study of Asthma and Allergies in Childhood (ISAAC) [24]. For this study, we used the validated Japanese version [25]. Based on the answers to the questions, the presence or absence of current (the most recent 12 months) symptoms of AD, bronchial asthma (BA) and allergic rhinitis (AR) was determined, and the volunteers were classified into the following 4 groups: healthy volunteers without AD, BA or AR (healthy group); those with BA but not AD (BA group); those with AR but not AD or BA (AR group) and those with AD (AD group 1). We also recruited patients who needed to be hospitalized because of severe AD (AD group 2). During hospitalization, patients were treated with emollients and topical corticosteroids according to clinical practice guidelines [1]. Blood samples were obtained from the study participants to measure the baseline serum levels of SCCA1 and SCCA2. Blood samples were obtained twice (i.e., at the beginning and end of hospitalization) from some patients with severe AD, and the serum levels of thymus and activation-regulated chemokine (TARC), as well as SCCA1 and SCCA2, were measured. TARC is a member of the Th2 type chemokine family and was reported to be a useful biomarker for AD [26–28]. The serum levels of TARC, SCCA1 and SCCA2 were measured using specific enzyme-linked immunosorbent assays (ELISAs) [21, 26]. The disease severity in the patients with severe AD was also assessed at the beginning and end of hospitalization using the severity scoring of atopic dermatitis (SCORAD) index [29]. Statistical analyses Page 4/15 Baseline serum levels of SCCA1 and SCCA2 were summarized as the geometric mean and standard deviation (SD). The logarithmically transformed means were compared among the participant groups by analysis of variance. Tukey’s multiple comparison test was used to assess all pairwise differences between the participant groups. For the severe AD patients, we calculated Spearman’s correlation coefficients between the SCORAD index and the logarithmically transformed values of TARC, SCCA1 and SCCA2 levels, and between the decrease in the SCORAD index and the decrease in each of the logarithmically transformed biomarker levels. All data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc.; CA, USA). All reported P values are two-sided. Characteristics of subjects A total of 576 participants (547 volunteers and 29 severe AD patients) were enrolled in the study (Fig. 1). All participants were included in the analyses. Table 1 summarizes the demographics, gender, age and allergic comorbidities of the subjects. The patients were relatively younger than the volunteers, with mean (SD) ages of 4.0 (4.9) years in AD group 2, and ranging from 6.7 (2.1) to 9.3 (3.2) years in the other groups. Table 1 Baseline characteristics of the study participants Volunteers Patients Healthy Group (n = 179) BA Group (n = 58) AR Group (n = 181) AD Group 1 (n = 129) AD Group 2 (n = 29) Male (n, %) 86 (48) 35 (60) 97 (54) 63 (49) 15 (52) Age (years, mean ± SD) 7.5 ± 2.8 6.7 ± 2.1 9.3 ± 3.2 8.0 ± 3.5 4.0 ± 4.9 Comorbidity Atopic dermatitis (n, %) 0 (0) 0 (0) 0 (0) — — Bronchial asthma (n, %) 0 (0) 58 (100) 0 (0) 6 (5) 2 (7) Allergic rhinitis (n, %) 0 (0) 38 (66) 65 (100) 65 (50) 4 (14) Abbreviations: n = the number of participants; SD = standard deviation; AD = atopic dermatitis; BA = bronchial asthma; AR = allergic rhinitis. Healthy group = healthy volunteers without allergic diseases; BA group = volunteers with BA; AR group = volunteers with AR; AD group 1 = volunteers with AD; AD group 2 = patients who needed to be hospitalized because of severe AD. Table 1 Table 1 Baseline characteristics of the study participants Volunteers Reference and cut-off values for SCCA1 and SCCA2 The healthy children were further divided into 3 age groups: 1–5, 6–10 and 11–17 years old. Table S2 summarizes the descriptive statistics of SCCA1 and SCCA2 in those groups. Although SCCA1 and SCCA2 were slightly higher in the 2 younger groups, there were no significant differences among the groups. Next, we calculated the geometric mean and SD for all healthy volunteers. As a result, the reference values (geometric mean − 2SD to geometric mean + 2SD) of SCCA1 and SCCA2 were 0.45 to 1.91 ng/mL and 0.21 to 1.93 ng/mL, respectively. ROC analysis was performed to determine the diagnostic performance of the biomarkers in identifying AD in the volunteer groups. The AUCs were 0.676 for SCCA1 and 0.715 for SCCA2. The cut-off levels based on the highest Youden index were 1.09 and 0.84 ng/ml, respectively. The cut-off levels at 95% specificity were 1.65 and 1.5 ng/ml, respectively (Table 2). Table 2 Diagnostic performance of SCCA1 and SCCA2 for AD in the volunteer population AUC (95%CI) Cut-off Sensitivity (95%CI) Specificity (95%CI) PPV † NPV † SCCA1 0.676 (0.613–0.738) 1.09 0.62 (0.53–0.70) 0.68 (0.61–0.75) 0.38 0.85 1.65 0.26 (0.19–0.35) 0.95 (0.91–0.98) 0.62 0.81 SCCA2 0.715 (0.657–0.774) 0.84 0.61 (0.52–0.70) 0.71 (0.64–0.77) 0.39 0.86 1.50 0.35 (0.27–0.44) 0.95 (0.91–0.98) 0.68 0.83 † PPV (positive predictive value) and NPV (negative predictive value) were calculated based on the prevalence of AD in the volunteer population. † PPV (positive predictive value) and NPV (negative predictive value) were c prevalence of AD in the volunteer population. † PPV (positive predictive value) and NPV (negative predictive value) were calculated based on the prevalence of AD in the volunteer population. Baseline characteristics of the study participants Volunteers Abbreviations: n = the number of participants; SD = standard deviation; AD = atopic dermatitis; BA = bronchial asthma; AR = allergic rhinitis. Healthy group = healthy volunteers without allergic diseases; BA group = volunteers with BA; AR group = volunteers with AR; AD group 1 = volunteers with AD; AD group 2 = patients who needed to be hospitalized because of severe AD. Page 5/15 Page 5/15 Serum levels of SCCA1 and SCCA2 in each allergic disease group Figure 2 and Table S1 show the serum levels of SCCA1 and SCCA2 in the 5 groups. Table S1 summarizes the pairwise comparisons between the groups. While the serum levels of SCCA1 and SCCA2 were similar among the healthy, BA and AR groups, they were significantly higher in AD groups 1 and 2 than in the healthy group (p < 0.0001). and also significantly higher (p < 0.0001) in AD groups 1 and 2 than in the BA and AR groups. Finally, those serum levels were significantly higher in AD group 2 than in AD group 1 (p < 0.0001). The differences in their geometric means were 6.6 ng/mL for SCCA1 and 20.0 ng/mL for SCCA2. SCCA1 and SCCA2 in severe AD patients Blood samples were obtained at the beginning and end of hospitalization of 18 severe AD patients. During the treatment in the hospital, the serum levels of TARC, SCCA1 and SCCA2 decreased significantly in all patients (Figs. 3A, 3B and 3C, respectively) and correlated significantly with the SCORAD index Page 6/15 Page 6/15 (Figs. 3D, 3E, and 3F, respectively). Spearman’s correlation coefficients were 0.6856, 0.7513 and 0.7885, respectively. We also assessed the relationship between the decrease in the SCORAD index and the decreases in the serum levels of TARC, SCCA1 and SCCA2 (Figs. 3G, 3H and 3I, respectively). Among these biomarkers, a decrease in the SCCA2 level most strongly predicted improvement in the severity, i.e., a decrease in the SCORAD index, with R2 = 0.476. Discussion In this study, children and adolescents with AD showed significantly higher serum levels of SCCA1 and SCCA2 compared with healthy volunteers. The levels were not elevated in those with BA or AR without AD, indicating that SCCAs are specific markers for AD in allergic children. Their levels were higher in AD group 2 (patients with severe AD) than in AD group 1 (volunteers with AD, presumably mild disease). We also found that the serum levels of SCCAs in AD group 2 correlated significantly with the SCORAD index, and decreases in their levels during treatment correlated well with decreases in the SCORAD index. The correlation indices were highest for SCCA2 compared with SCCA1 and TARC. These results indicate that SCCAs are reliable biomarkers for AD, and that the serum levels of SCCAs, especially SCCA2, predict the disease severity and treatment response. TARC has been regarded as the most reliable biomarker of the severity of AD [3]. TARC is a ligand for CC chemokine receptor 4, which is selectively expressed on Th2 type cells. It is produced by dendritic cells, endothelial cells and keratinocytes, and its overproduction leads to Th2 type cell accumulation at inflammation sites [30]. In contrast, SCCAs are induced by Th2 type cytokines such as IL-4 and IL-13 [16]. Therefore, SCCAs may reflect the “down-stream” of the immune response, which might lead to superiority of SCCAs in our study. The reason for the stronger correlation of the SCCA2 level with disease severity remains unclear. An earlier study showed that keratinocytes produced mainly SCCA2 upon stimulation with IL-4 or IL-13 [21]. This result may in part explain the superiority of SCCA2 over SCCA1 for disease activity. In addition, serum TARC levels are high in children, especially infants [26]. Thus, it is necessary to have different reference and cut-off values for TARC in different age groups for clinical use. On the contrary, the serum levels of SCCAs in healthy children were similar among the different age groups in our study. Although the levels were slightly higher in the 2 younger groups, the differences were not statistically significant and were much smaller than those found for TARC [26]. In our previous report [23], we demonstrated the validity of a single cut-off at 1.6 ng/ml for SCCA2. We also determined the normal ranges and cut-off levels of SCCA1 and SCCA2, irrespective of age, which may be helpful in clinical application. Discussion Because different subject groups were recruited for determination of cut-off levels, i.e., only mild AD in this study and more severe AD in the previous study, the cut-off levels of SCCA2 were different However, the cut-off level at 95% specificity was 1.5 ng/ml in this studyis close tothe 1.6 ng/ml in the previous study. In addition, serum TARC levels are high in children, especially infants [26]. Thus, it is necessary to have different reference and cut-off values for TARC in different age groups for clinical use. On the contrary, the serum levels of SCCAs in healthy children were similar among the different age groups in our study. Although the levels were slightly higher in the 2 younger groups, the differences were not statistically significant and were much smaller than those found for TARC [26]. In our previous report [23], we demonstrated the validity of a single cut-off at 1.6 ng/ml for SCCA2. We also determined the normal ranges and cut-off levels of SCCA1 and SCCA2, irrespective of age, which may be helpful in clinical application. Because different subject groups were recruited for determination of cut-off levels, i.e., only mild AD in this study and more severe AD in the previous study, the cut-off levels of SCCA2 were different. However, the cut-off level at 95% specificity was 1.5 ng/ml in this studyis close tothe 1.6 ng/ml in the previous study. Page 7/15 An important finding in this study was that the serum levels of SCCAs correlated with the severity of AD. It is sometimes difficult to assess the severity and evaluate the outcome of a certain treatment because physicians’ visual examinations are not always accurate [28]. Various instruments have been developed to measure the symptoms of AD, and several have been recommended as core outcome sets [31]. However, the main objective of the recommendation was to define the core outcome sets that should be used in clinical trials. These instruments are not suitable for use in daily clinical practice because their scoring systems are complex [32]. Accordingly, a simple and reliable biomarker for assessing disease severity would be a great breakthrough. The serum levels of SCCAs were similar between healthy volunteers and those with BA or AR. Discussion In a study that included children with acute asthma, serum SCCA levels increased only in the acute phase of asthma exacerbation, while in the recovery phase they were similar to those in age-matched healthy children [33]. Another study found the SCCA levels to be elevated in BA patients compared with controls, but the difference was only 1 ng/mL [34]. Furthermore, serum SCCA levels in adult patients with AR caused by cedar pollen were similar to those in healthy adult volunteers, and the median level in patients with AR caused by Dermatophagoides farinaen was only 0.20 ng/mL higher than that in healthy volunteers [35]. Our present findings are consistent with those earlier results. However, serum levels of SCCAs were reported to be elevated in patients with psoriasis [17], so special attention needs to be paid to differential diagnosis of AD from psoriasis. This study has several limitations. First, we used the SCORAD index as a severity scale, but that is not considered to be a gold-standard severity scale [4]. Second, we did not assess the relationship between the disease severity and the serum levels of biomarkers in patients with mild or moderate AD. However, we previously reported the SCCA2 levels in that population of AD patients [23], and one of the purposes of this study was to clarify utility of SCCAs in AD children with comorbidities In conclusion, the serum levels of SCCA1 and SCCA2 were elevated in children and adolescents with AD. In addition, the SCCA2 level correlated more strongly with the SCORAD index than the TARC or SCCA1 level. We think that SCCA2 has potential as a useful and reliable biomarker for assessing the severity of AD in children and adolescents and their responses to treatment. Abbreviations AD: atopic dermatitis Page 8/15 Page 8/15 TARC: thymus and activation-regulated chemokine Funding This research was supported by a grant-in-aid from the Japan Agency for Medical Research and Development (AMED). Authors’ contributions: Takao Fujisawa and Mizuho Nagao conceived the study. Junya Hirayama, Takao Fujisawa, Mizuho Nagao, Yu Kuwabara and Keigo Kainuma recruited the subjects and contributed to data collection. Yoshinori Azuma and Junya Ono conducted the assays. Junya Hirayama, Takao Fujisawa and Mizuho Nagao conducted the analyses of the data. Shoichiro Ohta and Kenji Izuhara contributed to interpretation of the data. Junya Hirayama and Takao Fujisawa wrote the manuscript. Masahiro Hirayama gave critical comments on the study design and manuscript. All the listed authors were involved in the review of the manuscript and approved the final content. Ethics approval and consent to participate The study protocol was approved by the Ethics Committee of Mie National Hospital (Approval No: 24-1). All guardians of the participants provided written informed consent. Consent for publication Consent for publication All guardians of the participants provided written informed consent. All guardians of the participants provided written informed consent. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests TF received lecture fees from Maruho Co., Ltd. and MSD Co., Ltd.. YA and JO are employees of Shino-Test Corporation. All other authors declare that they have no conflicts of interest. Declarations Ethics approval and consent to participate Acknowledgments The authors would like to thank Ms. Sumiko Yoshikawa, Ms. Manami Negoro and Ms. Kyoko Nishinaka for their excellent technical assistance. Writing and editing assistance was provided by Kenichi Hayashi (Alamedic Co., Ltd.; Tokyo, Japan) under contract with the principal authors (JH, MN and TF). Page 9/15 Page 9/15 References 1. Katoh N, Ohya Y, Ikeda M, Ebihara T, Katayama I, Saeki H, et al. Clinical practice guidelines for the management of atopic dermatitis 2018. The Journal of dermatology. 2019;46(12):1053-101. 2. Tang TS, Bieber T, Williams HC. Are the concepts of induction of remission and treatment of subclinical inflammation in atopic dermatitis clinically useful? JAllergy ClinImmunol. 2014;133(6):1615-25 e1. 3. Thijs J, Krastev T, Weidinger S, Buckens CF, de Bruin-Weller M, Bruijnzeel-Koomen C, et al. Biomarkers for atopic dermatitis: a systematic review and meta-analysis. Curr Opin Allergy Clin Immunol. 2015;15(5):453-60. 4. Eichenfield LF, Tom WL, Chamlin SL, Feldman SR, Hanifin JM, Simpson EL, et al. Guidelines of care for the management of atopic dermatitis: section 1. Diagnosis and assessment of atopic dermatitis. J Am Acad Dermatol. 2014;70(2):338-51. 5. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, et al. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. JBiolChem. 2001;276(36):33293-6. 6. Cataltepe S, Gornstein ER, Schick C, Kamachi Y, Chatson K, Fries J, et al. Co-expression of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and squamous cell carcinomas. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 2000;48(1):113-22. 7. Sivaprasad U, Kinker KG, Ericksen MB, Lindsey M, Gibson AM, Bass SA, et al. SERPINB3/B4 contributes to early inflammation and barrier dysfunction in an experimental murine model of atopic dermatitis. J Invest Dermatol. 2015;135(1):160-9. 7. Sivaprasad U, Kinker KG, Ericksen MB, Lindsey M, Gibson AM, Bass SA, et al. SERPINB3/B4 contributes to early inflammation and barrier dysfunction in an experimental murine model of atopic dermatitis. J Invest Dermatol. 2015;135(1):160-9. 8. Sakata Y, Arima K, Takai T, Sakurai W, Masumoto K, Yuyama N, et al. The squamous cell carcinoma antigen 2 inhibits the cysteine proteinase activity of a major mite allergen, Der p 1. JBiolChem. 2004;279(7):5081-7. 8. Sakata Y, Arima K, Takai T, Sakurai W, Masumoto K, Yuyama N, et al. The squamous cell carcinoma antigen 2 inhibits the cysteine proteinase activity of a major mite allergen, Der p 1. JBiolChem. 2004;279(7):5081-7. 9. Kato H, Torigoe T. Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma. Cancer. 1977;40(4):1621-8. 9. Kato H, Torigoe T. Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma. Cancer. 1977;40(4):1621-8. 10. References Shimura K, Mabuchi S, Yokoi T, Sasano T, Sawada K, Hamasaki T, et al. Utility of serum squamous cell carcinoma antigen levels at the time of recurrent cervical cancer diagnosis in determining the optimal treatment choice. J Gynecol Oncol. 2013;24(4):321-9. 10. Shimura K, Mabuchi S, Yokoi T, Sasano T, Sawada K, Hamasaki T, et al. Utility of serum squamous cell carcinoma antigen levels at the time of recurrent cervical cancer diagnosis in determining the optimal treatment choice. J Gynecol Oncol. 2013;24(4):321-9. 11. Williams M, Swampillai A, Osborne M, Mawdsley S, Hughes R, Harrison M, et al. Squamous cell carcinoma antigen: a potentially useful prognostic marker in squamous cell carcinoma of the anal canal and margin. Cancer. 2013;119(13):2391-8. 11. Williams M, Swampillai A, Osborne M, Mawdsley S, Hughes R, Harrison M, et al. Squamous cell carcinoma antigen: a potentially useful prognostic marker in squamous cell carcinoma of the anal canal and margin. Cancer. 2013;119(13):2391-8. 12. Yin M, Hou Y, Zhang T, Cui C, Zhou X, Sun F, et al. Evaluation of chemotherapy response with serum squamous cell carcinoma antigen level in cervical cancer patients: a prospective cohort study. PLoS One. 2013;8(1):e54969. 12. Yin M, Hou Y, Zhang T, Cui C, Zhou X, Sun F, et al. Evaluation of chemotherapy response with serum squamous cell carcinoma antigen level in cervical cancer patients: a prospective cohort study. PLoS One. 2013;8(1):e54969. Page 10/15 13. Chen IH, Liao CT, Wang HM, Huang JJ, Kang CJ, Huang SF. Using SCC antigen and CRP levels as prognostic biomarkers in recurrent oral cavity squamous cell carcinoma. PLoS One. 2014;9(7):e103265. 14. van Zijl F, Monserez DA, Korevaar TIM, Bugter O, Wieringa MH, Baatenburg de Jong RJ, et al. Postoperative value of serum squamous cell carcinoma antigen as a predictor of recurrence in sinonasal inverted papilloma. Clin Otolaryngol. 2017;42(3):528-35. 14. van Zijl F, Monserez DA, Korevaar TIM, Bugter O, Wieringa MH, Baatenburg de Jong RJ, et al. Postoperative value of serum squamous cell carcinoma antigen as a predictor of recurrence in sinonasal inverted papilloma. Clin Otolaryngol. 2017;42(3):528-35. 15. Campbell B, De'Ambrosis B. Squamous cell carcinoma antigen in patients with cutaneous disorders. J Am Acad Dermatol. 1990;22(4):639-42. 15. Campbell B, De'Ambrosis B. Squamous cell carcinoma antigen in patients with cutaneous disorders. J Am Acad Dermatol. 1990;22(4):639-42. 16. Mitsuishi K, Nakamura T, Sakata Y, Yuyama N, Arima K, Sugita Y, et al. References The squamous cell carcinoma antigens as relevant biomarkers of atopic dermatitis. ClinExpAllergy. 2005;35(10):1327-33. 16. Mitsuishi K, Nakamura T, Sakata Y, Yuyama N, Arima K, Sugita Y, et al. The squamous cell carcinoma antigens as relevant biomarkers of atopic dermatitis. ClinExpAllergy. 2005;35(10):1327-33. 17. Watanabe Y, Yamaguchi Y, Komitsu N, Ohta S, Azuma Y, Izuhara K, et al. Elevation of serum squamous cell carcinoma antigen 2 in patients with psoriasis: associations with disease severity and response to the treatment. Br J Dermatol. 2016;174(6):1327-36. 18. Horiuchi Y, Tsukahara T, Otoyama K. Immunohistochemical study of elevated expression of squamous cell carcinoma (SCC)-related antigens in erythrodermic epidermis. The Journal of dermatology. 1994;21(2):67-72. 19. Kawashima H, Nishimata S, Kashiwagi Y, Numabe H, Sasamoto M, Iwatsubo H, et al. Squamous cell carcinoma-related antigen in children with atopic dermatitis. Pediatr Int. 2000;42(4):448-50. 20. Yamane Y, Moriyama K, Yasuda C, Miyata S, Aihara M, Ikezawa Z, et al. New horny layer marker proteins for evaluating skin condition in atopic dermatitis. Int Arch Allergy Immunol. 2009;150(1):89- 101. 21. Ohta S, Shibata R, Nakao Y, Azuma Y, Taniguchi K, Arima K, et al. The usefulness of combined measurements of squamous cell carcinoma antigens 1 and 2 in diagnosing atopic dermatitis. Annals of clinical biochemistry. 2012;49(Pt 3):277-84. 22. Okawa T, Yamaguchi Y, Kou K, Ono J, Azuma Y, Komitsu N, et al. Serum levels of squamous cell carcinoma antigens 1 and 2 reflect disease severity and clinical type of atopic dermatitis in adult patients. Allergol Int. 2018;67(1):124-30. 23. Nagao M, Inagaki S, Kawano T, Azuma Y, Nomura N, Noguchi Y, et al. SCCA2 is a reliable biomarker for evaluating pediatric atopic dermatitis. JAllergy ClinImmunol. 2018;141(5):1934-6 e11. 24. Asher MI, Keil U, Anderson HR, Beasley R, Crane J, Martinez F, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. EurRespirJ. 1995;8(3):483-91. 25. Futamura M, Ohya Y, Akashi M, Adachi Y, Odajima H, Akiyama K, et al. Age-related prevalence of allergic diseases in Tokyo schoolchildren. Allergol Int. 2011;60(4):509-15. 25. Futamura M, Ohya Y, Akashi M, Adachi Y, Odajima H, Akiyama K, et al. Age-related prevalence of allergic diseases in Tokyo schoolchildren. Allergol Int. 2011;60(4):509-15. 26. Fujisawa T, Nagao M, Hiraguchi Y, Katsumata H, Nishimori H, Iguchi K, et al. Serum measurement of thymus and activation-regulated chemokine/CCL17 in children with atopic dermatitis: elevated normal levels in infancy and age-specific analysis in atopic dermatitis. Pediatr Allergy Immunol. 2009;20(7):633-41. Page 11/15 27. References Tamaki K, Kakinuma T, Saeki H, Horikawa T, Kataoka Y, Fujisawa T, et al. Serum levels of CCL17/TARC in various skin diseases. The Journal of dermatology. 2006;33(4):300-2. 28. Kataoka Y. Thymus and activation-regulated chemokine as a clinical biomarker in atopic dermatitis. The Journal of dermatology. 2014;41(3):221-9. 29. Severity scoring of atopic dermatitis: the SCORAD index. Consensus Report of the European Task Force on Atopic Dermatitis. Dermatology. 1993;186(1):23-31. 30. Saeki H, Tamaki K. Thymus and activation regulated chemokine (TARC)/CCL17 and skin diseases. J Dermatol Sci. 2006;43(2):75-84. 31. Gerbens LA, Prinsen CA, Chalmers JR, Drucker AM, von Kobyletzki LB, Limpens J, et al. Evaluation of the measurement properties of symptom measurement instruments for atopic eczema: a systematic review. Allergy. 2017;72(1):146-63. 32. de Bruin Weller MS, Knulst AC, Meijer Y, Bruijnzeel-Koomen CA, Pasmans SG. Evaluation of the child with atopic dermatitis. ClinExpAllergy. 2012;42(3):352-62. 33. Nishi N, Miyazaki M, Tsuji K, Hitomi T, Muro E, Zaitsu M, et al. Squamous cell carcinoma-related antigen in children with acute asthma. Ann Allergy Asthma Immunol. 2005;94(3):391-7. 34. Yuyama N, Davies DE, Akaiwa M, Matsui K, Hamasaki Y, Suminami Y, et al. Analysis of novel disease-related genes in bronchial asthma. Cytokine. 2002;19(6):287-96. 35. Suzuki K, Inokuchi A, Miyazaki J, Kuratomi Y, Izuhara K. Relationship between squamous cell carcinoma antigen and the clinical severity of allergic rhinitis caused by Dermatophagoides farinae and Japanese cedar pollen. Ann Otol Rhinol Laryngol. 2010;119(1):22-6. 35. Suzuki K, Inokuchi A, Miyazaki J, Kuratomi Y, Izuhara K. Relationship between squamous cell carcinoma antigen and the clinical severity of allergic rhinitis caused by Dermatophagoides farinae and Japanese cedar pollen. Ann Otol Rhinol Laryngol. 2010;119(1):22-6. Figures Page 12/15 Figure 1 Figure 1 Subjects enrolled in the study Subjects enrolled in the study j y Figure 2 Baseline serum levels of SCCA1 (Fig. 1A) and SCCA2 (Fig. 1B) in the study participants. The geometric mean serum levels of SCCA1 and SCCA2 differed among the 5 participant groups (p < 0.0001; analysis of variance). Figure 2 Figure 2 Baseline serum levels of SCCA1 (Fig. 1A) and SCCA2 (Fig. 1B) in the study participants. The geometric mean serum levels of SCCA1 and SCCA2 differed among the 5 participant groups (p < 0.0001; analysis of variance). Page 13/15 Figure 3 Page 14/15 Figure 3 Serum levels of TARC, SCCA1 and SCCA2 before and after treatment of severe AD patients Changes in the serum levels of TARC (A), SCCA1 (B) and SCCA2 (C) between the beginning and end of hospitalization. * P<0.0001 Wilcoxon matched-pairs signed rank test. Relationships between the decrease in the severity scoring of atopic dermatitis (SCORAD) index and the decreases in serum levels TARC (D), SCCA1 (E) and SCCA2 (F) during hospitalization. Closed and open circles indicate the values the beginning and end of hospitalization, respectively. * P<0.0001 Spearman’s correlation test. The X Figure 3 Page 14/15 Serum levels of TARC, SCCA1 and SCCA2 before and after treatment of severe AD patients Changes in the serum levels of TARC (A), SCCA1 (B) and SCCA2 (C) between the beginning and end of hospitalization. * P<0.0001 Wilcoxon matched-pairs signed rank test. Relationships between the decrease in the severity scoring of atopic dermatitis (SCORAD) index and the decreases in serum levels of TARC (D), SCCA1 (E) and SCCA2 (F) during hospitalization. Closed and open circles indicate the values at the beginning and end of hospitalization, respectively. * P<0.0001 Spearman’s correlation test. The X- Page 14/15 axes show the decrease in the SCORAD index, while the Y-axes show the decrease in the logarithmically transformed serum levels of TARC (G), SCCA1 (H) and SCCA2 (I), respectively. axes show the decrease in the SCORAD index, while the Y-axes show the decrease in the logarithmically transformed serum levels of TARC (G), SCCA1 (H) and SCCA2 (I), respectively. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTablesAACIfinal.docx Page 15/15
https://openalex.org/W2593708391	https://researchonline.lshtm.ac.uk/id/eprint/3599414/1/metaanalytical.pdf	English	null	Meta-analytical methods to identify who benefits most from treatments: daft, deluded, or deft approach?	BMJ. British medical journal	2,017	cc-by	6,569	Meta-analytical methods to identify who benefits most from treatments: daft, deluded, or deft approach? These complications are often overlooked by reviewers. The issues are independent of other aspects of meta-analysis, such as the choice of one stage or two stage model fitting, or whether aggre- gate data or individual participant data (IPD)1 is used. The three titular approaches discussed in this paper are derived from two independent quantities referred to in recent IPD literature as across-trial and within-trial interactions.2 3 We have used three descriptive terms to be memorable and to avoid ambiguity. “Daft” (meaning absurd or preposterous) refers to estimation of the across-trial interaction alone. “Deluded” (meaning mis- leading or deceiving) refers to an estimation of both the across-trial and within-trial interactions combined. “Deft” (demonstrating skill or cleverness) refers to esti- mation of the within-trial interaction alone. As their monikers suggest, daft and (to a lesser but non- negligible extent) deluded approaches are at risk of bias4—the extent of which is typically unknown— whereas deft approaches are not. Cite this as: BMJ 2017;356:j573 http://dx.doi.org/10.1136/bmj.j573 Accepted: 20 January 2017 open access open access open access Summary points Meta-analysis is often the only way to reliably detect whether treatment benefit differs between groups of participants—that is, to detect interactions between treatment efficacy and participant characteristics Of three general approaches, we advocate the deft approach, which avoids the risk of ecological bias over the deluded and daft approaches, which do not A systematic review of recently published meta-analyses of individual participant data shows that 89% of interaction analyses either used a deluded method (23%), or did not report sufficient details (66%) to tell which approach had been used. Further, graphical presentation often hindered appreciation of key data features These findings indicate poor appreciation of analysis and reporting issues surrounding interactions in the research community Of three general approaches, we advocate the deft approach, which avoids the risk of ecological bias over the deluded and daft approaches, which do not How to analyse and present participant level interactions data from a meta-analysis: daft, deluded, or deft approach Our illustrative example is a systematic review and IPD meta-analysis relating to the care of patients with acute stroke, which compared a strategy of early supported hospital discharge (ESD) to conventional hospital ser- vices and discharge arrangements.5 ESD reduced the mean duration of initial hospital stay. The authors investigated whether this effect varied according to whether a participant had a carer present—that is, whether there was an interaction between treatment and presence of a carer. Here, we describe the results from this published IPD meta-analysis using three approaches we previously identified.2 Research Methods and Reporting Research Methods and Reporting Meta-analytical methods to identify who benefits most from treatments: daft, deluded, or deft approach? David J Fisher,1 James R Carpenter,1,2 Tim P Morris,1 Suzanne C Freeman,1 Jayne F Tierney1 Identifying which individuals benefit most from particular treatments or other interventions underpins so-called personalised or stratified medicine. However, single trials are typically underpowered for exploring whether participant characteristics, such as age or disease severity, determine an individual’s response to treatment. A meta-analysis of multiple trials, particularly one where individual participant data (IPD) are available, provides greater power to investigate interactions between participant characteristics (covariates) and treatment effects. We use a published IPD meta-analysis to illustrate three broad approaches used for testing such interactions. Based on another systematic review of recently published IPD meta-analyses, we also show that all three approaches can be applied to aggregate data as well as IPD. We also summarise which methods of analysing and presenting interactions are in current use, and describe their advantages and disadvantages. We recommend that testing for interactions using within-trials information alone (the deft approach) becomes standard practice, alongside graphical presentation that directly visualises this. Meta-analysis of participant level treatment-covariate interactions raises additional complications that do not affect either standard meta-analysis or single trial inter- action analysis alone. These complications are often overlooked by reviewers. The issues are independent of other aspects of meta-analysis, such as the choice of one stage or two stage model fitting, or whether aggre- gate data or individual participant data (IPD)1 is used. The three titular approaches discussed in this paper are derived from two independent quantities referred to in recent IPD literature as across-trial and within-trial interactions.2 3 We have used three descriptive terms to be memorable and to avoid ambiguity. “Daft” (meaning absurd or preposterous) refers to estimation of the across-trial interaction alone. “Deluded” (meaning mis- leading or deceiving) refers to an estimation of both the across-trial and within-trial interactions combined. “Deft” (demonstrating skill or cleverness) refers to esti- mation of the within-trial interaction alone. As their monikers suggest, daft and (to a lesser but non- negligible extent) deluded approaches are at risk of bias4—the extent of which is typically unknown— whereas deft approaches are not. advantages and disadvantages. We recommend that testing for interactions using within-trials information alone (the deft approach) becomes standard practice, alongside graphical presentation that directly visualises this. Meta-analysis of participant level treatment-covariate interactions raises additional complications that do not affect either standard meta-analysis or single trial inter- action analysis alone. 1London Hub for Trials Methodology Research, MRC Clinical Trials Unit, University College London, London, UK 2Department of Medical Statistics, London School of Hygiene and Tropical Medicine, London, UK Correspondence to: D J Fisher d.fisher@ucl.ac.uk Additional material is published online only. To view please visit the journal online. Cite this as: BMJ 2017;356:j573 http://dx.doi.org/10.1136/bmj.j573 Accepted: 20 January 2017 Deft approach (within-trial interaction alone) This approach is presented in figure 1 . The left panel shows an attempt to make better use of the data by car- rying out a meta-analysis within the subgroup of partic- ipants who had a carer present, and another in those who did not. The right panel shows a simpler alternative presentation of this type of analysis that is often seen in the literature. The interaction test compares the treat- ment effects in the two carer subgroup level meta-anal- yses, but can also be extended to explore trends in effect across more than two ordered categories, such as stage of disease. As in the original review,5 this t pp This approach adheres to the underlying principles of meta-analysis by assessing the effect of interest as mea- sured within each relevant trial. The left panel in figure 2 shows the same data as those in figure 1 , but rearranged so as to demonstrate this approach. We test for an association between the effect of ESD and pres- ence of a carer within each trial, instead of testing for an association across carer subgroups. These interactions, interpretable as the difference in treatment effect for an individual participant with a carer present compared to Carer present Adelaide 2000 Belfast 2004 London 1999 Manchester 2001 Montreal 2000 Newcastle 1997 Oslo 2000 Stockholm 1998 Trondheim 2004 Subtotal: P=0.008, I2=61.5% No carer Adelaide 2000 Belfast 2004 London 1999 Manchester 2001 Newcastle 1997 Oslo 2000 Stockholm 1998 Trondheim 2004 Subtotal: P=0.402, I2=3.6% -3.88 (-5.96 to -1.80) -6.11 (-10.71 to -1.50) * Interaction (diference in mean diferences) 2.23 (95% CI -2.82 to 7.28) 82.99 17.01 -50 0 50 Subgroup and trial Favours ESD Favours no ESD Mean diference (95% CI) -10 0 * 10 Favours ESD Favours no ESD Mean diference (95% CI) Weight (%) Fig 1 \| Use of a deluded approach to analyse and present interactions in meta-analysis , illustrating how the effect of an early supported hospital discharge (ESD) strategy might vary by whether a carer is present.5 Left panel presents the effect of ESD for each subgroup within each trial, but ordered by subgroup; and right panel presents just the meta-analysed effects for each subgroup. The difference between the effects in right panel gives a deluded analysis (mean difference of 2.23, 95% confidence interval −2.82 to 7.28, P=0.39). Research Methods and Reporting approach suggests that the presence of a carer does not modify the effect of ESD (P=0.39). However, consider how the data is actually being used: trial data is split into participant subgroups, treatment effects are com- bined within these subgroups, and are then compared across subgroups. This process combines within-trial and across-trial interaction estimates.3 The analysis is again subject to ecological bias, although the addition of across-trial information can provide a gain in power. How the treatment effect varies at the individual parti cipant level (that is, the within-trial interaction) could be exaggerated or masked by the across-trial interaction; we are at risk of being deluded. In the large Montreal trial,w87 all participants had a carer, and hence this trial can contribute only an across-trial interaction (web fig). proportion of trial participants with a carer present increases, the benefit of ESD decreases (P<0.001, web fig 1). This analysis focuses solely on how the overall effect of ESD varies across trials with different propor- tions of carers present, relying on an aggregated sum- mary of the carer information for each trial rather than considering whether the effect for an individual partic- ipant varies according to the presence of a carer. Such an analysis is easily confounded and is at risk of ecolog- ical bias, whereby interactions at the aggregate (or “eco- logical”) level might not reflect the true interaction at the individual participant level.4 Given the available data, this is a daft approach. Daft approach (across-trial interaction alone) This approach can be visualised by plotting the mean difference in the length of hospital stay (ESD v standard care) for each trial against the proportion of trial partic- ipants who had a carer present. Fitting a meta- regression6 to the data points, we find that as the Where suitable data were reported in published systematic reviews, reanalysis showed that use of the different approaches can yield inconsistent results T ti f i t ti i d ft th d d i hi l t ti th t Testing for interactions using deft methods, and using a graphical presentation that directly visualises this, should become standard practice the bmj \| BMJ 2017;356:j573 \| doi: 10.1136/bmj.j573 1 Research Methods and Reporting Deft approach (within-trial interaction alone) Sizing of squares are in proportion to the inverse of the variance of the estimates. Note that the subgroup meta-analysis estimates do not match exactly those originally reported, because we used a fixed effect model for simplicity, rather than the random-effects model used by the original authors. See also web appendix A3 for references of studies and appendix B2 for details of statistical reanalysis Mean diference (95% CI) Weight (%) Weight (%) Mean diference (95% CI) Mean diference (95% CI) * Interaction (diference in mean diferences) 2.23 (95% CI -2.82 to 7.28) Fig 1 \| Use of a deluded approach to analyse and present interactions in meta-analysis , illustrating how the effect of an early supported hospital discharge (ESD) strategy might vary by whether a carer is present.5 Left panel presents the effect of ESD for each subgroup within each trial, but ordered by subgroup; and right panel presents just the meta-analysed effects for each subgroup. The difference between the effects in right panel gives a deluded analysis (mean difference of 2.23, 95% confidence interval −2.82 to 7.28, P=0.39). Sizing of squares are in proportion to the inverse of the variance of the estimates. Note that the subgroup meta-analysis estimates do not match exactly those originally reported, because we used a fixed effect model for simplicity, rather than the random-effects model used by the original authors. See also web appendix A3 for references of studies and appendix B2 for details of statistical reanalysis Fig 1 \| Use of a deluded approach to analyse and present interactions in meta-analysis , illustrating how the effect of an early supported hospital discharge (ESD) strategy might vary by whether a carer is present.5 Left panel presents the effect of ESD for each subgroup within each trial, but ordered by subgroup; and right panel presents just the meta-analysed effects for each subgroup. The difference between the effects in right panel gives a deluded analysis (mean difference of 2.23, 95% confidence interval −2.82 to 7.28, P=0.39). Sizing of squares are in proportion to the inverse of the variance of the estimates. Note that the subgroup meta-analysis estimates do not match exactly those originally reported, because we used a fixed effect model for simplicity, rather than the random-effects model used by the original authors. Research Methods and Reporting Adelaide 2000 Carer present No carer Belfast 2004 Carer present No carer London 1999 Carer present No carer Manchester 2001 Carer present No carer Montreal 2000 Carer present No carer Newcastle 1997 Carer present No carer Oslo 2000 Carer present No carer Stockholm 1998 Carer present No carer Trondheim 2004 Carer present No carer -6.50 (-25.65 to12.65) 29.50 (-2.82 to 61.82) -11.90 (-28.31 to 4.51) -63.90 (-123.59 to -4.21) Insufcient data -7.50 (-33.70 to 18.70) 4.90 (-10.31 to 20.11) -15.80 (-29.45 to -2.15) 0.90 (-40.42 to 42.22) Def analysis -6.47 (-13.65 to 0.71) P=0.077 Deluded analysis (See fg 1) 2.23 (-2.82 to 7.28) P=0.39 Daf analysis 18.43 (8.16 to 28.69) P<0.001 14.05 4.93 19.12 1.45 7.51 22.27 27.65 3.02 100.00 -50 0 50 Trial and subgroup Favours ESD Favours no ESD Interaction (diference in mean diferences) (95% CI) -50 0 50 Favours greater efect of ESD with carer present Favours greater efect of ESD with no carer present Interaction (diference in mean diferences) (95% CI) Weight (%) Fig 2 \| Use of a deft approach to analyse and present interactions in meta-analysis , illustrating how the effect of an early supported hospital discharge (ESD) strategy might vary by whether a carer is present.5 The left panel again presents the effect of ESD for each subgroup within each trial, but now ordered by trial. The right panel shows the interactions between the effect of ESD and presence of a carer for each trial, along with a meta-analysis of the interaction estimates (mean difference –6.64, 95% confidence interval –13.65 to 0.71, P=0.77; heterogeneity of interaction estimates: Q=12.8, df=7, I2=45%). Daft and deluded interaction estimates are presented alongside for comparison. Squares are used to depict treatment effect and circles the interaction effects, with sizing in proportion to the inverse of the variance of the estimates 50 Favours no ESD 50 Favours greater efect of ESD with carer present Favours greater efect of ESD with no carer present Fig 2 \| Use of a deft approach to analyse and present interactions in meta-analysis , illustrating how the effect of an early supported hospital discharge (ESD) strategy might vary by whether a carer is present.5 The left panel again presents the effect of ESD for each subgroup within each trial, but now ordered by trial. Research Methods and Reporting The right panel shows the interactions between the effect of ESD and presence of a carer for each trial, along with a meta-analysis of the interaction estimates (mean difference –6.64, 95% confidence interval –13.65 to 0.71, P=0.77; heterogeneity of interaction estimates: Q=12.8, df=7, I2=45%). Daft and deluded interaction estimates are presented alongside for comparison. Squares are used to depict treatment effect and circles the interaction effects, with sizing in proportion to the inverse of the variance of the estimates Deft approach (within-trial interaction alone) See also web appendix A3 for references of studies and appendix B2 for details of statistical reanalysis doi: 10.1136/bmj.j573 \| BMJ 2017;356:j573 \| the bmj Research Methods and Reporting Table 1 \| Systematic literature review: presentation and analysis of treatment-covariate interactions Primary method of analysis No (%) of meta-analyses Primary method of presentation Forest plot by subgroup only Forest plot by subgroup and trial Forest plot of interactions Kaplan-Meier plots by subgroup Line plots (for continuous covariates) No plot Total No (%) of meta-analyses 82 (100) 35 (43) 7 (9) 2 (2) 7 (9) 3 (4) 28 (34) Deft* 2 (2) — — — — — 2 (2) Deluded* 19 (23) 13 (16) § 3 (4) — — — 3 (4) Unclear† 54 (66) 20 (24) 3 (4) 2 (2)¶ 6 (7) 2 (2) 21 (26) Descriptive only‡ 7 (9) 2 (2) 1 (1) — 1 (1) 1 (1) 2 (2) Primary methods of analysis and presentation were considered to be those either described as such, or appearing first in the article. Generally, only one method was used per review; the exceptions are detailed below. *A statistical test was used that satisfies the definition of a deft or deluded approach as given in this article, including one stage models. †A statistical test was done (including one stage model fitting), but insufficient details were given for it to be definitely described as a daft, deluded, or deft analysis. ‡Results were presented by subgroup, but no statistical tests of interaction were reported or implied. §Two reviewsw54, w68 also stated that a deft analysis might be used for sensitivity; one did in fact present such a plot in an additional publication (see ¶). ¶Another revieww68 also presented a forest plot of interactions in their Cochrane Database review,8 but not in their peer reviewed journal article. Table 1 \| Systematic literature review: presentation and analysis of treatment-covariate interactions used a deluded approach primarily, but stated an inten- tion to carry out an additional deft analysis for sensitivity (see footnotes to table 1 ).8 Table 1 summarises the analysis and presentation methods used.f Efforts to advocate a deft approach2 9 (2% of analyses) seem to have been largely unheeded, while the deluded approach remains popular (23% of analyses). Encour- agingly, no daft analyses were explicitly reported. How- ever, in our sample, the majority of reviews (66% of analyses) provided insufficient information to identify whether a daft, deluded, or deft analysis was carried out. Reanalysis of published data from systematic literature review Six meta-analyses from our review5, w21, w28, w38, w47, w61 presented sufficient data to allow reanalysis of 31 inter- actions, providing useful insight into the implications of using the three approaches. This reanalysis (web appendix B) is itself a demonstration that aggregate data, albeit in this case derived from IPD, can be analysed by any approach. Our results suggest that, in practice, only deluded or deft analyses are used. How- ever, deluded analyses combine information from daft and deft analyses, which we found to be poorly cor- related (fig 2), demonstrating why a deluded analysis might be misleading. In practice, we found deluded analyses were more likely to be significant at the 5% level than deft analyses (16% v 6%). The majority of reviews (56% of 82) tested for interac- tion exclusively by calculating the treatment difference between just two participant level subgroups, even where the underlying data were continuous or ordinal. Such collapsing of data is known to be suboptimal, both in terms of power and bias, as well as biologically implausible.12 Considering the low power inherent in interaction testing, this suggests that the analysis of interactions could be better planned.13 We next made a descriptive assessment of levels of agreement between effect estimates from deluded and deft analyses.14 Our results (fig 4 ) show that deluded analysis did not result in a systematic bias in effect size, In our sample, other than presenting no plot at all (34% of reviews), the most common presentational approach (43%) by far was to present summary treat- ment effects within each covariate subgroup (fig 1 , right panel). As illustrated above, this approach invites read- ers to combine within-trial and across-trial interaction estimates, potentially deluding them about the strength of the evidence. A minority of reviews (9%) expanded on this, presenting data by trial within each subgroup (fig 1, left panel). This approach has more to commend it, because more information is displayed; indeed, it provides sufficient data to allow the reanalyses reported in the next section. Research Methods and Reporting This includes both so-called one stage modelling approaches (83% of insufficiently described analy- ses),10 which could be either deludedordeft,2 3 and other commonly used but deluded approaches.5 10 Given that such results might be used to inform clinical practice, this lack of detail is disturbing.11 Systematic literature review of published interactions between treatment effects and participant level covariates one without, can be combined and presented by use of standard meta-analytical techniques (fig 2, right panel).7 In this case, the results show that having a carer present increases the effect of ESD on duration of hospital stay relative to standard care (P=0.077). The right panel in figure 2, however, also shows some visual evidence of heterogeneity (confirmed statistically (I2=45%)) among the interaction estimates, suggesting that presence of a carer might not be a wholly reliable indicator of ESD efficacy. Appropriately, the Montreal trial is not included in this deft analysis, because a within-trial interaction is not estimable. Having highlighted what is at stake, we present the results of our systematic review of the literature. We searched Medline (2011-14) for IPD meta-analyses of treatment efficacy that included at least one investiga- tion of interaction between treatment and a partici- pant level covariate (web appendix A). Our search returned 184 unique results, of which 80 were eligible. However, one ineligible study cited two additional IPD meta-analyses, giving a total of 82 eligible results. the bmj \| BMJ 2017;356:j573 \| doi: 10.1136/bmj.j573 3 Reanalysis of published data from systematic literature review f d /b \| \| h b j P value from daf analysis P value from def analysis 0.0001 0.001 0.01 0.1 0.5 0.9 0.01 0.001 0.0001 0.1 0.5 0.9 Consistent direction of efect Illustrative example (see fg 1) Inconsistent direction of efect Fig 3 \| Scatter plot (logit scale) of P values from 31 deft reanalyses (see web appendix B) of treatment-covariate interactions against the corresponding daft reanalyses. Added lines are at P=0.1; arguably a suitable level of significance for an interaction test for which a trial was not powered P value from daf analysis P value from def analysis 0.0001 0.001 0.01 0.1 0.5 0.9 0.01 0.001 0.0001 0.1 0.5 0.9 Consistent direction of efect Illustrative example (see fg 1) Inconsistent direction of efect Consistent direction of efect Illustrative example (see fg 1) Inconsistent direction of efect P value from def analysis Nevertheless, we prefer that treatment effects by sub- group (fig 2 , left panel) or the resulting interactions (fig 2, right panel) are plotted within each trial, because this corresponds more directly with a deft analysis. Disap- pointingly, only 2% of reviews adopted such an approachw19, w55 by presenting within-trial interactions and meta-analyses of these interactions together in a for- est plot (fig 2, right panel). Two further reviewsw54, w68 Fig 3 \| Scatter plot (logit scale) of P values from 31 deft reanalyses (see web appendix B) of treatment-covariate interactions against the corresponding daft reanalyses. Added lines are at P=0.1; arguably a suitable level of significance for an interaction test for which a trial was not powered Research Methods and Reporting Shaded area=Bland-Altman 95% limits of agreement14; solid line represents mean difference (bias); dashed lines are 95% confidence intervals around the mean difference Fig 4 \| Bland-Altman14 plot showing level of agreement between treatment-covariate interactions from deluded and deft analyses. Thirty one interactions were reanalysed, but only 26 with outcomes measured by hazard ratios or odds ratios were plotted. The remaining five interactions, including our illustrative example, could not be included since their outcomes were measured by mean differences and were hence incompatible. Treatment-covariate interactions (measured on the log scale) might have a positive or a negative sign, but in this plot they have all been set to negative. Hence, differences in interaction effects below the zero line represent cases where a deluded analysis gives a result in the same direction as, but more extreme than, the equivalent deft analysis, and vice versa. Shaded area=Bland-Altman 95% limits of agreement14; solid line represents mean difference (bias); dashed lines are 95% confidence intervals around the mean difference Although a forest plot of treatment effects by covari- ate subgroup (eg, fig 1 , right panel) might be considered clinically useful, the visual and statistical comparison of such effects in the meta-analysis context remains deluded. This could invite claims regarding the efficacy of treatments for particular participant subgroups with- out appropriate regard to a test for interaction.19 Many reviewers continue to use deluded analysis methods— possibly because they have been widely used by well respected groups for many years10—which risks mis- leading conclusions and potentially inappropriate clin- ical recommendations. i However, within-trial (deft) identification of interac- tions is relatively rare,2 probably in part because of low power. If the across-trial (daft) interaction provides greater power,15 then both sources combined (deluded) could substantially improve the power of the interac- tion estimate over within-trial information alone, albeit with an increased risk of ecological bias. This may be particularly true where some trials, such as Montrealw87 in our illustrative example,5 only contribute data to one subgroup and therefore cannot be included in a deft analysis. Our reanalysis of the published data suggests that ecological bias might be present in deluded analy- ses even when based on a set of trials identical to that used in the deft (data not shown). Hence, although a deluded approach might have a role in exploratory analyses or hypothesis generation, we strongly recom- mend that this intention be made explicit. Research Methods and Reporting Moreover, in such cases, within-trial, across-trial, and combined interactions should be presented separately so that readers will not be deluded. Rather, they can make their own judgments about the usefulness or otherwise of the daft estimate. although any individual analysis could differ from its deft equivalent by up to 20% in either direction. There seemed to be two distinct patterns of discrepancy. Firstly, the effect size as estimated from the deluded analysis could be substantially larger than that from the deft analysis, as also demonstrated by our illustrative example (fig 2 ). This is likely a result of differences in the distribution of participant characteristics across tri- als,15 inducing a strong daft effect that confounded the deluded result. Secondly, the effect size as estimated from the deluded analysis could be comparable to that from the deft analysis, except that the deft analysis might not reach significance but the deluded analysis does, by virtue of gaining power from the daft. Research Methods and Reporting Research Methods and Reporting Average efect size (hazard ratio or odds ratio) % diference between deluded and def -30 -10 0 10 20 30 -20 0.6 0.7 0.8 0.9 1.0 Pdeluded>0.1 and Pdef>0.1 Pdeluded<0.1 and Pdef<0.1 Pdeluded<0.1 and Pdef>0.1 Pdeluded<0.05 and Pdef>0.05 Average efect size (hazard ratio or odds ratio) % diference between deluded and def -30 -10 0 10 20 30 -20 0.6 0.7 0.8 0.9 1.0 Pdeluded>0.1 and Pdef>0.1 Pdeluded<0.1 and Pdef<0.1 Pdeluded<0.1 and Pdef>0.1 Pdeluded<0.05 and Pdef>0.05 Going forward, we recommend that trial investigators routinely report all by-subgroup effect estimates gener- ated by trial analyses.f Plotting treatment effects in each participant group for each trial alongside a forest plot of the within-trial interactions (fig 2 ) focuses attention on those effects and their association with the interaction estimates. Use of circles instead of squares for the interaction esti- mates2 (fig 2 , right panel) helps distinguish such plots from those of main treatment effects. If space is limited, the summary within-trial interaction effect2 for a series of covariates can be displayed on one forest plot—for example, figure 3 in reference 17 , which may be accompanied by a more detailed plot for those analyses meriting further exploration—for example, web figure 2 in reference 17 ). Further detail, to provide greater clar- ity for what is currently a less familiar approach, could be provided by a plot similar to the entirety of figure 2.f Average efect size (hazard ratio or odds ratio) Fig 4 \| Bland-Altman14 plot showing level of agreement between treatment-covariate interactions from deluded and deft analyses. Thirty one interactions were reanalysed, but only 26 with outcomes measured by hazard ratios or odds ratios were plotted. The remaining five interactions, including our illustrative example, could not be included since their outcomes were measured by mean differences and were hence incompatible. Treatment-covariate interactions (measured on the log scale) might have a positive or a negative sign, but in this plot they have all been set to negative. Hence, differences in interaction effects below the zero line represent cases where a deluded analysis gives a result in the same direction as, but more extreme than, the equivalent deft analysis, and vice versa. doi: 10.1136/bmj.j573 \| BMJ 2017;356:j573 \| the bmj doi: 10.1136/bmj.j573 \| BMJ 2017;356:j573 \| the bmj Research Methods and Reporting doi:10.1016/S0140-6736(86)90837-8. 15 Simmonds MC, Higgins JP. Covariate heterogeneity in meta-analysis: criteria for deciding between meta-regression and individual patient data. Stat Med 2007;26:2982-99. doi:10.1002/sim.2768. 16 Fisher DJ. Two-stage individual participant data meta-analysis and generalized forest plots. Stata J 2015;15:369-96. 17 NSCLC Meta-analysis Collaborative Group. Preoperative chemotherapy for non-small-cell lung cancer: a systematic review an meta-analysis of individual participant data. Lancet 2014;383:1561- 71. doi:10.1016/S0140-6736(13)62159-5. 18 Vale CL, Tierney JF, Fisher D, et al. Does anti-EGFR therapy improve outcome in advanced colorectal cancer? A systematic review and meta-analysis. Cancer Treat Rev 2012;38:618-25. doi:10.1016/j. ctrv.2011.11.002. 19 Zhang S, Liang F, Li W, Hu X. Subgroup Analyses in Reporting of Phase III Clinical Trials in Solid Tumors. J Clin Oncol 2015;33:1697-702. doi:10.1200/JCO.2014.59.8862. 20 Tierney JF, Vale C, Riley R, et al. Individual participant data (IPD) meta-analyses of randomised controlled trials: Guidance on their use PLoS Med 2015;12:e1001855. doi:10.1371/journal.pmed.1001855. Appendix: Supplementary material commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ to judge whether models have been correctly specified so as to accomplish this.13 Furthermore, it is not obvi- ous how best to present graphically the results of a one stage model. We would recommend that it be used pri- marily for inference, while an additional two stage model (which can produce only deft results) is used as the basis of the forest plot. This approach has previ- ously been suggested in the context of main treatment effects.20 1 Stewart LA, Tierney JF. To IPD or not to IPD? Advantages and disadvantages of systematic reviews using individual patient data. Eval Health Prof 2002;25:76-97. doi:10.1177/0163278702025001006. f doi:10.1177/0163278702025001006. 2 Fisher DJ, Copas AJ, Tierney JF, Parmar MK. A critical review of methods for the assessment of patient-level interactions in individual participant data meta-analysis of randomized trials, and guidance for practitioners. J Clin Epidemiol 2011;64:949-67. doi:10.1016/j. jclinepi.2010.11.016. 3 Riley RD, Lambert PC, Staessen JA, et al. Meta-analysis of continuous outcomes combining individual patient data and aggregate data. Stat Med 2008;27:1870-93. doi:10.1002/sim.3165. Our example (fig 1 ) shows how to proceed with a binary covariate. Categorical participant level covari- ates, such as disease severity, also featured prominently in our literature review. Research Methods and Reporting If such covariates have a natural ordering, a deft meta-analysis of within‑trial interac- tions can be carried out assuming a linear trend across categories.2 Continuous variables such as age should not be categorised for statistical testing, because this loses power; worse, the choice of categorisation might affect the magnitude and statistical significance of the results.12 In the case of categorical covariates where a linear trend is not an appropriate assumption, it is pos- sible to do a global test of interaction. This simply tests for some variation in treatment effect across participant subgroups without specifying its nature; but the risk of ecological bias in this context is unclear. Recommendations for analysis and presentation of interaction data Poor reporting and presentation, particularly when com- bined with inappropriate methods, is a key shortcoming of interaction analyses. Given that a deft analysis is at the least risk of bias, and can be clearly presented and read- ily interpreted, it should be the preferred approach for assessing participant level interactions aimed at influ- encing clinical practice. This can be achieved (eg, using Stata16 ) with either IPD17 or aggregate data; although the aggregate data requires treatment effects to be reported by subgroup or similar.18 If these are not available, but interaction analysis remains an important component of the research project, we recommend requesting suitable aggregate data, or the full IPD, from the trial investigators. The two stage approach we recommend here has been criticised for being overly simple, inflexible, and possibly underpowered. Certainly, one stage models fitted to the entire dataset simultaneously allow for greater complexity; but an assessment of their relative power is confounded by their ability to produce either deluded or deft results depending on the separation of within-trial and across-trial effects.2 3 Unfortunately, we have found that the details required are rarely provided 5 the bmj \| BMJ 2017;356:j573 \| doi: 10.1136/bmj.j573 Research Methods and Reporting Conclusions 10 10 Simmonds MC, Higgins JPT, Stewart LA, Tierney JF, Clarke MJ, Thompson SG. Meta-analysis of individual patient data from randomized trials: a review of methods used in practice. Clin Trials 2005;2:209-17. doi:10.1191/1740774505cn087oa. Investigating associations between the effects of treat- ments or other interventions and participant level covari- ates can help identify who is most likely to benefit, and allow treatments to be targeted appropriately. In an era when such investigations are increasingly common, deft analysis and presentation should be the recommended approach for reliably informing clinical practice. 11 Donegan S, Williams L, Dias S, Tudur-Smith C, Welton N. Exploring treatment by covariate interactions using subgroup analysis and meta-regression in cochrane reviews: a review of recent practice. PLoS One 2015;10:e0128804. doi:10.1371/journal.pone.0128804. 11 Donegan S, Williams L, Dias S, Tudur-Smith C, Welton N. Exploring treatment by covariate interactions using subgroup analysis and meta-regression in cochrane reviews: a review of recent practice. PLoS One 2015;10:e0128804. doi:10.1371/journal.pone.0128804. 12 Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol 1999;28:964-74. doi:10.1093/ije/28.5.964. 12 Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol 1999;28:964-74. doi:10.1093/ije/28.5.964. 12 Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol 1999;28:964-74. doi:10.1093/ije/28.5.964. Contributors: JFT and DJF conceived the initial idea and drafted the manuscript. DJF carried out the systematic literature review, performed the reanalysis of published data, and created the figures. He is the guarantor. JRC provided additional statistical expertise, and JRC and JFT helped revise the manuscript structure. All authors contributed to critical discussions of the ideas, commented on versions of the manuscript, and approved the final submission. 13 Simmonds M, Stewart G, Stewart L. A decade of individual participant data meta-analyses: A review of current practice. Contemp Clin Trials 2015;45(Pt A):76-83. doi:10.1016/j.cct.2015.06.012. j 14 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307- 10. doi:10.1016/S0140-6736(86)90837-8. j 14 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307- 10. doi:10.1016/S0140-6736(86)90837-8. 15 Simmonds MC, Higgins JP. Covariate heterogeneity in meta-analysis: criteria for deciding between meta-regression and individual patient data. Stat Med 2007;26:2982-99. doi:10.1002/sim.2768. 15 Simmonds MC, Higgins JP. Covariate heterogeneity in meta-analysis: criteria for deciding between meta-regression and individual patient data. See rights and reprints http://www.bmj.com/permissions Research Methods and Reporting commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ 1 Stewart LA, Tierney JF. To IPD or not to IPD? Advantages and disadvantages of systematic reviews using individual patient data Eval Health Prof 2002;25:76-97. doi:10.1177/0163278702025001006. 2 Fisher DJ, Copas AJ, Tierney JF, Parmar MK. A critical review of method for the assessment of patient-level interactions in individual participant data meta-analysis of randomized trials, and guidance fo practitioners. J Clin Epidemiol 2011;64:949-67. doi:10.1016/j. jclinepi.2010.11.016. 3 Riley RD, Lambert PC, Staessen JA, et al. Meta-analysis of continuous outcomes combining individual patient data and aggregate data. Sta Med 2008;27:1870-93. doi:10.1002/sim.3165. 4 Berlin JA, Santanna J, Schmid CH, Szczech LA, Feldman HI. Anti-Lymphocyte Antibody Induction Therapy Study Group. Individua patient- versus group-level data meta-regressions for the investigation of treatment effect modifiers: ecological bias rears its ugly head. Stat Med 2002;21:371-87. doi:10.1002/sim.1023. 5 Fearon P, Langhorne P. Early Supported Discharge Trialists. Services for reducing duration of hospital care for acute stroke patients. Cochrane Database Syst Rev 2012;9:CD000443. 6 Thompson SG, Higgins JP. How should meta-regression analyses be undertaken and interpreted?Stat Med 2002;21:1559-73. doi:10.1002 sim.1187. 7 Deeks JJ, Higgins JPT, Altman DG, et al. Analysing data and undertakin meta-analyses. In: Higgins JPT, Green S, eds. Cochrane Handbook for Systematic Reviews of Interventions. 2008: 243- 96doi:10.1002/9780470712184.ch9. 8 Ronellenfitsch U, Schwarzbach M, Hofheinz R, et al. GE Adenocarcinoma Meta‐analysis Group. Perioperative chemo(radio) therapy versus primary surgery for resectable adenocarcinoma of the stomach, gastroesophageal junction, and lower esophagus. Cochran Database Syst Rev 2013;5:CD008107. 9 Thompson SG, Higgins JP. Treating individuals 4: can meta-analysis help target interventions at individuals most likely to benefit?Lancet 2005;365:341-6. doi:10.1016/S0140-6736(05)70200-2. 10 Simmonds MC, Higgins JPT, Stewart LA, Tierney JF, Clarke MJ, Thompson SG. Meta-analysis of individual patient data from randomized trials: a review of methods used in practice. Clin Trials 2005;2:209-17. doi:10.1191/1740774505cn087oa. 11 Donegan S, Williams L, Dias S, Tudur-Smith C, Welton N. Exploring treatment by covariate interactions using subgroup analysis and meta-regression in cochrane reviews: a review of recent practice. PLoS One 2015;10:e0128804. doi:10.1371/journal.pone.0128804. 12 Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials t model continuous risk variables in epidemiology. Int J Epidemiol 1999;28:964-74. doi:10.1093/ije/28.5.964. 13 Simmonds M, Stewart G, Stewart L. A decade of individual participan data meta-analyses: A review of current practice. Contemp Clin Trials 2015;45(Pt A):76-83. doi:10.1016/j.cct.2015.06.012. 14 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307- 10. Conclusions Stat Med 2007;26:2982-99. doi:10.1002/sim.2768. Funding: This work was supported by the UK Medical Research Council (MRC) via core funding for the MRC Clinical Trials Unit at University College London, and grant funding for the MRC London Hub for Trials Methodology Research (MC_EX_G0800814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 16 Fisher DJ. Two-stage individual participant data meta-analysis and generalized forest plots. Stata J 2015;15:369-96. 16 Fisher DJ. Two-stage individual participant data meta-analysis and generalized forest plots. Stata J 2015;15:369-96. 17 NSCLC Meta-analysis Collaborative Group. Preoperative chemotherapy for non-small-cell lung cancer: a systematic review and meta-analysis of individual participant data. Lancet 2014;383:1561- 71. doi:10.1016/S0140-6736(13)62159-5. Competing interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. 18 Vale CL, Tierney JF, Fisher D, et al. Does anti-EGFR therapy improve outcome in advanced colorectal cancer? A systematic review and meta-analysis. Cancer Treat Rev 2012;38:618-25. doi:10.1016/j. ctrv.2011.11.002. 19 Zhang S, Liang F, Li W, Hu X. Subgroup Analyses in Reporting of Phase III Clinical Trials in Solid Tumors. J Clin Oncol 2015;33:1697-702. doi:10.1200/JCO.2014.59.8862. 19 Zhang S, Liang F, Li W, Hu X. Subgroup Analyses in Reporting of Phase III Clinical Trials in Solid Tumors. J Clin Oncol 2015;33:1697-702. doi:10.1200/JCO.2014.59.8862. Provenance and peer review: Not commissioned; externally peer reviewed. 20 Tierney JF, Vale C, Riley R, et al. Individual participant data (IPD) meta-analyses of randomised controlled trials: Guidance on their use. PLoS Med 2015;12:e1001855. doi:10.1371/journal.pmed.1001855. This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for Appendix: Supplementary material Subscribe: http://www.bmj.com/subscribe See rights and reprints http://www.bmj.com/permissions
https://openalex.org/W4225641624	https://digibug.ugr.es/bitstream/10481/74358/1/8%20The%20productive%20vocabulary%20of%20multimodal%20and%20unimodal%20English%20as.pdf	English	null	The productive vocabulary of multimodal and unimodal English as a Foreign Language learners	Porta linguarum	2,022	cc-by	7,630	The productive vocabulary of multimodal and unimodal English as a foreign language learners Alejandra Montero-SaizAja Universidad de La Rioja Received: 30 May / Accepted: 7 January DOI: 10.30827/portalin.vi.21389 ISSN paper edition: 1697-7467, ISSN digital edition: 2695-8244 Received: 30 May / Accepted: 7 January DOI: 10.30827/portalin.vi.21389 ISSN paper edition: 1697-7467, ISSN digital edition: 2695-8244 ABSTRACT: This study investigated the productive vocabulary of EFL learners divided into two groups: multimodal (preference for two or three perceptual learning styles) and unimodal (preference for one perceptual learning style). The objectives of this research were twofold: (1) to identify the productive vocabulary of multimodal and unimodal EFL learners; and (2) to ascertain whether there were statistically significant differences between product- ive vocabulary and the preferences for learning (multimodality or unimodality). The sample consisted of 60 Spanish EFL learners (24 multimodal and 36 unimodal) in the 12th grade. The data collection instruments were the Learning Style Survey (Cohen et al., 2009) to divide the informants into multimodal and unimodal learners, and the 2,000-word version of the Productive Vocabulary Levels Test (Laufer & Nation, 1995, 1999) to measure their productive vocabulary. Then, data were coded and subjected to quantitative analyses. The findings indicated that multimodal learners had more productive vocabulary (1,186 words) than their unimodal peers (948 words). However, there were not statistically significant dif- ferences between multimodal and unimodal learners in their productive vocabulary. How- ever, both the effect size and the strength of association were large. Therefore, the results suggested that EFL learners employed different sensory modalities to learn vocabulary. Key words: multimodality, unimodality, productive vocabulary, perceptual learning style preferences, English as a Foreign Language. Key words: multimodality, unimodality, productive vocabulary, perceptual learning style preferences, English as a Foreign Language. 139-153 139-153 Monograph IV, 2022 139-153 Vocabulario productivo de estudiantes multimodales y unimodales aprendices de inglés como lengua extranjera RESUMEN: Este estudio investigó el vocabulario productivo de aprendices de inglés como lengua extranjera multimodales (preferencia por dos o tres estilos de aprendizaje) y uni- modales (preferencia por un estilo de aprendizaje). Los objetivos fueron: (1) identificar el vocabulario productivo de los estudiantes multimodales y unimodales; y (2) determinar si se encontraron diferencias estadísticamente significativas entre el vocabulario productivo y las preferencias en el aprendizaje (multimodalidad o unimodalidad). La muestra la formaron 24 alumnos multimodales y 46 unimodales, aprendices de inglés como lengua extranjera de 2º de Bachillerato. Los instrumentos utilizados fueron el Learning Style Survey (Cohen et al., 2009), para dividir a los alumnos en multimodales y unimodales, y el Productive Vocabulary Levels Test (Laufer & Nation, 1995, 1999) para medir el vocabulario productivo. A conti- nuación, los datos fueron codificados y sometidos a análisis cuantitativos. Los resultados in- dicaron que los alumnos multimodales presentaron un mayor vocabulario productivo (1.186 palabras) que los unimodales (948 palabras). No había diferencias estadísticamente signifi- cativas en el vocabulario productivo de los alumnos multimodales y unimodales, aunque el 139 Monograph IV January 2022 January 2022 tamaño del efecto y la fuerza de asociación fueron grandes. Estos resultados sugieren que los aprendices de inglés como lengua extranjera utilizan diferentes modalidades sensoriales para aprender vocabulario. Palabras clave: multimodalidad, unimodalidad, vocabulario productivo, estilos de apren- dizaje de percepción, inglés como lengua extranjera. 1. Introduction Vocabulary acquisition is a crucial element of learning a foreign language (FL) because, as Meara (1996) observed, “all other things being equal, learners with big vocabularies are more proficient in a wide range of language skills than learners with smaller vocabularies” (p. 37). It was not until the 1980s when L2 vocabulary acquisition research started to gradu- ally acquire relevance and when investigations began to proliferate (Meara, 1980). However, productive vocabulary knowledge has not been thoroughly investigated (e.g., Castro García, 2017; Laufer & Nation, 1999; Meara & Miralpeix, 2021). In an FL classroom, L2 vocabulary learning might be affected by learners’ individual differences. Language learners use different sensory modalities (visual, auditory, or tactile/kinesthetic), also called perceptual learning styles, to process information and learn vocabulary. These learners might use a specific sensory modality (unimodal learners), or a combination of sensory modalities in balance (multimodal learners). Although the relationship between L2 vocabulary and perceptual learning styles has been investigated (e.g., Hatami, 2018; Pouwels, 1992; Tight, 2010), to the best of the author’s knowledge, no study has examined the influence of perceptual learning styles on productive vocabulary, considering learners’ multimodal and unimodal learning preferences. The present study explores the productive vocabulary knowledge of EFL learners in the last course of Spanish post-secondary education (2nd of Baccalaureate, equivalent to the 12th grade), based on their multimodal and unimodal learning preferences. This research is of paramount importance for FL education, since it reveals the number of words available for communication, and it unveils whether the preference for one or several perceptual learning styles contributes to the development of productive vocabulary. EFL teachers could acknowledge their students’ learning preferences and whether more instruction on vocabulary is required. In this regard, teachers could plan their classes and teaching materials according to their students’ needs. 2.1. Vocabulary Vocabulary is a fundamental aspect in foreign language acquisition (FLA) (Laufer, 1998; Meara, 1990; Nation, 1990), since the knowledge of the vocabulary of a language would allow learners to communicate effectively. Researchers have investigated the number of words that are necessary to understand both written and spoken texts in a foreign language. However, a consensus has not been reached regarding the exact vocabulary size. For example, 3,000 140 Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... word families are required to comprehend a text, 5,000 word families to read for pleasure, 8,000 to 9,000 word families to understand a written text and 6,000 to 7,000 word families for a spoken text (Laufer & Aviad-Levitzky, 2017; Nation, 2006; van Zeeland & Schmitt, 2013). Research on word frequency has indicated that the knowledge of the 2,000-3,000 most frequent words would allow FL learners to communicate both orally and in written form (Nation & Waring, 1997; Schmitt & Schmitt, 2014; van Zeeland & Schmitt, 2013). Therefore, investigations on the vocabulary size of EFL learners would allow teachers and researchers to acknowledge their threshold vocabulary level and whether more instruction is necessary to read and comprehend texts (Laufer, 1998; Laufer & Aviad-Levitzky, 2017). It would also be beneficial for learners to know their most challenging aspects to be able to address them and enhance their learning. Two types of vocabulary can be distinguished: receptive and productive. Receptive vocabulary concerns the perception of a linguistic form and the understanding of its meaning in listening and reading (Meara, 1990). Productive vocabulary, on the other hand, involves the production of words in speaking and writing to convey meaning (Nation, 2001). Product- ive vocabulary can be classified into two types: controlled and free. Controlled productive vocabulary pertains to the production of words when they are triggered by a task (see Sec- tion 3.2.2.). In contrast, free productive vocabulary refers to the use of words at one’s free will (Laufer & Nation, 1999). In this regard, words are not prompted but used by learners by choice, such as in a writing task. It is widely acknowledged that receptive vocabulary precedes productive vocabulary and it is larger (e.g., Cheng & Matthews, 2018; Nation, 1990; Webb, 2008). Productive vocabulary has been underinvestigated, and few assessment methods exist to estimate it. 2.1. Vocabulary Among them, the Lexical Frequency Profile (Laufer & Nation, 1995) measures free productive vocabulary through a composition task, whilst the Productive Vocabulary Levels Test (PVLT) (Laufer & Nation, 1995, 1999) assesses controlled product- ive vocabulary. On the other hand, Webb (2008) used translation tests. Another method has been to use a word association task, in which prompts related to daily life situations act as cues so that test takers can write the words that first come to their minds (e.g., Jiménez Catalán, 2014; Jiménez Catalán & Montero-SaizAja, 2020). The present study is devoted to controlled productive vocabulary, since our aim is to ascertain the amount of words learners are ready to use for effective communication. Research on productive vocabulary is essential to acknowledge the vocabulary size of FL learners in a given educational level and context, and to tackle any issues that might arise in the acquisition of vocabulary. 2.2. Perceptual learning styles Learning styles are also fundamental elements in FLA, since they allow teachers and researchers to identify the different learning preferences that learners have. They can be defined as “an individual’s natural, habitual, and preferred way(s) of absorbing, processing, and retaining new information and skills” (Reid, 1995, p. viii). However, learning styles have been sharply criticized. Supporters (e.g., Barbe et al., 1979; Lovelace, 2005) advocate the matching of teaching and learning styles for effective learning. Other researchers (e.g., Pashler et al., 2009; Willingham et al., 2015) are against this idea because no conclusive evidence has been found. We agree with the latter viewpoint in that matching both styles will not ensure successful learning. Notwithstanding, we support a balanced instruction in which 141 Monograph IV January 2022 January 2022 teachers consider all learning styles in the classroom (not all at the same time as proponents argue) so that students can get the most out of their learning (e.g., Natividad & Batang, 2018; Payaprom & Payaprom, 2020). From the large number of models and classifications that have been proposed (e.g., Briggs Myers, 1962; Coffield et al., 2004; McCarthy, 1990), perceptual learning styles were selected because they are the most significant perceptual modalities that can be found in an FL classroom (Barbe et al., 1979; Tight, 2010). Perceptual learning styles, also referred to as modality preferences or learning preferences, are “the variations among learners in using one or more senses to understand, organize and retain experience” (Reid, 1987, p. 89). The senses of sight, hearing, and touch are thought to be the most relevant modalities found in an FL classroom (Barbe et al., 1979; Tight, 2010). Therefore, this study will be dedicated to the visual, auditory, and tactile/kinesthetic perceptual learning styles, as they make teachers aware of how their students prefer to learn. Visual learners like to receive information through the sense of sight. They enjoy reading, seeing charts, images, and taking detailed notes. Auditory learners like to receive information through hearing. They prefer listening to lectures, oral instructions, participating in discussions, and role-play activities. Tactile and kinesthetic styles are usually grouped together because, although they are not the same, they are somehow related. Tactile learners prefer to learn through the sense of touch, whereas kinesthetic learners like learning through movement. These learners like moving, doing experiments, or building things (Dörnyei, 2005; Dörnyei & Ryan, 2015; Oxford, 2003). All in all, FL learners can have a unique preference for learning (unimodal learners), or they can have a mixed-modality preference (multimodal learners) and use two or three styles in balance. Research on perceptual learning styles is crucial because it indicates the general preferences for learning a FL, and it sheds some light on the FL learning process. It also makes learners cognizant of their learning preferences, as well as their strengths and weaknesses. 2.3. Review of studies Few studies have examined the relationship between L2 vocabulary learning and percep- tual learning styles. For example, Shen (2010) investigated the effect of perceptual learning style preferences on L2 lexical inferencing with EFL university students in Taiwan. Before inferencing strategy instruction, results suggested that group learners achieved the best out- comes in the lexical inferencing test, followed by individual, kinesthetic, tactile, auditory, and visual learners. In the lexical inferencing posttest, auditory and visual learners outper- formed the other participants. Similarly, Tight (2010) researched the relationship between perceptual learning style matching and the acquisition and retention of L2 Spanish nouns with American university students. Regarding unimodal learners, visual was reported to be their most favored modality (38%), followed by auditory (16%), and tactile/kinesthetic (9 %) modalities. Visual was also the preferred modality of multimodal learners (15%), fol- lowed by auditory (12%), and tactile/kinesthetic (5%) modalities. In fact, the rest (4%) were multimodal learners who had a tie between their two highest modalities. Findings indicated that mixed modality instruction was the most beneficial for the acquisition and retention of L2 vocabulary. Moreover, style matching seemed to result in a greater retention than style mismatching. Other studies found that the relationship between L2 vocabulary learning and perceptual learning styles was not effective. Yeh and Wang (2003) studied whether Taiwanese EFL 142 Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... university students’ perceptual learning styles influenced the effectiveness of three types of vocabulary annotations (text annotation only, text plus picture, and text plus picture and sound). Results suggested that the text plus picture vocabulary annotation was the most effective. However, perceptual learning styles did not appear to influence the effectiveness of vocabulary annotations. They tended to prefer visual to auditory annotations. Likewise, Kassaian (2007) examined whether matching the instructional method (visual or aural) with Iranian EFL university students’ perceptual learning styles enhanced their vocabulary acquis- ition and retention. Findings indicated that the words which were presented visually were acquired and retained better than auditory ones, despite being visual or auditory learners. Results also showed that perceptual learning styles did not have an effect on the retention of the material instructed. 2.3. Review of studies In the same vein, Hatami (2018) explored whether there was a relationship between Iranian EFL university students’ perceptual learning styles and L2 in- cidental vocabulary acquisition and retention through reading, when matched to their input mode. Most of the participants were visual learners (47%) followed by mixed-modality preference (33%), tactile/kinesthetic (11%), and auditory (9%) learners. Significant differ- ences between the reading group and incidental vocabulary acquisition and retention were not found. Findings also revealed that perceptual learning style matching did not influence incidental word learning through reading. g g g On the other hand, Pouwels’ (1992) research showed mixed results. He investigated the impact of perceptual learning styles on a vocabulary test using pictorial, verbal and pictorial-verbal aids in L2 university students in the United States. Most informants were visual learners (41.77%), followed by auditory (29.11%) and parity (29.11%) learners. Res- ults confirmed that parity learners obtained better results in the vocabulary test, followed by visual and auditory learners. Findings also indicated a statistically significant positive correlation between visual learners and the combination of picture and verbal aids, but the correlation was negative with auditory learners. As it can be inferred from this review of studies, no concluding evidence has been found to support the relationship between perceptual learning styles and L2 vocabulary learning. Furthermore, these studies only investigated intentional (Kassaian, 2007; Pouwels, 1992; Tight, 2010), and incidental (Hatami, 2018) L2 vocabulary acquisition, lexical inferencing (Shen, 2010), or vocabulary annotations (Yeh & Wang, 2003). However, investigations into the productive vocabulary size of multimodal (mixed-modality preference) and unimodal (single modality preference) EFL learners are lacking in the literature. The current study aims at contributing to fill this gap in L2 vocabulary and perceptual learning style studies by examining the productive vocabulary size of multimodal and unimodal EFL learners and identifying whether perceptual learning style preferences influence the knowledge of productive vocabulary. This investigation would determine whether the preference for one or several modalities when learning EFL vocabulary contributes to a higher productive vocabulary knowledge. It would make EFL teachers aware of their students’ vocabulary size and learning preferences, and it would indicate whether new instructional approaches are needed. Therefore, this study addresses the following research questions: 1. What is the productive vocabulary knowledge of multimodal and unimodal EFL learners?i 1. What is the productive vocabulary knowledge of multimodal and unimodal EFL learners?i 2. Monograph IV Monograph IV January 2022 January 2022 3. Methodology This study is a quantitative, cross-sectional, descriptive, and correlational research This study is a quantitative, cross-sectional, descriptive, and correlational research. 3.1. Participants A group of 60 EFL learners participated in this investigation. This group comprised 24 multimodal (mixed-modality preference) and 36 unimodal (single modality preference) students. They attended the last course of Spanish post-secondary education (equivalent to the 12th grade) in a state school in La Rioja, a monolingual autonomous community in the north of Spain. Their mean age was 17.1 years old, and all of them were learning English as a Foreign Language, which was taught as a curricular subject. Their instructional level was B1, which was the level assigned to this course by the educational board of this com- munity. Respondents differed in their mother tongue. The majority of them (78.33%) had Spanish as their L1, but the rest (21.67%) had other languages as their mother tongue, which were only spoken at home. Their L1 was Arabic (10 %), Bulgarian (1.67%), Macedonian (3.33%), and Romanian (6.6 %). 3.2. Data collection instruments 3.2.1. Learning Style Survey (LSS) 3.2.1. Learning Style Survey (LSS) The Learning Style Survey (LSS) was developed by Cohen et al. (2009) to determine eleven learning style preferences. However, only the first part of this questionnaire “How I use my physical senses” was selected for this study. This part pertains to the perceptual learning styles (visual, auditory, and tactile/kinesthetic), which are the styles we use to divide learners into multimodal and unimodal. It is composed of 30 behavioral statements: 10 each correspond to the visual, auditory, and tactile/kinesthetic modalities. Reflecting on their behavior in learning, the participants had to circle their answer based on a five-point Likert scale (ranging from 0 = never to 4 = always). For example, item one reads as follows: “I remember something better if I write it down.” This questionnaire was selected for four reasons. Firstly, it is appropriate for language learning. Secondly, items are more L2 specific than in the Perceptual Learning Style Preference Questionnaire (Reid, 1987) and the Style Analysis Survey (SAS) (Oxford, 1995), since “it contains some L2-learning specific items, mixed with non-subject-specific ones” (Dörnyei, 2005, p. 146). Thirdly, it includes one part which examines perceptual learning styles specifically, it is based on Oxford’s SAS (1995) and it is an improved version (Cohen & Weaver, 2005). And finally, the test re-test reliab- ility of the first part of this survey is reported to have a correlation of .74 (Tight, 2010). 2.3. Review of studies Are there any statistically significant differences between productive vocabulary and the preferences for learning of EFL learners? 143 Monograph IV 3.2.2. Productive Vocabulary Levels Test (PVLT) The Productive Vocabulary Levels Test (PVLT), designed by Laufer and Nation (1995, 1999), was used to determine the controlled productive vocabulary knowledge of the inform- ants. It is a quantitative measure which examines vocabulary growth by analyzing discrete, selective, and context dependent vocabulary (Moreno Espinosa, 2010). Particularly, the 144 Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... 2,000-word parallel version (version A + version C) was selected, since the knowledge of the 2,000 most frequent words seems to allow learners to communicate both orally and in written form in a foreign language (Nation & Waring, 1997; Schmitt & Schmitt, 2014; van Zeeland & Schmitt, 2014). This version consists of 30 different sentence contexts where students have to complete the word that fits in each particular sentence. The first letters are provided in each sentence in order to trigger the target word. It is considered to be a reliable and valid measure of productive vocabulary size (Laufer & Nation, 1999). For example, the first item reads as follows: “They will restore the house to its orig_______ state.” We decided to choose this instrument because our objective was to measure controlled productive vocabulary, specifically the 2,000-word version. These are high-frequency words which belong to the basic vocabulary of the language (Nation, 2006). Knowing those words would imply that learners would be able to use them effectively in different communicative contexts. Monograph IV Monograph IV January 2022 January 2022 the PVLT, zero was the lowest score and 30 was the highest. Following Nation (1990, p. 78), the productive vocabulary knowledge was calculated as the number of correct answers multiplied by the total number of words of the test (2,000) and divided by the number of items (30). We decided that a word was correct if it was well-written both grammatically and orthographically. The first letters of the target word and context are given as a clue, so it is easier to find out to which word it refers. Besides, knowing a productive word entails the knowledge of its form (pronunciation, writing, spelling, word parts), meaning (word form, concepts, references, associations), and use (grammatical function, collocations, constraints on use) (Nation, 2001; Qian & Sun, 2019). The sample was analyzed using RStudio version 1.2.5019 to perform descriptive and inferential statistics. The Kolmogorov-Smirnov test was run to determine the normality of the sample, and the Levene Test was conducted to identify whether there was equality of variances. The Wilcoxon Rank Sum Test was also performed to ascertain statistically signi- ficant differences between productive vocabulary and the perceptual preferences for learning. Finally, the effect size was calculated using Becker’s (1998) Effect Size Calculators. 3.3. Procedure and analysis Data were collected in one session during school time. Participants were presented with a background questionnaire, the LSS and the PVLT tests. The headmaster of the par- ticipating school signed a written consent for the administration of these questionnaires to participants. Students, their parents, and tutors were informed of the research purpose of these tasks and its voluntary basis. The background questionnaire included questions to obtain information on learners’ age, gender, nationality, mother tongue, other languages spoken at home, their instruction in EFL, and their previous experience with English. Part one of the LSS was distributed in Spanish after being granted the permission to use the questionnaire and translate it into Spanish by The Center for Advanced Research on Language Acquisition (CARLA), University of Minnesota. It was translated into Spanish because this was the L1 of the majority of the respondents (78.33%) and the L2 of the rest (21.67%), who both spoke and used Spanish in their daily life with a native-like fluency. It was thought that as they did not have the same command in the English language, their better understanding of this test would imply more accurate responses. They had 10 minutes to complete the LSS. In contrast, the PVLT test was administered in English, since our aim was to determine the informants’ productive vocabulary knowledge in EFL. The time allotted to complete this test was 10 minutes as well. At the beginning of the tests, written instructions were given in English or Spanish, depending on the test, and they were also given orally in Spanish to clarify what students were being asked to do. Once data were collected, responses were coded and entered into a Microsoft Excel file. Afterwards, all the tests were corrected and marked. Regarding the LSS, scores for each perceptual learning style were obtained by summing the points of each item: zero was the lowest point per item and four was the highest. As there were 10 items per modality, 40 was the maximum score in each. The modality which had the highest overall score was established as informants’ perceptual learning style preference, and these types of learners were regarded as unimodal. Following Tight (2010), if there was not a difference of at least three points between the highest and the following highest modality, informants were considered to have a mixed-modality preference, that is, multimodal learners. Concerning 145 4. Results The first research question aimed to identify the productive vocabulary knowledge of EFL multimodal and unimodal learners. Table 1 presents the descriptive statistics for multimodal and unimodal learners in the study of their productive vocabulary knowledge, particularly their word estimates. Out of the 2,000 most frequent words that the PVLT measured, mul- timodal learners obtained a mean of 1,186 words, whilst their unimodal peers’ mean was 948 words. Table 1 also shows that both multimodal and unimodal EFL learners reached a maximum of 1,733.33 words. However, the minimum of word estimates was different. It was of 400 words for multimodal learners and 200 for the unimodal group. Therefore, the overall productive vocabulary knowledge of this sample was lower than 1,000 words in the case of unimodal students, and a little higher in multimodal learners. Table 1. Descriptive statistics for multimodal and unimodal learners’ productive vocabulary Learning preferences N Mean SD MIn. Max. Multimodal 24 1,186.11 394.64 400 1,733.33 Unimodal 36 948.15 469.5452 200 1,733.33 Table 1. Descriptive statistics for multimodal and unimodal learners’ productive vocabulary Table 2 shows multimodal and unimodal learners’ productive vocabulary classified by each perceptual modality preference. Regarding multimodals, visual, auditory, and tactile/ kinesthetic learners appeared to have the highest productive vocabulary available, since their mean was 1,400 words. They were followed by visual and tactile/kinesthetic (1,224 words), auditory and tactile/kinesthetic (947 words), and visual and auditory (933 words) learners. In contrast, findings suggested that visual learners had the highest productive vocabulary of the unimodal group, since they were reported to have a vocabulary size of 1,009 words. They were followed by tactile/kinesthetic (872 words), and auditory (822 words) learners. 146 Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... Table 2. Productive vocabulary of multimodal and unimodal learners according to their perceptual learning styles (T/K stands for tactile/kinesthetic) Multimodal learners Unimodal learners Perceptual styles N Mean SD Perceptual styles N Mean SD Visual and auditory 2 933.33 94.281 Visual 21 1,009.52 479.484 Visual and T/K 11 1,224.24 389.042 Auditory 3 822.22 214.303 Auditory and T/K 5 946.67 512.944 T/K 12 872.22 507.287 Visual, audi- tory, and T/K 6 1,400 245.855 le 2. Productive vocabulary of multimodal and unimodal learners according to their perceptua learning styles (T/K stands for tactile/kinesthetic) The second research question aimed to ascertain whether there were statistically sig- nificant differences between productive vocabulary knowledge and EFL learners’ learning preferences. 5. Discussion The first research question aimed to identify the productive vocabulary of multimodal and unimodal EFL learners in the 12th grade. Our results revealed that the overall productive vocabulary of multimodal learners (1,186 words) was higher than that of unimodal learners (948 words). However, neither of the two groups of learners had a productive vocabulary size of 2,000 words, so they might have issues communicating in English orally and in written form (Nation & Waring, 1997; Schmitt & Schmitt, 2014; van Zeeland & Schmitt, 2013). These findings seem to be consistent with the study conducted by Pouwels (1992), which showed that the parity group outperformed visual and auditory learners in a vocabulary test. The difference lies in that Pouwels (1992) investigated intentional L2 vocabulary acquisition, whilst the present study focused on EFL productive vocabulary knowledge. Likewise, Fadel and Lemke (2012) also claimed that multimodal learners achieved better results in learning than unimodal learners. Based on their perceptual learning styles, those multimodal learners who used the visual, auditory, and tactile/kinesthetic modalities in balance appeared to have the highest productive vocabulary size (1,400 words). An interpretation of this finding might be that if in a particular case these learners find it difficult to learn vocabulary with a specific style (e.g., visual), as they can use the three of them equally well, they can resort to the other two styles (e.g., auditory, and tactile/kinesthetic). In this regard, they seem to have better opportunities of success in vocabulary learning than unimodal learners, who depend on a single modality. Brain-related evidence has confirmed our interpretation: “A word network consisting of many components, i.e., visual, aural, kinetic, olfactory, etc. […] stores and retrieves information more efficiently than a small network” (Macedonia, 2015, p. 2). Our results also indicated that visual and auditory learners had the lowest productive vocabulary size of the multimodal group. This finding might be related to the conclusion Mansourzadeh (2014) reached, which pointed to the ineffectiveness of learning English vocabulary through audio-visual aids. On the other hand, visual unimodal learners seemed to have the highest vocabulary size (1,009 words). This might be because textbooks, apart from being the main medium of instruction in EFL learning (e.g., Gibbons, 2015; Hutchinson & Torres, 1994), are also the major source of vocabulary input (e.g., Jiménez Catalán & Mancebo Francisco, 2008; Nordlund & Norberg, 2020). Monograph IV Monograph IV January 2022 4. Results The data were normally distributed in the multimodal group (p= .7128), but not in the unimodal group (p = .074) according to the Kolmogorov-Smirnov test. A Levene Test confirmed the homogeneity of variance, as the Pr(>F) was higher than 0.05 (0.07417). The Wilcoxon Rank Sum Test was then implemented to identify whether there were statistically significant differences between productive vocabulary and learning preferences (multimodality and unimodality). Findings indicated that there were not statistically significant differences in the productive vocabulary of multimodal and unimodal learners (see Table 3). Table 3. Wilcoxon Rank Sum Test Wilcoxon Rank Sum Test W p-value 558.5 .05677 Table 3. Wilcoxon Rank Sum Test Regarding the effect size for the comparison between two means, results suggested that there was a large effect size, as Cohen’s d was 1.057 (see Table 4). Similarly, the strength of association was both positive and large. Table 4. Effect size Cohen’s d Effect-size r 1.057 .467 147 Monograph IV 5. Discussion EFL textbooks usually include readings which introduce the vocabulary to be learnt in the unit and then exercises to practice that vocabulary. Therefore, EFL students primarily learn vocabulary from visual materials, which might explain why visual learners had a higher productive vocabulary. Our results also suggested that audit- ory learners had the lowest productive vocabulary size (822 words). This finding supports the studies conducted by Brown et al. (2008) and van Zeeland and Schmitt (2013) which reported the difficulty EFL learners have to recognize the meaning and recall vocabulary learnt through listening. Another plausible explanation might be that listening is thought to be the most difficult skill in EFL learning (e.g., Goh, 2002; Nushi & Orouji, 2020), what might also hinder vocabulary acquisition. The second research question aimed to ascertain whether there were statistically sig- nificant differences between productive vocabulary and learning preferences (multimodality and unimodality). The results of the present study indicated that there were not statistically significant differences. However, the effect size and the strength of association were large. 148 Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... Alejandra Montero-SaizAja The productive vocabulary of multimodal and unimodal English as... In this respect, this finding corroborates the results obtained by Yeh and Wang (2003), Kassaian (2007), and Hatami (2018), since they confirmed that perceptual learning styles did not influence L2 vocabulary learning. These studies differed from ours because they investigated the relationship between perceptual learning styles and vocabulary annotation, intentional and incidental vocabulary acquisition, respectively. They did not research the impact of learning preferences (multimodality and unimodality) on the productive vocabulary knowledge of EFL learners. This outcome implies that learning preferences, that is, being multimodal or unimodal, do not influence the learning of vocabulary. An interpretation of this finding might be that 12th grade multimodal and unimodal informants have been ex- posed to the same method of vocabulary instruction in EFL since they started the Spanish education system. This vocabulary input predominantly comes from the activities found in the textbooks (e.g., reading, listening, role-play) (Jiménez Catalán & Mancebo Francisco, 2008; Nordlund & Norberg, 2020). Although there were not significant differences, the productive vocabulary size of Spanish 12th graders was very low (around 1,000 words), after studying English for at least 11 years. 6. Conclusions This study investigated the productive vocabulary knowledge of EFL learners in the last course of Spanish post-secondary education in La Rioja (Spain), based on their multimodal and unimodal learning preferences. The results indicated that the productive vocabulary of multimodal learners was slightly higher than that of unimodal learners, although both had a knowledge of around 1,000 words. Accordingly, these 12th grade Spanish EFL learners would not be able to use the language effectively for communicative purposes. Considering their perceptual learning styles, those multimodal students who used the three modalities in balance seemed to have the highest vocabulary knowledge, whereas visual and auditory learners had the lowest. Thus, learning EFL vocabulary through the three modalities appeared to result in a higher vocabulary size. In the case of unimodal students, visual learners appeared to have the highest productive vocabulary available, whilst auditory students had the lowest. This result might be explained by the predominance of textbooks in Spanish EFL education (Jiménez Catalán & Mancebo Francisco, 2008). Finally, no statistically significant differences were observed in the productive vocabulary knowledge of both groups (multimodal and unimodal learners) regarding their learning preferences. In fact, the findings showed that there was a large effect size and a positive and large strength of association. Therefore, this implies that 12th grade multimodal and unimodal EFL learners’ productive vocabulary knowledge did not differ significantly, regardless of their different learning preferences (similar to Hatami, 2018; Kassaian, 2007, see Section 2.3.). However, this might only be the case of our multimodal and unimodal informants, who have been exposed to similar EFL vocabulary input in the Spanish educational system. More research is needed to determine whether this is the norm for Spanish EFL learners. The present study presents several limitations. Firstly, it is limited by its small sample size, as only 60 students participated in this research. The second limitation of this study 149 Monograph IV January 2022 January 2022 was that it was only conducted in one state school of post-secondary education. Accordingly, the findings reported cannot be taken as representative of either the population of 12th grade students, or the autonomous community of La Rioja. Another constraint was that the division into multimodal and unimodal learners was made only considering the results obtained in the LSS questionnaire. 6. Conclusions We did not contrast these results with other tests that also identified perceptual learning styles, or with oral interviews with the students to compare whether the learning preferences they believed to have are the ones they actually use in the process of learning. Likewise, their productive vocabulary knowledge was only measured with one instrument. It was not contrasted with other productive vocabulary tests or tasks, so this might have affected the findings.i Several implications for EFL instruction arise from this investigation. Our findings indicated that the productive vocabulary of 12th grade Spanish EFL learners was around 1,000 words. Therefore, more instruction on EFL vocabulary would be necessary so that learners would be able to communicate in the English language, since the knowledge of at least 2,000-3,000 words is required for that (Nation & Waring, 1997; Schmitt & Schmitt, 2014; van Zeeland & Schmitt, 2013). Furthermore, the instruction of EFL vocabulary could also include tasks based on authentic input to practice and strengthen the vocabulary learnt from the textbooks and curricular materials (e.g., Rajendran, 2020). Although Spanish EFL students’ learning preferences did not seem to influence their productive vocabulary know- ledge, teachers could accommodate their teaching materials to their learners’ perceptual learning styles to improve their learning (balanced instruction). In this regard, students would become more positive and motivated, since the instruction would be more learner- centered and inclusive of all learning preferences, not favoring one particular learning style (e.g., Natividad & Batang, 2018; Payaprom & Payaprom, 2020). All in all, new teaching methodologies and a more learner-centered approach would be beneficial for Spanish EFL learners in their language learning in general and in vocabulary learning specifically. Some interesting areas of future research could be to conduct a longitudinal study at the beginning and at the end of the academic year in the 12th grade. At the beginning, PVLT and LSS questionnaires could be distributed to determine EFL learners’ productive vocabulary and perceptual learning styles, respectively. Then, teachers and learners could be instructed in perceptual teaching and learning styles respectively to become familiar with other ways of teaching and learning and enhance it. After knowing their learners’ learning styles, teachers could teach them vocabulary not only from textbooks but also from authentic exposures. They could design vocabulary exercises that cater for all the different learning styles (e.g., readings, videos, role-plays) to increase their EFL vocabulary. 7. Acknowledgements This work is framed under a research project grant number PGC2018-095260-B-100, financed by the Spanish Ministry of Science, Innovation and Universities. 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https://openalex.org/W2569750833	https://www.rbciamb.com.br/Publicacoes_RBCIAMB/article/download/146/113	Portuguese	null	Políticas públicas relacionadas ao transporte individual e coletivo e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais	Revista Brasileira de Ciências Ambientais	2,016	cc-by	6,145	Endereço para correspondência: Endereço para correspondência: Boscolli Barbosa Pereira – Avenida João Naves de Ávila, 2121, Bloco 3E, Laboratório de Vigilância em Saúde Ambiental – Santa Mônica – 38400‑902 – Uberlândia (MG), Brasil – E‑mail: boscolli@ufu.br DOI: 10.5327/Z2176-947820160027 DOI: 10.5327/Z2176-947820160027 RESUMO Paolla Brandão da Cunha Curso de Gestão em Saúde Ambiental pela Universidade Federal de Uberlândia (UFU) – Uberlândia (MG), Brasil. Paolla Brandão da Cunha Curso de Gestão em Saúde Ambiental pela Universidade Federal de Uberlândia (UFU) – Uberlândia (MG), Brasil. O trânsito na área urbana de Uberlândia torna‑se a cada dia mais desorganizado e inseguro, comprometendo a qualidade de vida da população e afetando a segurança e a saúde das pessoas devido aos diversos conflitos e às emissões de poluentes oriundos do elevado tráfego de automóveis. O presente trabalho objetivou habilitar o Modelo FPEEEA (OMS) como instrumento de análise dos impactos das políticas públicas relacionadas direta e/ou indiretamente aos sistemas de transporte coletivo e individual na saúde da população de Uberlândia, MG. Os dados para elaboração da matriz foram levantados em bases de dados da literatura científica e de domínio público virtual. Foram selecionados indicadores para a identificação das forças motrizes (FM) e pressões (P). Para cada nível da matriz foram identificadas e relacionadas ações (A) de atenção e vigilância em saúde ambiental, que podem contribuir para as decisões e gerenciamento de riscos associados à vigilância em saúde ambiental. Guilherme Gomes Silva Curso de Enfermagem pela Universidade Federal de Uberlândia (UFU) – Uberlândia (MG), Brasil. Boscolli Barbosa Pereira Programa de Pós‑graduação em Genética e Bioquímica pela Universidade Federal de Uberlândia (UFU) – Uberlândia (MG), Brasil. Palavras‑chave: vigilância em saúde ambiental; indicadores de contaminação; qualidade do ar; políticas públicas. INTRODUÇÃO Uberlândia é considerada a capital da logística por pos‑ suir o maior centro atacadista da América Latina. A lo‑ calização geográfica da cidade contribui para isso, pois sua malha rodoviária faz com que os maiores centros econômicos do Brasil (São Paulo, Rio de Janeiro, Belo Horizonte, Goiânia e Brasília) contem com Uberlândia como ponto de intersecção (PEREIRA et al., 2014). Como os estudos experimentais são geralmente limita‑ dos por questões éticas, dificuldades de financiamento e metodologias que não conseguem isolar os efeitos específicos dos poluentes, investigações observacio‑ nais têm sido amplamente incentivadas e utilizadas (CASTRO; GOUVEIA; ESCAMILLA‑CEJUDO, 2003). Nesse tipo de estudo, os resultados da avaliação dos efeitos da poluição na saúde da população são avalia‑ dos de maneira generalizada, ou seja, o grupo de indi‑ víduos investigado pode ser definido em bairros, cida‑ des, regiões ou países. A rápida urbanização de Uberlândia trouxe como con‑ sequência um crescimento significativo no número de veículos que trafegam na cidade nos últimos anos. Na realidade, a frota veicular praticamente dobrou no município. De acordo com dados fornecidos pela Se‑ cretaria de Trânsito e Transportes (SETTRAN, 2012), enquanto em 2001 circulavam pela cidade 171.829 veí‑ culos, em 2011, o número de veículos em Uberlândia atingiu 341.364 unidades. Contudo, a maior parte dos estudos dessa natureza tende a concentrar seu foco de investigação no eixo ex‑ posição‑efeito, desconsiderando fatores determinan‑ tes e condicionantes da situação de saúde da popula‑ ção avaliada, como condição socioeconômica, acesso aos serviços de saúde e outras situações que poderiam aumentar o impacto das contribuições desses estudos para gestores dos setores ambiental e da saúde, para a tomada de decisões e implementação de políticas pú‑ blicas racionalizadas (TEIXEIRA, 2005). Diante desse cenário, o trânsito na área urbana de Uberlândia torna‑se a cada dia mais desorganizado e inseguro, comprometendo a qualidade de vida da po‑ pulação e afetando a segurança e a saúde das pessoas, devido aos diversos conflitos e às emissões de poluen‑ tes oriundos do elevado tráfego de automóveis. Pensando nessa relação complexa entre meio ambien‑ te e saúde humana, no início da década de 1990, a Or‑ ganização Mundial de Saúde (OMS) criou um modelo para caracterizar e avaliar os problemas ocasionados por essas inter‑relações, com o objetivo de propor ações de prevenção e identificação de indicadores (KJELLSTRÖM & CORVALÁN, 1995). ABSTRACT Traffic in the urban area of Uberlândia becomes increasingly disorganized and unsure, compromising the population’s quality of life and affecting the safety and health of people, due to various conflicts and the emissions of pollutants coming from high automobile traffic. This study aimed to enable the Model DPSEEA (WHO) as an analytical tool of the impacts of direct related public policies and/or indirectly to the collective and individual transport systems in the health of the population of Uberlândia, MG. The data for the preparation of the matrix were collected in databases of scientific literature and virtual public domain. Indicators were selected to identify the Driving Forces (FM) and Pressures (P). For each level of the matrix, have been identified and related actions (A) to provide care and environmental health surveillance, which can contribute to decisions and managing risks associated with environmental health surveillance. Keywords: environmental health surveillance; pollution indicators; air quality; public policies. 16 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Políticas públicas relacionadas ao transporte individual e coletivo e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais Políticas públicas relacionadas ao transporte individual e coletivo e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais INTRODUÇÃO Embora a qualidade do ar seja avaliada por meio de medições físico‑químicas, que aferem com precisão a concentração dos poluentes, os resultados dessas me‑ dições não permitem conclusões a respeito do impac‑ to desses contaminantes sobre seres vivos (PEREIRA; CAMPOS JÚNIOR; MORELLI, 2013) De fato, os efeitos simultâneos da associação de outros poluentes que caracterizam a mistura complexa que constitui o ar das grandes cidades sobre a biota não po‑ dem ser conhecidos a partir de informações baseadas somente na concentração de cada um desses poluentes. Esse modelo foi chamado de quadro de força motriz – pressão – estado – exposição – efeito – ação (FPEEEA), o qual tem a finalidade de identificar e organizar os dados para construção de indicadores sugeridos para a vigilân‑ cia em saúde de populações e ambientes, que permitem inferir sobre os diferentes processos que determinam e condicionam a saúde, podendo desencadear possíveis efeitos danosos ao bem‑estar físico e mental da popula‑ ção (ARAUJO‑PINTO; PERES; MOREIRA, 2012). Diversas abordagens metodológicas e conceituais têm sido empregadas em estudos epidemiológicos brasilei‑ ros que visam analisar a associação entre poluição at‑ mosférica e desfechos na saúde humana (WHO, 2006). Os estudos realizados com esse fim têm sido delineados a partir de enfoques observacionais e experimentais (AMANCIO et al., 2012; ANDRADE FILHO et al., 2013; ARBEX et al., 2009; BRAGA et al., 2007; IGNOTTI et al., 2010; JUNGER; PONCE DE LEON, 2007; MASCARENHAS et al., 2008; MOURA et al., 2011; NASCIMENTO, 2011; OLIVEIRA et al., 2011). De acordo com esse modelo, as forças motrizes propi‑ ciam uma visão macro sobre os problemas que podem gerar algum dano a saúde e ao ambiente (MINISTÉRIO DA SAÚDE, 2011). As pressões são decorrentes dos elementos de for‑ ça motriz, ou seja, por meio dessa pressão originada 17 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Cunha, P.B.; Silva, G.G.; Pereira, B.B. te, as ações são os indicativos de possibilidade de in‑ tervenção, que podem atuar em quaisquer dos demais eixos (MINISTÉRIO DA SAÚDE, 2011). da força motriz é que o ambiente será alterado. Já o Estado está relacionado às possibilidades de um im‑ pacto no ambiente, resultado das diversas alterações produzidas nesse ambiente pelos dois primeiros níveis (MINISTÉRIO DA SAÚDE, 2011). METODOLOGIA METODOLOGIA Tipo de estudo metodologia utilizada, este estudo pode ser caracteri‑ zado como descritivo‑exploratório. A presente investigação configura‑se como estudo de caso e utiliza abordagem quanti‑qualitativa. Segundo a OBJETIVOS Fornecer subsídios para ações e tomadas de deci‑ sões relacionadas aos serviços de vigilância em saú‑ de ambiental. Habilitar o Modelo FPEEEA (OMS) como instrumento de análise dos impactos das políticas públicas relacionadas di‑ reta e/ou indiretamente aos sistemas de transporte coleti‑ vo e individual na saúde da população de Uberlândia, MG. Identificar, por meio da análise de determinantes de forças motrizes e de pressão, quais indicadores estão disponíveis e em condições de contribuir para o pro‑ cesso decisório e de gerenciamento de riscos associa‑ do à vigilância em saúde ambiental. Estruturar sistemas de indicadores que permitam ana‑ lisar os efeitos e impactos, positivos ou não, de políticas públicas propostas aos sistemas de transporte coletivo e individual na saúde da população de Uberlândia, MG. INTRODUÇÃO Nessa direção, a realização da pesquisa é justificada pela necessidade de estudos que proponham a estru‑ turação de sistemas de indicadores que permitam ana‑ lisar os efeitos e impactos, positivos ou não, de políticas públicas propostas para os diversos setores da adminis‑ tração pública sobre as condições de saúde da popula‑ ção contribuindo para o fortalecimento das ações de Saúde Pública no Brasil (CARNEIRO et al., 2006). Esses estudos permitem inferir sobre os diferentes processos que determinam e condicionam a saúde e fornecem informações que podem subsidiar o planejamento de ações de prevenção de possíveis efeitos danosos ao bem‑estar físico e mental da população. Em decorrência disso, a exposição humana é a condi‑ ção necessária para o processo de adoecimento. Desse modo, a exposição significa o contato do homem com os agentes nocivos (poluentes atmosféricos no caso da análise da qualidade do ar) à saúde, resultantes do Estado, das pressões e das forças motrizes. Os efeitos relacionam‑se diretamente com a saúde dos indiví‑ duos, que estão vinculados ao bem‑estar e ao padrão de morbimortalidade de uma população. Estes efeitos dependerão do local, da duração, da exposição, da sus‑ cetibilidade individual, entre outros fatores. Finalmen‑ Levantamento e organização dos dados no modelo FPEEEA O modelo FPEEEA foi empregado para análise dos im‑ pactos das políticas públicas envolvidas nos setores de transporte individual e coletivo na cidade de Uberlân‑ dia (MG) sobre indicadores de qualidade de saúde e ambiente. fia e Estatística (IBGE), o Departamento de Informáti‑ ca do Sistema Único de Saúde (DATASUS), Instituto de Pesquisa Econômica Aplicada (IPEA), Departamento Nacional de Trânsito (DENATRAN) e Prefeitura Munici‑ pal de Uberlândia. Os elementos para elaboração da matriz analítica do modelo FPEEEA foram levantados em bases de dados de literatura científica, incluindo as bases de indexação PubMed, LILACS, MEDLINE e SCIELO e de domínio pú‑ blico virtual, incluindo o Instituto Brasileiro de Geogra‑ Os dados foram analisados com base na organização dos determinantes e indicadores associados a cada eixo na matriz FPEEEA. Também foram identificadas e relacionadas ações (A) de atenção e vigilância em saú‑ de ambiental. 18 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Políticas públicas relacionadas ao transporte individual e coletivo e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais Como critérios de inclusão foram adotados: Como critérios de inclusão foram adotados: Os indicadores definidos para os eixos “força motriz” e “pressão”, que compõem a matriz do modelo FPEEEA produzida, foram listados e avaliados. Esses índices analisados são restritos à cidade de Uberlândia e o período avaliado está compreendido entre janeiro de 2003 e dezembro de 2013. • disponibilidade de dados para o município de Uberlândia, • disponibilidade de dados para organização e dis‑ tribuição nos níveis de complexidade, conforme o modelo FPEEEA. e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais RESULTADOS A partir do levantamento dos dados e informações, foi possível elencar possíveis indicadores para a matriz FPEEEA (Tabela 1), enfatizando os eixos de forças motrizes e pressões (Tabela 2). Análise de determinantes, indicadores e ações relacionados aos problemas de mobilidade urbana do município de Uberlâ Análise de determinantes, indicadores e ações relacionados aos problemas de mobilidade urbana do município de Uberlândia, Minas Gerais usuários do transporte coletivo, alto crescimento do interesse pelo transporte individual motorizado; as‑ pectos socioeconômicos; fatores ambientais, culturais e de saúde. A análise de dados e informações disponíveis na Se‑ cretaria de Trânsito e Transporte da Prefeitura Muni‑ cipal de Uberlândia e nas bases do DENATRAN permi‑ tiu acessar uma série de possíveis indicadores para o período de 2003 a 2013. Conforme mostra a Tabela 1, os determinantes considerados incluem políticas eco‑ nômicas, como a redução do Imposto sobre Produtos Industrializados (IPI) e o aumento da tarifa para trans‑ porte público; políticas específicas para a mobilidade urbana, como criação de ciclovias, corredores de ôni‑ bus, aumento da taxa de motorização, diminuição da taxa de ocupação, baixo crescimento do número de As emissões de poluentes e seus respectivos efeitos sobre a saúde humana vêm trazendo diversos desafios para a gestão e a tomada de decisão na formulação e implementação de políticas públicas e ações (A). Assim, para cada nível da matriz, foram identificadas as ações, tais como medidas de intervenção na po‑ Nível Determinantes Ações Indicadores Forças motrizes Redução do IPI para carros novos Revisão das políticas econômicas para o setor automotivo Emplacamentos Aumento da tarifa (transporte público) Melhoria da qualidade do transporte; Subsídios fiscais para o transporte Variação do valor da tarifa Aumento da Urbanização Políticas e ações de planejamento urbano Ruas e Avenidas Crescimento populacional Criação de ciclovias Estímulo ao transporte individual não motorizado Quilômetros de ciclovias Sistema integrado de transporte – corredores de ônibus Estímulo ao transporte público coletivo Quilômetros de corredores Tabela 1 – Determinantes, indicadores e ações para vigilância em saúde ambiental do município de Uberlândia em relação aos problemas associados à mobilidade urbana. Continua... Tabela 1 – Determinantes, indicadores e ações para vigilância em saúde ambiental do município de Uberlândia em relação aos problemas associados à mobilidade urbana 19 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Cunha, P.B.; Silva, G.G.; Pereira, B.B. Tabela 1 – Continuação. RESULTADOS Determinantes Ações Indicadores Aumento da frota de automóveis Fiscalização sobre emissões veiculares Frota de automóveis Aumento da taxa de motorização Fiscalização sobre emissões veiculares Taxa de motorização Diminuição da taxa de ocupação Estímulo ao transporte público coletivo Taxa de ocupação Redução do número de usuários do transporte coletivo Estímulo ao transporte público coletivo Usuários do transporte Crescimento do interesse pelo transporte individual motorizado Estímulo ao transporte individual não motorizado; Estímulo ao transporte público coletivo Número de CNHs emitidas Ineficiente cobertura do serviço de transporte coletivo Estímulo ao transporte público coletivo Quilometragem realizada Aumento da frota de veículos do transporte coletivo Estímulo ao transporte público coletivo Frota Deterioração da qualidade do ar Estudos de biomonitoramento ambiental; Monitoramento ambiental de parâmetros físico‑químicos para qualidade do ar Indicadores físico‑químicos Indicadores biológicos Aumento dos congestionamentos Estudos de biomonitoramento ambiental; Estímulo ao transporte individual não motorizado; Estímulo ao transporte público coletivo Número de congestionamentos Tempo de viagem o Aumento do número de habitações em áreas de risco Políticas e ações de planejamento urbano; Manejo ambiental; Estímulo à percepção de riscos Habitações em áreas de risco Aumento do número de trabalhadores expostos à atmosfera contaminada Avaliação clínica dos trabalhadores; Atividades educativas; Estímulo à percepção de riscos Número de trabalhadores expostos Aumento da morbimortalidade associada à poluição atmosférica Inquéritos epidemiológicos considerando DSS Indicadores epidemiológicos Aumento do número de acidentes de trânsito Fiscalização sobre emissões veiculares Acidentes de trânsito Tabela 1 – Continuação. RESULTADOS Aumento dos congestionamentos 20 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Políticas públicas relacionadas ao transporte individual e coletivo e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais e seus pactos sob e d cado es de saúde e a b e te e Ube â d a, as Ge a s 21 Indicadores Ano 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Emplacamentos 10.426 13.092 13.981 16.017 20.953 26.089 22.695 26.971 27.518 23.254 19.751 Variação do valor da tarifa 1,50 1,50 1,90 1,90 1,90 1,90 2,20 2,25 2,40 2,60 2,85 Ruas 3.547 3.555 3.585 3.585 3.610 3.636 3.745 3.809 3.850 3.874 ‑ Avenidas 382 424 424 424 424 422 431 434 441 451 ‑ Crescimento populacional 529.061 538.690 548.496 558.478 568.648 579.000 589.548 604.013 611.904 619.536 646.673 km de ciclovias 0 0 0 0 0 0 31,95 31,95 31,95 31,95 ‑ km de corredores 0 0 0 15,0 15 15 15 15 15 15 15 Frota de automóveis 118.668 123.549 129.554 134.539 140.837 153.933 171.243 190.670 210.778 229.947 247.941 Taxa de motorização automotiva* 0,22 0,23 0,24 0,24 0,25 0,27 0,29 0,32 0,34 0,37 0,38 Taxa de motorização veicular 0,34 0,35 0,36 0,38 0,40 0,44 0,47 0,52 0,56 0,59 0,60 Taxa de ocupação automotiva* 4,46 4,36 4,23 4,15 4,04 3,76 3,44 3,17 2,90 2,69 2,61 Taxa de ocupação veicular**** 2,96 2,87 2,75 2,64 2,50 2,28 2,11 1,94 1,79 1,68 1,65 Passageiros transportados 55.694.462 54.733.131 53.317.611 54.708.315 57.759.575 60.228.871 60.308.127 62.972.458 64.311.682 64.323.916 64.517.470 Número de CNHs emitidas 6.470 7.376 9.562 12.852 13.026 14.743 18.957 16.851 17.166 15.083 15.653 Quilometragem realizada pelo transporte público coletivo 28.801.213 27.081.635 28.782.310 29.657.418 30.010.301 30.032.834 30.331.313 30.268.592 30.625.643 30.409.557 30.063.250 Frota de ônibus para o transporte público coletivo 351 355 353 329 332 337 352 362 368 365 361 Tabela 2. Indicadores e informações disponíveis em condições de contribuir para o processo decisório e de gerenciamento de riscos associado à vigilância em saúde ambiental do município de Uberlândia em relação aos problemas referentes à mobilidade urbana. – Dados não disponíveis. * É a relação entre o número de automóveis por habitantes; É a relação entre o número de veículos por habitantes; * É a relação entre o número de habitantes por automóvel (caminhonete, camioneta e utilitário); ** É a relação entre o número de habitantes por veículo. Análise e seleção dos indicadores para os eixos “for os indicadores para os eixos “força motriz” e “pressão”, segundo o modelo FPEEEA Análise e seleção dos indicadores para os eixos “força motriz” e “pressão”, segundo o modelo FPEEEA “força motriz” e “pressão”. A Tabela 2 apresenta o rol de indicadores selecionados que serão detalha‑ dos a seguir. A partir do levantamento e da organização das in‑ formações obtidas, foi possível selecionar, propor e analisar indicadores e informações para os eixos Forças motrizes Os dados levantados com base nos emplacamentos de veículos revelam que houve aumento no número de automóveis adquiridos ao longo de todo período avaliado, sendo que a partir de 2006 essa elevação foi notavelmente maior. população do município, uma vez que, segundo a es‑ timativa do ano de 2003, a população de Uberlândia era de 529.061 habitantes, e, em 2013, houve um aumento para 646.673 habitantes, representando um crescimento de 22,2% da população residente no município. Em relação à variação do valor da tarifa para o trans‑ porte coletivo é notória a elevação do preço da passa‑ gem, com aumento de 1,35 reais de 2003 a 2013. Em relação aos dados levantados sobre o indicador de quilômetros (km) de ciclovias, revela‑se que somente em 2009 foram implementadas ciclovias, com exten‑ são total de 31,95 km, sendo que nos últimos quatro anos esse cenário não sofreu alteração. Quanto ao terceiro indicador (Ruas e Avenidas), foi possível observar crescimento durante os últimos 10 anos, segundo o qual, em 2003, o município apre‑ sentava 3.547 ruas construídas e 382 avenidas e, em 2013, este número cresceu, apresentando 3.874 ruas e 451 avenidas construídas no município de Uberlândia, segundo dados disponibilizados pela SETRAN (2014). Por fim, o último indicador analisado refere‑se aos qui‑ lômetros de corredores exclusivos para o transporte pú‑ blico coletivo que, segundo os dados do SETRAN (2014), somente no ano de 2006 foram implementadas 13 es‑ tações ao longo da Avenida João Naves com extensão total de 15 km, sendo 7,5 km no sentido Bairro‑Centro e 7,5 km no sentido Centro‑Bairro. Com base no indicador de crescimento populacional, é possível notar uma forte tendência de aumento da RESULTADOS e veículos por habitantes; * É a relação entre o número de habitantes por automóvel eículo Indicad Emplaca Variação tarifa Ruas Avenida Crescim populac km de c km de c Frota de Taxa de automo Taxa de veicular Taxa de automo Taxa de veicular Passage transpo Número emitida Quilom realizad transpo coletivo Frota d o trans coletivo – Dados * É a rela (caminho 21 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Cunha, P.B.; Silva, G.G.; Pereira, B.B. Cunha, P.B.; Silva, G.G.; Pereira, B.B. e ações de planejamento urbano adequados ao cres‑ cimento populacional. lítica econômica, considerando, inclusive, a revisão dessas políticas para o setor automotivo; interven‑ ções na mobilidade urbana como estímulo ao uso do transporte público coletivo; melhoria da qualidade do transporte público e subsídios fiscais para o se‑ tor; estímulo ao transporte individual não motoriza‑ do; fiscalização sobre emissões veiculares; políticas lítica econômica, considerando, inclusive, a revisão dessas políticas para o setor automotivo; interven‑ ções na mobilidade urbana como estímulo ao uso do transporte público coletivo; melhoria da qualidade do transporte público e subsídios fiscais para o se‑ tor; estímulo ao transporte individual não motoriza‑ do; fiscalização sobre emissões veiculares; políticas Para os eixos estado, exposição e efeito, as principais ações elencadas se concentram em estudos de biomo‑ nitoramento ambiental para avaliação de parâmetros físico‑químicos para qualidade do ar; ações de manejo ambiental e atividades educativas. Análise e seleção dos indicadores para os eixos “força motriz” e “pressão”, segundo o modelo FPEEEA Análise e seleção dos indicadores para os eixos “força motriz” e “pressão”, segu DISCUSSÃO Por meio da organização e análise dos dados existentes, foi possível observar diferentes situações e cenários que podem contribuir para o aumento da exposição da população de Uberlândia aos poluentes atmosféricos, aumentando o risco de comprometimento da saúde desses indivíduos expostos. Desta forma, segundo Lemes (2005), as cidades de mé‑ dio a grande porte do Brasil vêm lidando com inúmeros problemas devido ao seu crescimento acelerado e sem planejamento. Assim, com a rápida urbanização, pro‑ blemas como poluição, congestionamentos, a falta de transporte público de qualidade capacitado para aten‑ der a alta demanda são consequências ou mesmo ine‑ xistência de um planejamento urbano eficaz e de trans‑ portes integrados. Como foi possível perceber, em relação às forças motri‑ zes (FM), as políticas públicas têm um papel significati‑ vo no quadro da qualidade do ar no município pesqui‑ sado. A redução dos IPIs, especialmente na indústria automobilística, e o aumento no valor da tarifa do transporte público serviram de incentivo para ampliar a procura pelo transporte individual motorizado, como foi mostrado nos indicadores de emplacamentos e da variação do valor da tarifa. Em resposta, é necessário, segundo o mesmo autor, que haja um planejamento eficiente dos sistemas de transporte, capaz de analisar os fatores intervenientes, tais como: o uso atual do solo, projeções para conhecer o comportamento da demanda futura, entre outros. Deste modo, a partir dos estudos realizados é possível coordenar o crescimento e o desenvolvimento das ci‑ dades com os investimentos necessários para atender com qualidade a demanda exigida (LEMES, 2005). De acordo com o IPEA (2011), essa migração para o transporte individual deu‑se pela abundante oferta de crédito para aquisição de veículos e uma política tribu‑ tária (IPI) que reduziu impostos de veículos populares que, como consequência disso, diminuiu o interesse da população pelo uso do transporte coletivo, aumentan‑ do assim o uso do transporte individual motorizado. Em relação à proposta de criação de sistema integrado de transporte, que tem como objetivo melhorar aces‑ so ao transporte público pela população, observamos que além da construção dos terminais, a instalação dos corredores exclusivos para o transporte coletivo e tam‑ bém a criação de ciclovias, em tese, poderiam atuar no desestímulo do uso do transporte individual não mo‑ torizado, mas avançaram muito pouco e ainda não são suficientes para atender à grande e crescente popula‑ ção de Uberlândia. Pressão A partir dos dados levantados sobre a frota de automó‑ veis (automóvel, camioneta, caminhonete e utilitário), observa‑se que, nos 10 anos avaliados, houve um au‑ mento de 129.273 automóveis na cidade de Uberlândia. geral, incluindo automóveis, motocicletas, caminhões, ônibus e outros assemelhados. Para esse indicador, foi possível observar um aumento de 0,26 ao longo do período avaliado. Quanto à taxa de motorização auto‑ motiva, caracterizada pelos automóveis, camionetas, caminhonetes e utilitários, também houve uma eleva‑ ção de 0,16 entre 2003 e 2013. A taxa de motorização, que estabelece o número de meios de transporte por habitantes, neste estudo, foi avaliada de forma a considerar separadamente a taxa de motorização veicular e a taxa de motorização automotiva. A taxa de motorização veicular foi, des‑ se modo, caracterizada por meios de locomoção em Em relação à taxa de ocupação, a qual estabelece o nú‑ mero de habitantes em relação aos meios de transpor‑ te, também foram consideradas separadamente a taxa 22 RBCIAMB \| n.40 \| jun 2016 \| 16-26 Políticas públicas relacionadas ao transporte individual e coletivo e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais Políticas públicas relacionadas ao transporte individual e coletivo s impactos sobre indicadores de saúde e ambiente em Uberlândia Minas Gerais Políticas públicas relacionadas ao transporte individual e coletivo p p e seus impactos sobre indicadores de saúde e ambiente em Uberlândia, Minas Gerais vo no período de 2003 a 2009, sendo que somente de 2010 a 2013 foi possível observar uma redução na pro‑ cura para emissão de novas CNHs. de ocupação veicular e a taxa de ocupação automotiva. De acordo com a análise desse indicador, a taxa de ocu‑ pação veicular mostrou uma redução de 1,31 ao longo dos anos avaliados, enquanto a taxa de ocupação auto‑ motiva reduziu 1,85 no período avaliado. de ocupação veicular e a taxa de ocupação automotiva. De acordo com a análise desse indicador, a taxa de ocu‑ pação veicular mostrou uma redução de 1,31 ao longo dos anos avaliados, enquanto a taxa de ocupação auto‑ motiva reduziu 1,85 no período avaliado. Em relação ao indicador de quilometragem realizado pelo transporte público coletivo, foi possível observar um baixo crescimento de oferta desse serviço ao longo dos anos analisados. Pressão Sobre o indicador de passageiros transportados no ser‑ viço público de transporte coletivo, foi possível obser‑ var baixo crescimento de interesse/utilização desses usuários para o uso do transporte público no município de Uberlândia no período avaliado. Por fim, a partir dos dados sobre a frota do transporte coletivo, observa‑se que houve um baixo crescimento dessa frota nos últimos 10 anos, sendo que nos anos de 2005, 2006, 2012 e 2013 a frota de ônibus foi reduzida. Em contrapartida, os dados levantados sobre o número de CNHs emitidas mostraram um aumento significati‑ DISCUSSÃO É possível observar também que o município de Uber‑ lândia apresenta um contínuo aumento da urbanização, conforme revelam os indicadores de ruas, avenidas e o crescimento populacional, fazendo com que haja a ne‑ cessidade de políticas e ações de planejamento urbano na cidade que racionalizem o crescimento da malha ur‑ bana e reduzam impactos na qualidade do ar. É importante destacar, no que se refere às pressões (P), que com o crescimento da frota de veículos, simulta‑ 23 RBCIAMB \| n.40 \| jun 2016 \| 16-26 neamente ocorreu aumento na taxa de m e ocupação veicular, revelando, consequen baixo interesse pelo transporte coletivo, pr te pela falta de qualidade e superlotação d Segundo Rodrigues e Sorratini (2005), o coletivo possui uma grande importância de deslocamento urbano entre os meio moção, uma vez que este consegue ame problemas, tais como: diminuir as viagen automóveis, colaborando na redução dos namentos, da poluição ambiental, dos a trânsito, entre outros. Entretanto, várias cias fazem a população preferir o uso do individual em vez do transporte coletiv que este gera muita insatisfação em vár assim como lotação, tempo de viagem, vias, segurança, características dos veícu de parada. Sendo assim, fazem‑se necessários estudos tema operacional que atendam às expecta A análise dos dados que foram apresentad dos direciona para o promissor uso do mod (OMS) como instrumento de trabalho para a principalmente para a vigilância em saúde a cidade de Uberlândia, em particular na sua a qualidade do ar. Uma vez que esse model olhar para as diversas situações problemas e tintos, com destaques para os determinante res e as ações apresentadas, representa um te ferramenta de subsídio à elaboração e pl das ações de vigilância que envolvem ambie Cunha, P.B.; Silva, G.G.; Pereira, B.B. pulação com o propósito de melhorar o nível de serviço do transporte coletivo (RODRIGUES & SORRATINI, 2005). neamente ocorreu aumento na taxa de motorização e ocupação veicular, revelando, consequentemente, o baixo interesse pelo transporte coletivo, principalmen‑ te pela falta de qualidade e superlotação desse modal. A análise da matriz de indicadores disponíveis possibili‑ tou uma visão mais detalhada sobre a situação atual da qualidade da mobilidade urbana em Uberlândia que, conforme o exposto, traz prejuízos à locomoção e de‑ teriora a atmosfera do município. CONCLUSÕESi Associada a essa situação, foi possível observar que ainda não existem indicadores, nem ações exe‑ cutadas em relação ao monitoramento da qualidade do ar de Uberlândia. Buscam‑se, portanto, estimu‑ lar, a partir do modelo apresentado e discutido ao longo do presente estudo, novas experiências rela‑ cionadas à sua aplicação, como subsídio a ações de vigilância em saúde ambiental, para que possa ga‑ rantir melhor qualidade de vida para a população e maior conservação do ambiente. A análise dos dados que foram apresentados e discuti‑ dos direciona para o promissor uso do modelo FPEEEA (OMS) como instrumento de trabalho para as vigilâncias, principalmente para a vigilância em saúde ambiental da cidade de Uberlândia, em particular na sua relação com a qualidade do ar. Uma vez que esse modelo favorece o olhar para as diversas situações problemas em níveis dis‑ tintos, com destaques para os determinantes, indicado‑ res e as ações apresentadas, representa uma importan‑ te ferramenta de subsídio à elaboração e planejamento das ações de vigilância que envolvem ambiente e saúde. DISCUSSÃO Com isso, para cada nível da matriz foi proposto um conjunto de ações para prevenção e mitigação dos danos associados aos po‑ luentes atmosféricos, destacando‑se alguns dos prin‑ cipais indicadores para possível uso nas atividades da vigilância em saúde ambiental. Segundo Rodrigues e Sorratini (2005), o transporte coletivo possui uma grande importância no cenário de deslocamento urbano entre os meios de loco‑ moção, uma vez que este consegue amenizar vários problemas, tais como: diminuir as viagens feitas por automóveis, colaborando na redução dos congestio‑ namentos, da poluição ambiental, dos acidentes de trânsito, entre outros. Entretanto, várias circunstân‑ cias fazem a população preferir o uso do transporte individual em vez do transporte coletivo, uma vez que este gera muita insatisfação em vários fatores, assim como lotação, tempo de viagem, estado das vias, segurança, características dos veículos e locais de parada. No âmbito das políticas públicas (FM), é relevante des‑ tacar a necessidade de estímulo e subsídios fiscais para melhoria do transporte público; incentivo ao transpor‑ te não motorizado, com a criação de ciclovias e revi‑ são das políticas econômicas para o setor automotivo. Para o nível de pressão, são necessárias ações de fis‑ calização sobre as emissões veiculares e melhoria nos serviços de planejamento urbano do município. Sendo assim, fazem‑se necessários estudos em seu sis‑ tema operacional que atendam às expectativas da po‑ REFERÊNCIAS REFERÊNCIAS AMANCIO, C. T.; NASCIMENTO, L. F. C. Asma e poluentes ambientais: um estudo de séries temporais. Revista da Associação Médica Brasileira, v. 58, n. 3, p. 302‑307, 2012. ANDRADE FILHO, V. S.; ARTAXO, P.; HACON, S.; CARMO, C. N.; CIRINO, G. Aerossóis de queimadas e doenças respiratórias em crianças, Manaus, Brasil. Revista de Saúde Pública, v. 47, n. 2, p. 239‑247, 2013. ARAUJO‑PINTO, M.; PERES, F.; MOREIRA, J. C. Utilização do modelo FPEEEA (OMS) para a análise dos riscos relacionados ao uso de agrotóxicos em atividades agrícolas do estado do Rio de Janeiro. 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https://openalex.org/W3165489823	https://advancesindifferenceequations.springeropen.com/counter/pdf/10.1186/s13662-021-03429-2	English	null	An accurate approach based on the orthonormal shifted discrete Legendre polynomials for variable-order fractional Sobolev equation	Advances in difference equations	2,021	cc-by	7,420	© The Author(s) 2021. 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/. An accurate approach based on the orthonormal shifted discrete Legendre polynomials for variable-order fractional Sobolev equation M.H. Heydari1* and A. Atangana2,3 *Correspondence: heydari@sutech.ac.ir 1Department of Mathematics, Shiraz University of Technology, Shiraz, Iran Full list of author information is available at the end of the article Abstract This paper applies the Heydari–Hosseininia nonsingular fractional derivative for deﬁning a variable-order fractional version of the Sobolev equation. The orthonormal shifted discrete Legendre polynomials, as an appropriate family of basis functions, are employed to generate an operational matrix method for this equation. A new fractional operational matrix related to these polynomials is extracted and employed to construct the presented method. Using this approach, an algebraic system of equations is obtained instead of the original variable-order equation. The numerical solution of this system can be found easily. Some numerical examples are provided for verifying the accuracy of the generated approach. Keywords: Variable-order time fractional Sobolev equation; Orthonormal shifted discrete Legendre polynomials; Nonsingular variable-order fractional derivative ( 2021) 2021:272 ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations https://doi.org/10.1186/s13662-021-03429-2 1 Introduction Over the past decades, the subject of fractional calculus (as a generalization of the classical calculus) has been widely studied [1–3]. In fact, fractional derivative and integral opera- tors, due to higher degree of freedom in comparison to the classical operators as well as their memory and nonlocal properties, have received many applications in various prob- lems [4]. For instance, some important works related to recent developments in fractional calculus and its applications can be found in [5–10]. The reader should note that the most important issue about problems involving such operators is ﬁnding their exact solutions, which is often very diﬃcult and may even be impossible. This fact has led to the use of numerical methods as a convenient alternative to solve this drawback. Some numerical methods that have recently been applied to solve such problems can be found in [11–18]. Given that the order of fractional operators is permissible to take any value, a more general generalization is that the order of fractional operators be a deﬁnite function of the variables in the problem [19]. In fact, fractional operators of variable order (VO) can be utilized for more accurate modeling of real-world phenomena [20, 21]. The remark- able point about such operators is that their memory property is more evident [22]. Some Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Page 2 of 12 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 ( 2021) 2021:272 problems that have recently been modeled by such operators can be found in [23, 24]. However, similar to constant-order fractional equations, the major challenge in dealing with VO fractional equations is ﬁnding their analytical solutions, which is often impossi- ble. For this reason, in recent years, many numerical approaches have been constructed to solve this category of problems. For instances, see [25–29]. The Sobolev equation is a well-studied partial diﬀerential equation which has been fre- quently utilized in the ﬂuid dynamics to express the ﬂuid motion through rock or soil, and other media [30]. This equation is a special form of the Benjamin–Bona–Mahony– Burgers problem, where the coeﬃcients of nonlinear term and both ﬁrst-order derivatives are zero [31]. Many applications of the Sobolev equation have been reported in moisture migration in soil [32], thermodynamics [33], and ﬂuid motion [33]. There are many ap- proaches that have been applied to solve various types of the Sobolev equation in recent years. 1 Introduction For instances, see [30, 31, 34–37]. Recently, the author of [38] introduced a new nonsingular VO fractional derivative, where the Mittag-Leﬄer function is its kernel. As far as we know, there is no previous VO fractional version of the Sobolev problem. This motivates us to pursue the following goals: • Deﬁning a VO fractional prescription of the Sobolev equation using the nonsingular • Deﬁning a VO fractional prescription of the Sobolev equation using the nonsingular fractional derivative expressed in [38]. fractional derivative expressed in [38]. • Constructing a highly accurate method based upon the orthonormal shifted discrete • Constructing a highly accurate method based upon the orthonormal shifted discrete Legendre polynomials (DLPs) for this equation g g y p Legendre polynomials (DLPs) for this equation. So, we concentrate on the problem Legendre polynomials (DLPs) for this equation. Legendre polynomials (DLPs) for this equation. So, we concentrate on the problem HH 0 ∂ζ(τ) τ θ(y,τ) – μHH 0 ∂ζ(τ) τ θyy(y,τ) – νθyy(y,τ) = ϕ(y,τ), ζ(τ) ∈(0,1),(y,τ) ∈[0,yb] × [0,τb], (1.1) (1.1) under the initial and boundary conditions under the initial and boundary conditions θ(y,0) = ˆθ(y), θ(0,τ) = ˜θ0(τ), θ(yb,τ) = ˜θ1(τ), (1.2) (1.2) where θ(·,·) is the undetermined solution, μ and ν are positive constants, ζ(·) is a con- tinuous function in its domain, and ϕ(·,·), ˆθ(·), ˜θ0(·), and ˜θ1(·) are given functions. Also, HH 0 ∂ζ(τ) τ θ(y,τ) is the VO fractional derivative of order ζ(τ) with respect to τ in the Heydari– Hosseininia (HH) sense of the functions θ(y,τ) [38]. This equation can have useful appli- cations in many applied problems, such as the transport phenomena of humidity in soil, the heat conduction phenomena in diﬀerent media, and the porous theories concerned with percolation into rocks with cracks. Note that in the case of ζ(τ) = 1, this problem reduces to the classical Sobolev problem. One good idea for solving fractional functional equations is employing polynomials as basis functions to construct numerical methods. This is important for two reasons: First, the computation of the fractional derivative and integral of these functions is easy; and second, if the solution of the problem under study is suﬃciently smooth, high-precision solutions can be achieved. Basis orthogonal polynomials are classiﬁed into discrete and continuous kinds regarding the method of calculating their expansion coeﬃcients [39]. 2 Preliminaries Here, we review the deﬁnition of the VO fractional diﬀerentiation used in this study. First of all, we express the deﬁnition of the Mittag-Leﬄer function that is given in [4] by Ea,b(τ) = ∞ j=0 τ j (ja + b), a,b ∈R+,τ ∈C. (2.1) (2.1) Please remember that for b = 1 it is considered as Ea(τ) = Ea,1(τ). The VO fractional derivative of order ζ(τ) ∈(0,1) (where ζ(τ) is a continuous function on its domain) in the HH sense of the function θ(τ) is given in [38] as follows: HH 0 Dζ(τ) τ θ(τ) = 1 1 – ζ(τ) τ 0 E1 –ζ(τ)(τ – s) 1 – ζ(τ) θ′(s)ds, τ > 0. (2.2) (2.2) The above deﬁnition results in The above deﬁnition results in he above deﬁnition results in HH 0 Dζ(τ) τ τ r = ⎧ ⎪⎨ ⎪⎩ 0, r = 0, r! 1 – ζ(τ)trE1,r+1(– τζ(τ) 1 – ζ(τ)), r ≥1, (2.3) (2.3) where r ∈Z+ {0}. where r ∈Z+ {0}. where r ∈Z+ {0}. 1 Introduction Unlike continuous polynomials, the expansion coeﬃcients of which are calculated by in- tegrating (in most cases numerically), the expansion coeﬃcients of discrete polynomials Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations Page 3 of 12 ( 2021) 2021:272 ( 2021) 2021:272 are calculated accurately using a ﬁnite summation. In recent years, discrete polynomials have been extensively applied for solving diverse problems. For instances, see [39–47]. This study applies the orthonormal shifted DLPs for solving the Sobolev equation (1.1) subject to conditions (1.2). To this end, a new fractional matrix related to the VO fractional diﬀerentiation of these polynomials is obtained and applied for generating a numerical technique for this problem. The intended approach is constructed using these polynomi- als expansion and the tau technique. This technique converts the VO fractional problem into an algebraic system of equations that readily can be handled. Note that since it is eas- ier to obtain the operation matrix of VO fractional derivative of the orthonormal shifted DLPs than continuous polynomials, we have considered these discrete polynomials as ba- sis functions for solving this VO fractional problem. Organization of this article is as follows: The VO fractional derivative in the HH sense is reviewed in Sect. 2. The orthonormal shifted DLPs are reviewed in Sect. 3. Some matrix equalities are obtained in Sect. 4. The computational approach is explicated in Sect. 5. Numerical examples are given in Sect. 6. Conclusion of this study is provided in Sect. 7. 3 Orthonormal shifted discrete Legendre polynomials (3.8) 3 Orthonormal shifted discrete Legendre polynomials The orthonormal shifted DLPs are deﬁned over [0,τb] as follows [44]: Lτb,i(τ;N) = 1 √σ(i,N) i k=0 k m=0 (–1)k i k i + k k N τb m S(m) k N(k) τ m, i = 0,1,...,N, (3.1) where σ(i,N) = (N + i + 1)(i+1) (2i + 1)N(i) , σ(i,N) = (N + i + 1)(i+1) (2i + 1)N(i) , (3.2) (3.2) Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 ( 2021) 2021:272 Page 4 of 12 Heydari and Atangana Advances in Diﬀerence Equations S(m) k s are the ﬁrst type Stirling numbers, S(m) k s are the ﬁrst type Stirling numbers, S(m) k s are the ﬁrst type Stirling numbers, N(k) = ⎧ ⎨ ⎩ 1, k = 0, N(N – 1)(N – 2)...(N – k + 1), k ≥1, (3.3) (3.3) and i k is the binomial coeﬃcient. These polynomials can be utilized for approximating any continuous function θ(τ) over [0,τb] as follows: θ(τ) ≃ N i=0 eiLτb,i(τ;N) ≜ET τb,N(τ), (3.4) (3.4) where E = [e0 e1 ... eN]T, E = [e0 e1 ... eN]T, in which ei = N r=0 θ τb N r Lτb,i τb N r;N , ei = N r=0 θ τb N r Lτb,i τb N r;N , (3.5) (3.5) and τb,N(τ) = Lτb,0(τ;N) Lτb,1(τ;N) ... Lτb,N(τ;N) T. (3.6) (3.6) Likely, a continuous function θ(y,τ) deﬁned over [0,yb] × [0,τb] can be approximated by the orthonormal shifted DLPs as Likely, a continuous function θ(y,τ) deﬁned over [0,yb] × [0,τb] can be approximated by the orthonormal shifted DLPs as θ(y,τ) ≃ M i=0 N j=0 θijLyb,i(y;M)Lτb,j(τ;N) ≜ yb,M(y)T τb,N(τ), (3.7) (3.7) in which = [θ(i–1)(j–1)] is a matrix with (M + 1) × (N + 1) entries as in which = [θ(i–1)(j–1)] is a matrix with (M + 1) × (N + 1) entries as θ(i–1)(j–1) = M r=0 N l=0 θ yb Mr, τb N l Lyb,i yb Mr;M Lτb,j τb N l;N , θ(i–1)(j–1) = M r=0 N l=0 θ yb Mr, τb N l Lyb,i yb Mr;M Lτb,j τb N l;N , 1 ≤i ≤M + 1,1 ≤j ≤N + 1. (3.8) 1 ≤i ≤M + 1,1 ≤j ≤N + 1. 4 Matrix relationships Here and in what follows, we give some matrix relationships related to the orthonormal shifted DLPs. Theorem 4.1 ([44]) Diﬀerentiation of the vector τb,N(τ) introduced in (3.6) satisﬁes the relation d τb,N(τ) dτ = D(1,τb) N τb,N(τ), τb,N(τ) dτ = D(1,τb) N τb,N(τ), (4.1) (4.1) uations ( 2021) 2021:272 Page 5 of 12 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Page 5 of 12 ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations where D(1,τb) N = [d(1,τb) ij ] is a matrix of order (N + 1) with entries where D(1,τb) N = [d(1,τb) ij ] is a matrix of order (N + 1) with entries d(1,τb) ij = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎩ 1 √σ(i – 1,N) N l=0 i–1 k=1 k r=1 (–1)k i – 1 k i + k – 1 k × N τb S(r) k N(k) rlr–1Lτb,j–1 τb N l;N , 2 ≤i ≤N + 1, 1 ≤j ≤i – 1, 0, otherwise. Moreover, for any integer n, we have Moreover, for any integer n, we have dn τb,N(τ) dτ n = D(1,τb) N × D(1,τb) N × ··· × D(1,τb) N n times τb,N(τ) ≜D(n,τb) N τb,N(τ). (4.2) (4.2) Theorem 4.2 Suppose that ζ : [0,τb] −→(0,1) is a given continuous function and τb,N(τ) is the vector expressed in (3.7). Then we have HH 0 Dζ(τ) τ τb,N(τ) ≃Q(ζ,τb) N τb,N(τ), (4.3) HH 0 Dζ(τ) τ τb,N(τ) ≃Q(ζ,τb) N τb,N(τ), (4.3) where Q(ζ,τb) N = [q(ζ,τb) ij ] is a matrix of order (N + 1) with entries where Q(ζ,τb) N = [q(ζ,τb) ij ] is a matrix of order (N + 1) with entries q(ζ,τb) ij = ⎧ ⎨ ⎩ a(ζ,τb) ij , 2 ≤i ≤N + 1,1 ≤j ≤N + 1, 0, otherwise, q(ζ,τb) ij = ⎧ ⎨a(ζ,τb) ij , 2 ≤i ≤N + 1,1 ≤j ≤N + 1, otherwise, in which in which a(ζ,τb) ij = 1 √σ(i – 1,N) N l=0 i–1 k=1 k m=1 (–1)k i – 1 k i + k – 1 k × S(m) k N(k) m!lm 1 – ζ( τb N l)E1,m+1 –( τb N l)ζ( τb N l) 1 – ζ( τb N l) Lτb,j–1 τb N l;N . For example, whenever ζ(τ) = 0.5 + 0.25sin(τ), we obtain Q(ζ,2) 5 = ⎡ ⎢⎢⎢⎢⎢⎢⎢⎣ 0.0 0.0 0.0 0.0 0.0 0.0 –1.45297090 0.61950634 0.34505555 0.10604946 –0.02074431 –0.01425841 –0.07721547 –1.86890658 1.68369319 0.62293619 0.00763093 –0.05902572 –0.63012499 0.20031328 –1.56749609 2.41761593 0.51704726 –0.09569810 0.16799373 –0.53602371 0.52366685 –1.58744491 2.84614045 0.31438495 –0.10105451 0.93727364 –1.13652072 0.69242364 –1.92755571 3.29194139 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎦ . The above result can be approximated as The above result can be approximated as The above result can be approximated as HH 0 Dζ(τ) τ Lτb,ˆi(τ;N) ≃ N ˆj=0 ˆq(ζ,τb) ˆiˆj Lτb,ˆj(τ;N), (4.4) HH 0 Dζ(τ) τ Lτb,ˆi(τ;N) ≃ N ˆj=0 ˆq(ζ,τb) ˆiˆj Lτb,ˆj(τ;N), (4.4) where, regarding (3.5), we have where, regarding (3.5), we have ˆq(ζ,τb) ˆiˆj = N l=0 HH 0 Dζ(τ) τ Lτb,ˆi(τ;N) Lτb,ˆj(τ;N)\|τ= τb N l = 1 σ(ˆi,N) N l=0 ˆi k=1 k m=1 (–1)k ˆi k ˆi + k k S(m) k N(k) lmm! 1 – ζ( τb N l) × E1,m+1 –( τb N l)ζ( τb N l) 1 – ζ( τb N l) Lτb,ˆj τb N l;N . Eventually, via the change of indices ˆi = i – 1 and ˆj = j – 1, and considering q(ζ,τb) ij instead of ˆq(ζ,τb) i–1j–1, we obtain q(ζ,τb) ij = 1 √σ(i – 1,N) N l=0 i–1 k=1 k m=1 (–1)k i – 1 k i + k – 1 k S(m) k N(k) m!lm 1 – ζ( τb N l) × E1,m+1 –( τb N l)ζ( τb N l) 1 – ζ( τb N l) Lτb,j–1 τb N l;N for 2 ≤i ≤N + 1 and 1 ≤j ≤N + 1. Thus, the expressed claim is proved. □ for 2 ≤i ≤N + 1 and 1 ≤j ≤N + 1. Thus, the expressed claim is proved. □ □ For example, whenever ζ(τ) = 0.5 + 0.25sin(τ), we obtain 4 Matrix relationships Proof Regarding (2.3), we have HH 0 Dζ(τ) τ Lτb,0(τ;N) = 0. So, in the matrix Q(ζ,τb) N , the ﬁrst row should be zero. Assume ˆi ≥1 and ζ : [0,τb] −→(0,1) be a continuous function. From (2.3) and (3.1), we get HH 0 Dζ(τ) τ Lτb,ˆi(τ;N) = 1 σ(ˆi,N) ˆi k=0 k m=0 (–1)k ˆi k ˆi + k k N τb m S(m) k N(k) HH 0 Dζ(τ) τ τ m HH 0 Dζ(τ) τ Lτb,ˆi(τ;N) = 1 σ(ˆi,N) ˆi k=0 k m=0 (–1)k ˆi k ˆi + k k N τb m S(m) k N(k) HH 0 Dζ(τ) τ τ m = 1 σ(ˆi,N) ˆi k=1 k m=1 (–1)k ˆi k ˆi + k k N τb m S(m) k N(k) m! 1 – ζ(τ) × τ mE1,m+1 – τζ(τ) 1 – ζ(τ) . Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Page 6 of 12 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 5 Computational method In order to use the orthonormal shifted DLPs for problem (1.1) with initial and boundary conditions (1.2), we express the unknown solution as In order to use the orthonormal shifted DLPs for problem (1.1) with initial and boundary conditions (1.2), we express the unknown solution as θ(y,τ) ≃ yb,M(y)T τb,N(τ), (5.1) (5.1) where is an (M + 1) × (N + 1) matrix, and its elements are undetermined. Theorem 4.1 results in where is an (M + 1) × (N + 1) matrix, and its elements are undetermined. Theorem 4.1 results in θyy(y,τ) ≃ yb,M(y)T D(2,yb) M T τb,N(τ). (5.2) Besides, Theorem 4.2 together with the above relations yields Besides, Theorem 4.2 together with the above relations yields Besides, Theorem 4.2 together with the above relations yields ides, Theorem 4.2 together with the above relations yields HH 0 ∂ζ(τ) τ θ(y,τ) ≃ yb,M(y)TQ(ζ,τb) N τb,N(τ) (5.3) HH 0 ∂ζ(τ) τ θ(y,τ) ≃ yb,M(y)TQ(ζ,τb) N τb,N(τ) (5.3) ence Equations ( 2021) 2021:272 Page 7 of 12 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Page 7 of 12 and HH∂ζ(τ)θ (y τ) ≃ M(y)T D(2,yb)TQ(ζ,τb) N(τ) (5 4) and and and HH 0 ∂ζ(τ) τ θyy(y,τ) ≃ yb,M(y)T D(2,yb) M TQ(ζ,τb) N τb,N(τ). (5.4) (5.4) In addition, we represent ϕ(y,τ) using the orthonormal shifted DLPs as follows: ϕ(y,τ) ≃ yb,M(y)TΦ τb,N(τ), (5.5) (5.5) where Φ is an (M + 1) × (N + 1) given matrix, and its elements are evaluated like in (3.8). By inserting (5.2)–(5.5) into (1.1), we obtain where Φ is an (M + 1) × (N + 1) given matrix, and its elements are evaluated like in (3.8). By inserting (5.2)–(5.5) into (1.1), we obtain yb,M(y)T Q(ζ,τb) N – μ D(2,yb) M TQ(ζ,τb) N – ν D(2,yb) M T – Φ τb,N(τ) ≃0. 5 Computational method (5.6) (5.6) The functions given in (1.2) can also be approximated via the orthonormal shifted DLPs as The functions given in (1.2) can also be approximated via the orthonormal shifted DLPs as The functions given in (1.2) can also be approximated via the orthonormal shifted DLPs as as ˆθ(y) ≃ yb,M(y)T ˆ, (5.7) nd ˆθ(y) ≃ yb,M(y)T ˆ, (5.7) ˆθ(y) ≃ yb,M(y)T ˆ, (5.7) and ˜θ0(τ) ≃˜ T 0 τb,N(τ), ˜θ1(τ) ≃˜ T 1 τb,N(τ), (5.8) (5.8) in which ˆ is an (M + 1)-order column vector, ˜0 and ˜1 are (N + 1)-order column vec- tors, and their elements are evaluated like in (3.5). Now, from (1.2), (5.1), (5.7), and (5.8), we obtain yb,M(y)T τb,N(0) – ˆ 1 ≃0 (5.9) (5.9) 1 and yb,M(0)T – ˜ T 0 2 τb,N(τ) ≃0, yb,M(yb)T – ˜ T 1 3 τb,N(τ) ≃0. (5.10) (5.10) Utilizing (5.6), (5.9), and (5.10), we generate the following system: ⎧ ⎪⎪⎨ ⎪⎪⎩ []ij = 0, i = 1,2,...,M – 1,j = 2,3,...,N + 1, (1)i = 0, i = 1,2,...,M + 1, (2)j = 0, (3)j = 0, j = 2,3,...,N + 1. (5.11) (5.11) Finally, by solving (5.11) and ﬁnding the elements of the matrix , we ﬁnd a numerical solution for the primary VO fractional problem by inserting into (5.1). Finally, by solving (5.11) and ﬁnding the elements of the matrix , we ﬁnd a numerical solution for the primary VO fractional problem by inserting into (5.1). Page 8 of 12 ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Heydari and Atangana Advances in Diﬀerence Equations 6 Numerical examples The approach generated using the orthonormal shifted DLPs is applied in this section for solving some numerical examples. The L2-error of the numerical results is measured as eθ = τb 0 yb 0 θ(y,τ) – ˜θ(y,τ) 2 dydτ 1/2 , eθ = τb 0 yb 0 θ(y,τ) – ˜θ(y,τ) 2 dydτ 1/2 , where θ and ˜θ are the analytic and numerical solutions, respectively. The convergence order (CO) of this approach is computed as follows: where θ and ˜θ are the analytic and numerical solutions, respectively. The convergence order (CO) of this approach is computed as follows: CO = log ¯N1 ¯N2 ε2 ε1 , CO = log ¯N1 ¯N2 ε2 ε1 , where εi and ε2 are the ﬁrst and second L2-error values, respectively. Furthermore, ¯Ni = (Mi + 1) × (Ni + 1) for i = 1,2 is the number of the orthonormal shifted DLPs utilized in the ith implementation. In addition, we have applied Maple 18 (with 15 digits precision) for obtaining the results. Meanwhile, the series generating the Mittag-Leﬄer function is applied for 25 terms. Example 1 Consider problem (1.1) on [0,3] × [0,1] with μ = ν = 1 and Example 1 Consider problem (1.1) on [0,3] × [0,1] with μ = ν = 1 and ϕ(y,τ) = sin(τ)sinh(y – 3). This example has the analytic solution This example has the analytic solution θ(y,τ) = sin(τ)sinh(3 – y). So, we have θ(y,0) = 0, θ(0,τ) = sin(τ)sinh(3), θ(3,τ) = 0. We have applied the expressed method for this example with three choices of ζ(τ). The ex- tracted results are listed in Table 1. This table shows the high-precision of the proposed ap- proach in solving this example. It also conﬁrms that the results have a high degree of con- vergence. The last column of this table conﬁrms the low computational works of the pre- sented algorithm. Graphical behaviors of the extracted results for ζ(τ) = 0.50 + 0.25sin(τ) where (M = 9,N = 8) are illustrated in Fig. 1. This ﬁgure shows the high accuracy of the presented method for obtaining a smooth solution for this example. This example has the analytic solution Table 1 Results extracted via the presented approach for Example 1 with three choices of ζ(τ) M N ζ(τ) = 0.50 + 0.25sin(τ) ζ(τ) = 0.85 – 0.25e–τ ζ(τ) = 0.65 + 0.25τ 3 cos(τ) CPU time eθ CO eθ CO eθ CO 5 4 4.8716E-03 – 4.8703E-03 – 4.8704E-03 – 02.54 6 5 4.3022E-04 07.2127 4.3018E-04 07.2122 4.3018E-04 07.2122 05.29 7 6 3.2230E-05 09.0078 3.2171E-05 09.0139 3.2179E-05 09.0130 12.71 8 7 2.6413E-06 09.9541 2.6412E-06 09.9470 2.6379E-06 09.9529 31.04 9 8 1.9235E-07 11.7400 2.1424E-07 11.2568 2.1424E-07 11.2512 64.64 Table 1 Results extracted via the presented approach for Example 1 with three choices of ζ(τ) Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 ( 2021) 2021:272 Page 9 of 12 Heydari and Atangana Advances in Diﬀerence Equations Figure 1 Achieved results for θ(y,t) whenever ζ(τ) = 0.50 + 0.25sin(τ) with (M = 9,N = 8) in Example 1 Figure 1 Achieved results for θ(y,t) whenever ζ(τ) = 0.50 + 0.25sin(τ) with (M = 9,N = 8) in Example 1 Table 2 Results extracted via the presented approach for Example 2 with three choices of ζ(τ) M N ζ(τ) = 0.50 + 0.25sin(τ) ζ(τ) = 0.85 – 0.25e–τ ζ(τ) = 0.65 + 0.25τ 3 cos(τ) CPU time eθ CO eθ CO eθ CO 4 4 1.6710E-03 – 1.6731E-03 – 1.6713E-03 – 05.70 5 5 1.6538E-04 06.3430 1.6555E-04 06.3436 1.6540E-04 06.3431 12.96 6 6 6.7121E-06 10.3935 6.7288E-06 10.3887 6.7221E-06 10.3890 23.78 7 7 6.8627E-07 08.5388 6.8598E-07 08.5496 6.8298E-07 08.5623 51.53 8 8 1.5974E-08 15.9628 1.5855E-08 15.9928 1.5414E-08 16.0939 92.15 Figure 2 Achieved results for θ(y,t) whenever ζ(τ) = 0.65 + 0.25τ 3 cos(τ) with (M = N = 8) in Example 1 Table 2 Results extracted via the presented approach for Example 2 with three choices of ζ(τ) M N ζ(τ) = 0.50 + 0.25sin(τ) ζ(τ) = 0.85 – 0.25e–τ ζ(τ) = 0.65 + 0.25τ 3 cos(τ) CPU time eθ CO eθ CO eθ CO 4 4 1.6710E-03 – 1.6731E-03 – 1.6713E-03 – 05.70 5 5 1.6538E-04 06.3430 1.6555E-04 06.3436 1.6540E-04 06.3431 12.96 6 6 6.7121E-06 10.3935 6.7288E-06 10.3887 6.7221E-06 10.3890 23.78 7 7 6.8627E-07 08.5388 6.8598E-07 08.5496 6.8298E-07 08.5623 51.53 8 8 1.5974E-08 15.9628 1.5855E-08 15.9928 1.5414E-08 16.0939 92.15 Figure 2 Achieved results for θ(y,t) whenever ζ(τ) = 0.65 + 0.25τ 3 cos(τ) with (M = N = 8) in Example 1 Table 2 Results extracted via the presented approach for Example 2 with three choices of ζ(τ) Figure 2 Achieved results for θ(y,t) whenever ζ(τ) = 0.65 + 0.25τ 3 cos(τ) with (M = N = 8) in Example 1 Example 2 Consider problem (1.1) on [0,1] × [0,2] with μ = 1 2, ν = 1 and Example 2 Consider problem (1.1) on [0,1] × [0,2] with μ = 1 2, ν = 1 and ϕ(y,τ) = 4e–τ – 1 1 – ζ(τ) ∞ l=0 (–τ)l+1E1,l+2 – τζ(τ) 1 – ζ(τ) sin(2y). Author details 1Department of Mathematics, Shiraz University of Technology, Shiraz, Iran. 2Faculty of Natural and Agricultural Sciences, University of the Free State, Bloemfontein, South Africa. 3Department of Medical Research China Medical University Hospital, China Medical University, Taichung, Taiwan. Received: 21 April 2021 Accepted: 21 May 2021 eived: 21 April 2021 Accepted: 21 May 2021 This example has the analytic solution This example has the analytic solution This example has the analytic solution This example has the analytic solution θ(y,τ) = e–τ sin(2y). Thus, we have Thus, we have θ(y,0) = sin(2y), θ(0,τ) = 0, θ(1,τ) = e–τ sin(2). The technique established upon the orthonormal shifted DLPs is implemented for this ex- ample. The gained results are provided in Table 2, and they conﬁrm the high-precision and Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 Page 10 of 12 Heydari and Atangana Advances in Diﬀerence Equations ( 2021) 2021:272 ( 2021) 2021:272 low computations of the approach. It can also be seen that as the number of the orthonor- mal shifted DLPs increases, the accuracy of the results increases rapidly. The obtained results with (M = N = 8) whenever ζ(τ) = 0.65 + 0.25τ 3 cos(τ) are shown in Fig. 2. This ﬁgure illustrates that the proposed method can provide a highly accurate solution for this example across the domain. low computations of the approach. It can also be seen that as the number of the orthonor- mal shifted DLPs increases, the accuracy of the results increases rapidly. The obtained results with (M = N = 8) whenever ζ(τ) = 0.65 + 0.25τ 3 cos(τ) are shown in Fig. 2. This ﬁgure illustrates that the proposed method can provide a highly accurate solution for this example across the domain. Authors’ contributions All authors contributed equally to the writing of this paper. All authors read and approved the ﬁnal manuscript. Funding Funding The ﬁnancial assistance is not applicable. Funding The ﬁnancial assistance is not applicable. Availability of data and materials Data sharing is not applicable to this study. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and in Received: 21 April 2021 Accepted: 21 May 2021 References 1. 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Li, M.: Multi-fractional generalized Cauchy process and its application to teletraﬃc. Physica A (2020). https://doi.org/10.1016/j.physa.2019.123982 7 Conclusion In this study, the Heydari–Hosseininia fractional diﬀerentiation as a kind of nonsingu- lar variable-order (VO) fractional derivative was utilized for generating a VO fractional version of the Sobolev equation. The orthonormal shifted discrete Legendre polynomials (DLPs) as a convenient family of basis functions were employed to generate a numerical algorithm for this equation. A new fractional operational matrix related to VO fractional diﬀerentiation of these polynomials was obtained. The established scheme converts solv- ing the problem under consideration into solving an algebraic system of equations. The validity of this technique was investigated by solving two numerical examples. The ob- tained results conﬁrmed that the established method is able to generate numerical so- lutions with high accuracy for such problems even by applying a small number of the orthonormal shifted DLPs. As future research direction, the VO fractional derivative ap- plied in this study can be utilized for generating VO fractional version of other applicable problems, such as Schrödinger equation and advection-diﬀusion equation. Acknowledgements The authors would like to thank the anonymous reviewers for their valuable comments and suggestions on this paper. Funding The ﬁnancial assistance is not applicable. Acknowledgements Th h ld l k Acknowledgements The authors would like to thank the anonymous reviewers for their valuable comments and suggestions on this paper. Funding References 2017, 295 (2017) 9. Veeresha, P., Prakasha, D.G., Singh, J., Kumar, D., Baleanu, D.: Fractional Klein-Gordon-Schrödin Mittag-Leﬄer memory. Chin. J. Phys. 68, 65–78 (2020) 9. 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https://openalex.org/W1991718146	https://europepmc.org/articles/pmc3543441?pdf=render	English	null	Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution	PloS one	2,013	cc-by	10,766	Abstract Background: Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. a-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO. Methodology/Principal Finding: The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCD15). Structure was determined by molecular replacement method and refined to 2.8 A˚ resolution. The orthorhombic crystal exhibits P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 A˚. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure. Conclusions/Significance: We determined the tetrameric crystal structure of EhODCD15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI. Citation: Preeti, Tapas S, Kumar P, Madhubala R, Tomar S (2013) Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution. PLoS ONE 8(1): e53397. doi:10.1371/journal.pone.0053397 Editor: Dan Zilberstein, Technion-Israel Institute of Technology, Israel Editor: Dan Zilberstein, Technion-Israel Institute of Technology, Israel Received October 10, 2012; Accepted November 28, 2012; Published January 11, 2013 Received October 10, 2012; Accepted November 28, 2012; Published January 11, 2013 Copyright: 2013 Preeti 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: Preeti thanks CSIR (Council of Scientific & Industrial Research), S. Abstract Tapas thanks DRDO (Defence Research and Development Organisation, Government of India), PK thanks DST (Department of Science and Technology, Government of India), and S. Tomar thanks ICMR (Indian Council of Medical Research) for financial support. 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. * E-mail: shailfbt@iitr.ernet.in Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution Preeti1, Satya Tapas1, Pravindra Kumar1, Rentala Madhubala2, Shailly Tomar1* 1 Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India, 2 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Preeti1, Satya Tapas1, Pravindra Kumar1, Rentala Madhubala2, Shailly Tomar1* Cloning of C-terminal truncated EhODC Polymerase Chain Reaction (PCR) amplification was carried out using forward primer 59-ATATCCATATGAAACAAA- CATCTCTAGAAG-39 and reverse primer 59- GAACCTC- GAGTCATTCAATTGACTTAGGGATTTGAAT-39 with NdeI and XhoI restriction enzyme sites respectively to obtain DNA fragment encoding the C-terminal 15 residues truncated EhODC (EhODCD15). The previously cloned full-length EhODC was used as a template in the PCR reaction [29,36]. PCR was performed in a 50 ml reaction mixture containing 10 ml of 56HF phusion buffer supplied with the enzyme, 300 mM of dNTP mix, 6.25 pmol of each of forward and reverse primers, 10 ng of template DNA, 1 ml of 2.5 U/ml phusion polymerase and water. The reaction was performed with initial denaturation at 95uC for 30 s, followed by 30 PCR cycles of denaturation at 95uC for 30 s, annealing at 51uC for 60 s and extension at 72uC for 1 min and 15 s. A final extension was carried out at 72uC for 15 min. The resultant PCR product was subcloned into NdeI and XhoI sites of pET-28c with His6-tag preceding the N-terminal and tobacco etch virus (TEV) protease cleavage site to allow the removal of tag from recombinant protein. Ligated product was transformed into freshly prepared E. coli DH5a competent cells. Kanamycin resistant transformants were selected and grown in LB broth supplemented with 50 mg/ml kanamycin. The pET28- EhODCD15 plasmid was isolated and right size insert in the construct was confirmed by DNA sequencing from TCGA, New Delhi. Furthermore, in mammals, the activity of antizyme is negatively regulated by a protein called antizyme inhibitor (AZI). AZI binds to antizyme and blocks the binding of antizyme to ODC which down regulates ODC degradation as well as leads to ODC activation. AZI has higher binding affinity for antizyme as compared to ODC which results in antizyme sequestration and elevation of ODC levels [18,19,20,21,22,23]. Previously, it has been reported that AZI is homologous to ODC and the major residues involved in catalytic activity of ODC are conserved in AZI [24]. However, AZI does not possess enzymatic activity due to changes in the sequence that lead to protein inability to bind cofactor PLP along with the failure in decarboxylation activity [24,25,26]. In E. histolytica, ODC is the only enzyme of polyamine biosynthetic pathway that has been reported to exist in the organism [27]. The analysis of polyamine content shows that considerable amount of putrescine is present in E. histolytica. Introduction which are involved in various cellular processes that govern cell growth and proliferation [9]. Subsequently, the actively prolifer- ating cells have higher concentrations of polyamines. The intracellular concentrations of polyamines are tightly regulated by different mechanisms including biosynthesis, inter-conversion, degradation, and uptake from the surrounding through polyamine transporter. The failure in regulation of polyamine levels in cells has been linked to various cancers. Hence, polyamine metabolic pathway is also a potential target for cancer treatment [10,11,12]. Consequently, not only the polyamine biosynthetic pathway but also the key components of polyamine homeostasis are potential therapeutic targets [8]. The two enzymes of polyamine biosyn- thesis pathway, ornithine decarboxylase (ODC) and S-adenosyl- methionine decarboxylase (SAMDC) are highly-regulated and have a very short half-life by which cells quickly alter the levels of polyamines [13]. Entamoeba histolytica is responsible for causing amoebiasis, amoebic liver abscess and amoebic colitis in humans. It is the third major and a dangerous public health problem in the world [1,2]. Though a small number of drugs including metronidazole, emetine, tinidazole, chloroquine and nitrazoxanide are used for the treatment of the disease, most of them are associated with numerous side effects. In some cases, frequent use of these drugs has led to the development of clinical drug resistance in the pathogen [3,4]. Thus, it is crucial to identify and elucidate a potent metabolic pathway in E. histolytica which could be set as a therapeutic target for development of new anti-amoebic drugs. In last few decades, the polyamine metabolic pathway in protozoan diseases including African sleeping sickness [5], giardiasis [6] and leishmaniasis [7] has emerged as a potential therapeutic target [8]. The polyamines such as putrescine, spermidine and spermine are essential polycationic compounds, Ornithine decarboxylase catalyzes the first and rate-limiting step of polyamine biosynthetic pathway. L-ornithine is decarbox- January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 1 January 2013 \| Volume 8 \| Issue 1 \| e53397 Crystal Structure of ODC from E. histolytica ylated by ODC enzyme in the presence of cofactor pyridoxal-59- phosphate (PLP) to produce putrescine. The enzymatic activity of ODC is tightly regulated by a distinct mechanism in which polyamines induce the expression of a regulatory protein called antizyme (AZ) by +1 ribosomal frameshifting [14]. AZ inhibits ODC enzyme activity by binding and disrupting active ODC homodimers, and subsequently marks the enzyme for ubiquitin- independent degradation by the 26S proteasome [15,16]. Expression and purification p p The pET28-EhODCD15 plasmid containing truncated EhODC gene was transformed into E. coli BL21 (DE3) competent cells. For protein expression, transformed BL21 (DE3) cells were grown at 37uC to an optical density of ,0.6 at 600 nm (OD600) and induced with 0.5 mM isopropyl-ß-thiogalactopyranoside (IPTG). Induced cultures were transferred to 18uC and cells were grown for ,14 h. Cells were harvested by centrifugation at 5,000 rpm at 4uC and cell pellets were stored at 220uC until further use. For protein purification, cell pellets from 1 litre culture were re- suspended in 20 ml of ice cold binding buffer containing 50 mM Tris HCl (pH 7.5), 40 mM imidazole, 250 mM sodium chloride, 2 mM phenylmethylsuphonyl fluoride (PMSF) and 5% glycerol (v/v). Lysozyme was added to a final concentration of 100 mg/ml and kept on rocking platform at 4uC for 45 min. Cells were disrupted by sonication on ice with 50% amplitude and a pulse of 20 sec on and 60 sec off for 15 min. The lysate was centrifuged at 18,000 rpm for 45 min at 4uC to separate supernatant from cell debris. The supernatant was loaded onto 5 ml HisTrap HP affinity column pre-equilibrated with the binding buffer. Protein was eluted by running a linear gradient of 40–1000 mM imidazole in 60 ml of buffer A [50 mM Tris HCl (pH 7.5), 1 M imidazole, 250 mM sodium chloride and 5% glycerol (v/v)] at a flow rate of 1 ml/min. Eluted fractions were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and frac- tions containing pure protein were pooled together. To remove the N-terminal His-tag, TEV protease was added to the sample with protein to TEV ratio 1:20 and incubated for ,12 h at 4uC and simultaneously dialyzed against buffer A without imidazole. To remove uncleaved His-tag protein and His-tag TEV protease, the sample was again loaded onto 5 ml HisTrap HP column. Flow- through containing EhODCD15 without His-tag was collected and In this study, we have determined the crystal structure of EhODC to elucidate the structural features responsible for DFMO insensitivity and low substrate binding affinity. Furthermore, detailed comparative sequence and structural analysis was performed with functional ODCs and non-functional ODC homologue i.e. AZI to investigate the evolutionary status of EhODC. Introduction Addi- tionally, AZ negatively regulates the uptake of polyamines by repressing polyamine transporter [17]. Thus, polyamine homeo- stasis is maintained in a cell through polyamines themselves via a negative feedback system, by governing the synthesis of AZ protein. screens were obtained from Hampton Research (Hampton Research Inc. Aliso Viejo, CA). The plasmid pET30a containing full length of EhODC was taken as template for sub-cloning [29]. Cloning of C-terminal truncated EhODC While, very low levels of spermidine and no spermine is detected supporting the absence of other genes of polyamine biosynthetic pathway in E. histolytica genome [28,29]. Interestingly, the comparison of EhODC kinetic parameters with other well characterized ODCs indicates that it has low substrate affinity and catalytic efficiency [29]. Moreover, DFMO, a suicide substrate inhibitor of ODC is used for the treatment of African sleeping sickness, a protozoan disease caused by Trypanosome brucei gambiense [30,31]. Interestingly, DFMO being an effective drug against T. brucei gambiense is reported to have relatively poor effect on the more virulent strain T. brucei rhodesiense [32,33]. Further- more, the ODC of E. histolytica, being a pathogenic strain from protozoa kingdom, is insensitive to DFMO due to sequence divergence in the substrate binding residues [29,34,35,36]. Natural resistance to DFMO within the same Trypanosome species as well as within the protozoa kingdom draws attention towards the sequence and structural divergence for their evolutionary adap- tation. Reagents Restriction enzymes NdeI, XhoI, T4-DNA ligase and phusion polymerase were purchased from NEB. Primers were ordered from Integrated DNA Technology. HisTrap HP Ni Sepharose column and Hiload 16/60 Superdex 200 pg size exclusion column were obtained from GE healthcare. For crystallization, PEG ION January 2013 \| Volume 8 \| Issue 1 \| e53397 January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 2 Crystal Structure of ODC from E. histolytica method using Molrep program of CCP4-6.0 suite [39]. The model was generated using previously reported crystal structure of human ODC (PDB ID: 2ON3) [40]. Non-crystallographic symmetry restraints were applied throughout the refinement stages using four EhODCD15 molecules in the asymmetric unit. Structure refinement was performed using CNS v.1.2, Phenix v 1.7.2-869, and REFMAC 5.2 refinement tools [41,42,43]. Rounds of model building were carried out using program Coot v 0.6.2 [44]. The quality of the model was evaluated by PROCHECK [45]. concentrated using a 10 kDa cut-off Amicon Ultra-15 concentra- tor (Millipore, Bedford, Massachusetts, USA). The concentrated protein was loaded onto HiLoad 16/60 prep grade Superdex 200 size-exclusion chromatography column pre-equilibrated with buffer B containing 30 mM HEPES-Na (pH 7.5), 250 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol and 1 mM DTT. The major peak fractions containing pure protein were pooled and concen- trated to 5 mg/ml. Homogeneity of purified EhODCD15 protein was analysed on 12% SDS-PAGE. Protein concentration and yield were determined using the Bio-Rad protein assay kit with bovine serum albumin (BSA) as a standard. Gel filtration analysis The average molecular weight of EhODCD15 was determined using size exclusion chromatography and compared with previ- ously characterized full-length EhODC [36]. In brief, the purified protein was concentrated to 5 mg/ml and was injected onto HiLoad 16/60 Superdex 200 gel filtration chromatography column using A¨ KTA purification system (GE Healthcare). Protein was allowed to pass through the column at a rate of 0.5 ml/min. For the molecular weight estimation of EhODCD15, the elution profile of the major peak containing purified protein was compared with the elution profile of the standard Gel Filtration HMW Calibration kit molecular weight markers (GE healthcare). Table 1. Statistical representation of data collection and structure refinement parameters along with quality of the model accessed by Ramachandran plot. Data collection Space group P212121 Unit cell parameters a (A˚), b (A˚), c (A˚) 76.66, 119.28, 179.28 Resolution (A˚) 99.5–2.87 (2.92–2.87)a Number of reflections 35570 Completeness (%) 92.1(59.0)a Mean redundancy 3.4 (2.1)a I/s 4.82 (2.0)a Rmerge b (%) 0.150 (0.670)a Refinement Resolution (A˚) 99.5–2.87 (2.92–2.87)a Number of non-H atoms in asymmetric unit Protein 10484 Water molecules 101 R-factor (%) 25.3 Rfree c value (%) 29.9 Average B-factor (A˚2) 54.4 Rms deviations bond lengths (A˚) 0.005 bond angles (u) 0.831 Ramachandran plot Residues in favored region (%) 88.7 Residues in allowed region (%) 10 Residues in generously allowed region (%) 0.9 Residues in outlier region (%) 0.4 avalue in parentheses are for the highest resolution shell. bRmerge = S \| I2I¯ \|/S I \| where I = observed intensity and I¯= average intensity. cRfree = S (\|F\|obs2\|F\|calc\|)/S \|F\|obs where \|F\|obs are observed structure factor amplitudes for a given reflection and \|F\|calc are calculated structure factor amplitude. doi:10.1371/journal.pone.0053397.t001 Model generation for active site analysis In the crystal structure of EhODCD15, the flexible loops missing in one subunit were present in the other subunits. Therefore, coordinates for missing loops near active site in the structure were EhODCD15 enzymatic activity The sequence of EhODC, along with other functional ODCs and AZI were retrieved from NCBI database [46]. Multiple sequence alignment and phylogenetic tree of these sequences were obtained using ClustalW [47] for evolutionary variation analysis. To confirm that the truncation of 15 residues from C-terminus does not inactivate EhODC, ornithine decarboxylation activity of purified protein and production of putrescine was spectrophoto- metrically determined using the method developed by Badolo et al [37]. Enzymatic activity of the purified EhODCD15 protein was compared with full-length EhODC [36]. Crystallization For crystallization, purified EhODCD15 protein was concen- trated to 12.5 mg/ml in 30 mM HEPES-NaOH buffer (pH 7.5) containing 1 mM EDTA, 0.25 M NaCl, 1 mM DTT and 10% (v/v) glycerol. Crystallization trials were performed using the sitting drop vapour diffusion method in 96 well plates (Hampton Research) at 20uC and 4uC. The drops were prepared by mixing 2 ml of protein solution with 1 ml of reservoir solution and equilibrated against 80 ml reservoir solution. Hampton Research crystallization screens Crystal screen, Crystal screen 2 and PEG/ ION screen (Hampton Research, USA) were used to explore the initial crystallization conditions. Crystals were obtained in PEG ION screen containing 20% PEG 3350 in 0.2 M LiCl solution maintained at pH 6.8. Diamond shaped crystals of EhODCD15 appeared in four months at 20uC. Prior to data collection, crystal was cryo-protected by bathing it in mother liquor containing 3% (v/v) ethylene glycol for 10 s. The crystal was flash-frozen under cryogenic conditions at 100 K using liquid nitrogen stream to prevent radiation damage during data collection. C-terminal Truncation and Purification of EhODC C-terminal Truncation and Purification of EhODC The ODC enzyme from E. histolytica belongs to fold type III group IV decarboxylase of a B6-dependent family, having eukaryotic ornithine decarboxylase characteristics [50,51]. Under this classification, crystal structures are only available from three different sources including human, mouse and Trypanosome brucei ODC [52,53,54]. For crystal structure determination of EhODC, full-length protein was purified using the previously established protocol [36] and was used for crystallization experiments. However, extensive crystallization trials of full-length EhODC were unsuccessful. In order to decrease the conformational heterogeneity, it is a common practice to truncate the flexible N and/or C-terminal residues to facilitate the crystallization process. Therefore, EhODC sequence was examined to identify disordered regions using bioinformatics tools DisEMBL and GlobPlot [55,56]. These programs predicted a fragment of approximately 13–17 residues at the C-terminus of EhODC to be flexible. Additionally, it has been reported that the truncation of 37 residues from the C-terminus of mouse ODC resulted in protein stability and has been crystallized successfully for structure determination [53,57,58]. The C-terminal sequence of EhODC shows similarity with mouse ODC in having a PEST like sequence [36]. Based on these observations, 15 residues were deleted from the C-terminus of EhODC. Expression and solubility of EhODCD15 construct was optimized by varying induction temperature (37uC, 25uC, and 18uC). Maximum solubility was observed at 18uC when induced with 0.5 mM IPTG for ,14 h. Recombinant EhODCD15 was purified in three sequential purification steps, with yield of ,5 mg per liter of E. coli culture. Elution profiles from the gel filtration column demonstrated that EhODCD15 exists in the dimeric form similar to full-length EhODC [36]. The purified protein exhibited a single band of approximately ,45 kDa in 12% SDS-PAGE gel (Figure 1). The enzymatic activities of EhODCD15 and wild-type proteins were compared using previously established protocol [36]. The comparative analysis of both the full-length and EhODCD15 forms didn’t show any notable difference in the activity indicating that the truncation of 15 residues from the C-terminus of EhODC does not affect its activity. In the tetrameric structure, residues Phe91 and Leu87 of chain A interact with Ser388 of chain C through a water molecule. In addition, Glu90 of chain A interacts to Ser388 of chain C through polar interaction. Similarly, Asp88 of chain A is forming direct interaction with residue Leu386 of chain C and vice versa. Overall structure and folding g Each monomer consists of b/a barrel and b-sheet domain which are arranged identical to previously known ODC structures (Figure 4). However, the tetramer arrangement displays a number of unusual features. Residues from barrel involved in contact and dimer formation are located at the surface or in proximity to sheet domain of opposite monomer. Interface residues of helices a5, a7, a8 and a9 of chain A barrel form extensive contacts with sheet domain S2 of chain B. All four chains in asymmetric unit showed similar structures and are involved in similar interactions. The analysis of dimer-dimer interactions exhibited large intermolecular distances of ,4.0 A˚ . In a monomer, helix a1 is connected to sheet b1 (Gly27-Phe31) through a loop and enters the barrel. The barrel is composed of eight helices i.e. a2 (T33-N46), a3 (P62-L71), a4 (L80-L89), a5 (Y105-L114), a6 (I124-Y133), a7 (D163-K175), a8 (E194-F213) and a9 (F232-L246) followed by eight alternate b- strands b2 (R51-A55), b3 (G74-C77), b4 (I96-Y98), b5 (H118- V121), b6 (G138-R142), b7 (V182-F184), b8 (L219-D221) and b9 (R253-A256). The sheet domain comprises of eight randomly arranged b-strands which can be further divided into S1 and S2 b- sheets that are perpendicular to each other. Sheet S1 consisted of four sheets b10 (F267-S271), b15 (L355-F357), b16 (I381-T383) in addition to b1, which are roughly perpendicular to S2 containing b11 (H274-Q281), b12 (K284-S291), b13 (Y325-Y330) and b14 (A341-L345) (Figure 4). However, both domains are connected by two loops in between b1-a2 and a10-b10. The barrel and b-sheet domains of the monomeric subunits are associated in head to tail manner in the dimer. In addition, various polar interactions at the dimer interface including salt bridges and hydrophobic interac- tions are involved in the formation of dimer. The structure of EhODC has several highly mobile loop moieties that are depicted by dashed lines in Figure 4. Crystal Structure of ODC from E. histolytica Crystal Structure of ODC from E. histolytica generated by MODELLER 9.10 [48] using the solved crystal structure of one subunit of EhODC as a template. Evaluation of the steriochemical properties of obtained structure having built-in loops was performed using PROCHECK [45]. All the figures of structure and active sites were generated using PyMol [49]. The four monomers in the asymmetric unit of crystal are arranged as two separate dimers (subunits A, B and subunits C, D) facing each other at the convex surfaces. Each monomer in a dimer makes side to side contacts with each other forming an overall bent structure. Further, as the loops in a dimer interface are disordered and clear density was not observed, the central part of dimer forms a hollow structure. In the dimer, chain A and chain B are arranged in head to tail manner at origin (0,0,0) of orthorhombic unit cell (Figure 2). The b/a barrel of chain A and b sheet of chain B pose at origin and their counterpart extends along X-direction. Dimer of AB is situated along with X-axis by an angle of 30u approximately; whereas other dimer CD is situated at rotation angle of 180u with a screw distance of 19.1 A˚ that occupies approximately one quarter of unit cell. The crystallized structure of EhODCD15 consists of a tetramer. The asymmetric unit contains two dimers comprising of chain A, B, C, D. The total area of the molecule of EhODC containing four molecules was estimated to be 61227.6 A˚ 2. Each dimer interacts with its symmetry mate to form dimer-dimer interfaces as A–B dimer interacts with C–D dimer (Figure 2). Interface area evaluated by PISA web server was averaged to 1373.4 A˚ 2 which was 1599.8 A˚ 2 and 1147.0 A˚ 2 between B, A and D, C respectively [59]. C-terminal Truncation and Purification of EhODC Residues Glu110 and His113 of chain B are at a distance of 3.1 A˚ and 3.3 A˚ from Lys84 and Asp88 of chain D showing polar interactions and vice versa (Figure 3). Data Collection and structure determination The diffraction data of EhODCD15 were collected at 100 K using Cu Ka radiation generated by a Bruker Microstar-H rotating-anode generator assembled with MAR 345 imaging-plate system. The data were collected at 1.54 A˚ with a crystal-to- detector distance of 200 mm and 1u oscillation per image with 20 min exposure per frame. Crystal diffracted to 2.8 A˚ resolution. The data were indexed, integrated and scaled using HKL2000 program [38]. Table 1 summarizes data collection and processing statistics. The structure was solved by molecular replacement January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 3 January 2013 \| Volume 8 \| Issue 1 \| e53397 Crystal packing Crystallization of purified EhODCD15 was performed using sitting drop vapor diffusion method. Crystals were obtained at 20uC in Hampton PEG ion screen 4 containing 20% (v/v) PEG 3350, 0.2 M LiCl maintained at pH 6.8. Crystals belonged to the orthorhombic space group exhibiting P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 A˚ and a = b = c = 90u. The crystal diffracted to 2.8 A˚ resolution, possess- ing four molecules per asymmetric unit and the solvent content was calculated to be 46.69% with a Matthews coefficient of 2.2 A˚ 3 Da21. Quality of the obtained structure was assessed with the PROCHECK program showing 88.7% of the residues in the favored region, whereas 10% in allowed, 0.9% in generously allowed and only 0.4% residues are observed in the disallowed region of Ramachandran Plot (Table 1). January 2013 \| Volume 8 \| Issue 1 \| e53397 January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 4 Crystal Structure of ODC from E. histolytica Figure 1. Purification and gel filtration profile of EhODCD15. A) 12% SDS-PAGE gel showing the affinity purified protein Lane 1: Molecular weight markers shown in kDa. Lane 2–3: Protein purified by affinity chromatography. B) Elution profile of the EhODC1D15 protein. The protein was eluted at a volume of 74 ml corresponding to molecule weight of ,87 kDa. Insert shows the purified protein in 12% SDS-PAGE after gel filtration chromatography. doi:10.1371/journal.pone.0053397.g001 Crystal Structure of ODC from E. histolytica Figure 1. Purification and gel filtration profile of EhODCD15. A) 12% SDS-PAGE gel showing the affinity purified protein Lane 1: Molecular weight markers shown in kDa. Lane 2–3: Protein purified by affinity chromatography. B) Elution profile of the EhODC1D15 protein. The protein was eluted at a volume of 74 ml corresponding to molecule weight of ,87 kDa. Insert shows the purified protein in 12% SDS-PAGE after gel filtration chromatography. doi:10.1371/journal.pone.0053397.g001 Figure 1. Purification and gel filtration profile of EhODCD15. A) 12% SDS-PAGE gel showing the affinity purified protein Lane 1: Molecular weight markers shown in kDa. Lane 2–3: Protein purified by affinity chromatography. B) Elution profile of the EhODC1D15 protein. The protein was eluted at a volume of 74 ml corresponding to molecule weight of ,87 kDa. Insert shows the purified protein in 12% SDS-PAGE after gel filtration chromatography. doi:10.1371/journal.pone.0053397.g001 Figure 2. Crystal packing Schematic representation of overall structure of the model obtained after molecular replacement. A) Cartoon diagram of tetrameric model of EhODC showing AB-CD, dimer-dimer interface; B) Active site of EhODC at the interface of dimer where (9) denotes the residues from the other subunit. doi:10.1371/journal.pone.0053397.g002 Figure 2. Schematic representation of overall structure of the model obtained after molecular replacement. A) Cartoon diagram of tetrameric model of EhODC showing AB-CD, dimer-dimer interface; B) Active site of EhODC at the interface of dimer where (9) denotes the residues from the other subunit. doi:10.1371/journal.pone.0053397.g002 January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Crystal Structure of ODC from E. histolytica Figure 3. Tetrameric structure with dimer-dimer interaction. A–C) shows the interaction between chain A and chain C. B–D) indicates the interaction between chain B and chain D. Pink dashes shows the interaction of residues through water molecule and green dashes indicates the polar interactions. Symbol (0) and (9) denotes the residues of chain C and chain D, respectively. doi:10.1371/journal.pone.0053397.g003 Figure 3. Tetrameric structure with dimer-dimer interaction. A–C) shows the interaction between chain A and chain C. B–D) indicates the interaction between chain B and chain D. Pink dashes shows the interaction of residues through water molecule and green dashes indicates the polar interactions. Symbol (0) and (9) denotes the residues of chain C and chain D, respectively. doi:10.1371/journal.pone.0053397.g003 Comparative analysis of active site architecture Furthermore, alpha-carbon backbone consisting residues Tyr331 and Asp332 in TbODC shows direct interactions with bound DFMO and these interactions play a role in proper DFMO molecule orientation in the active site pocket. However, these residues are mutated to Phe305 and Glu306 respectively in EhODC. Also, EhODC crystal structure reveals that the loop consisting of Phe305 and Glu306 residues is not located close to the active site thus may not contribute to DFMO binding. Moreover, residue Tyr’323 from other subunit of TbODC also supports the favourable orientation of DFMO by side chain hydroxyl group interaction with DFMO through a water molecule. Tyr’323 is replaced with His’296 in EhODC and the loop containing His’296 residue is positioned away from the active site (Figure 5). In contrast, the cofactor PLP and substrate L- ornithine are accommodated in EhODC active site with polar interactions to facilitate the catalysis (Figure 2) [29,35]. These structural details indicate that amino acid substitutions in the active site of EhODC create a novel architecture which not only makes it resistant to DFMO but also lowers its catalytic efficiency by weakening substrate binding, as the reported Km values for L- ornithine for active but DFMO sensitive T. brucei and mouse ODC binding residues (His197, Gly276, Arg277 and Tyr389 respec- tively) of TbODC (54, PDB ID: 2TOD). The side chains of these conserved residues interacting with PLP through polar interactions in TbODC are also expected to bind PLP and correctly orientation it into the active site of EhODC. Interestingly, Ser200 of TbODC is present in a loop and is seen interacting with PLP in TbODC structure (54, PDB ID: 2TOD). However, this residue (Ser188) is also conserved in EhODC, but shows small displacement from its expected position and has opposite orientation in the crystal structure of EhODC. Though, at this point it cannot be ruled out that the flexible loop of EhODC possessing Ser188 may approach the active site and may orient Ser188 in favourable position in the presence of PLP, whereas apo-enzyme might not restrict its position. Addition to this, Gly225 of EhODC shares exact position of Gly237 of TbODC, whose backbone carbon chain contributes to polar interactions. Apart from this, few residues interact with PLP through water molecules which include residues Phe238, Tyr278, Arg154 and Ala111 in TbODC. Comparative analysis of active site architecture In contrast, Cys’334 the conserved residue of EhODC that is expected to form a covalent bond with DFMO is slightly displaced from its position and has distinct orientation that is structurally unfavourable for covalent linkage with DFMO. In addition, the residue Asp’361 of TbODC is substituted by Asn’335 in EhODC, which is not expected to interact with DFMO. Furthermore, alpha-carbon backbone consisting residues Tyr331 and Asp332 in TbODC shows direct interactions with bound DFMO and these interactions play a role in proper DFMO molecule orientation in the active site pocket. However, these residues are mutated to Phe305 and Glu306 respectively in EhODC. Also, EhODC crystal structure reveals that the loop consisting of Phe305 and Glu306 residues is not located close to the active site thus may not contribute to DFMO binding. Moreover, residue Tyr’323 from other subunit of TbODC also supports the favourable orientation of DFMO by side chain hydroxyl group interaction with DFMO through a water molecule. Tyr’323 is replaced with His’296 in EhODC and the loop containing His’296 residue is positioned away from the active site (Figure 5). In contrast, the cofactor PLP and substrate L- ornithine are accommodated in EhODC active site with polar interactions to facilitate the catalysis (Figure 2) [29,35]. These structural details indicate that amino acid substitutions in the active site of EhODC create a novel architecture which not only makes it resistant to DFMO but also lowers its catalytic efficiency by weakening substrate binding, as the reported Km values for L- ornithine for active but DFMO sensitive T. brucei and mouse ODC subunit plays the most critical role in DFMO binding by making a permanent covalent bond with the enzyme. However, covalent bond formation of Cys’360 with substrate L-ornithine has not been reported for any ODC enzyme. In addition to this, the next residue of TbODC Asp’361 helps to position the Cys’360 residue in proper orientation and also interacts with DFMO through a water molecule. In contrast, Cys’334 the conserved residue of EhODC that is expected to form a covalent bond with DFMO is slightly displaced from its position and has distinct orientation that is structurally unfavourable for covalent linkage with DFMO. In addition, the residue Asp’361 of TbODC is substituted by Asn’335 in EhODC, which is not expected to interact with DFMO. Comparative analysis of active site architecture both the subunits. For comparative analysis of active site, we superimposed the EhODC structure over TbODC complexed with DFMO. The super-imposition of monomers shows root mean square deviation (rmsd) of 1.18 A˚ whereas super-imposition of dimer shows rmsd of 1.7 A˚ . Active site superimposition of TbODC and EhODC shows that most of the conserved residues in active site of EhODC share same positions as in TbODC, however few residues pose in different orientation (Figure 5). His185, Gly259, Arg260 and Tyr363, the well conserved PLP binding residues of EhODC share the position and have orientation similar to the PLP The ODC enzyme is an obligate homodimer with two symmetry-related active sites located at the dimer interface. According to our previous report, EhODC enzyme is functionally active in the dimeric form [36]. As expected, the crystal structure of EhODC contains two equivalent active site pockets at the dimer interface formed by residues that are contributed from both the subunits (Figure 2). The proper orientation of active site formed by two subunits is highly essential for functionality of the enzyme. The active site in EhODC is mainly contributed by loops from Figure 4. Crystal structure of EhODC monomeric subunit. A) Cartoon diagram of the monomer showing arrangement of barrel and sheet domain. B) Topology diagram of monomer of EhODC where helices are represented with cylinder and sheets with the arrows connected with loops, dashed line indicates the sequence missing in the structure. doi:10.1371/journal.pone.0053397.g004 Figure 4. Crystal structure of EhODC monomeric subunit. A) Cartoon diagram of the monomer showing arrangement of barrel and sheet domain. B) Topology diagram of monomer of EhODC where helices are represented with cylinder and sheets with the arrows connected with loops, dashed line indicates the sequence missing in the structure. doi:10.1371/journal.pone.0053397.g004 January 2013 \| Volume 8 \| Issue 1 \| e53397 January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 6 Crystal Structure of ODC from E. histolytica subunit plays the most critical role in DFMO binding by making a permanent covalent bond with the enzyme. However, covalent bond formation of Cys’360 with substrate L-ornithine has not been reported for any ODC enzyme. In addition to this, the next residue of TbODC Asp’361 helps to position the Cys’360 residue in proper orientation and also interacts with DFMO through a water molecule. EhODC sequence and structural comparison with ODC homologs From various mutational studies, it is reported that a conserved Lys (Lys57 in EhODC) is important as it forms Schiff base with PLP which is later displaced by L-ornithine that undergoes decarboxylation through nucleophilic attack via a conserved Cys (Cys3349 in EhODC) [36,63,64,65]. However, both the residues are well conserved in both functional ODCs and AZI (except in few AZIs). In all functional ODCs including ODCs from T. brucei, Homo sapiens (HsODC) and mouse, residue Ala111 and Arg154 (HsODC) are highly conserved and interact with PLP through water. Interestingly, in EhODC, though Arg142 is conserved, however Ala111 is uniquely substituted with Ser99. AZI possesses substitution at both the positions with Ala to Thr/Ile/Ser and Arg to His/Gln that make AZI incapable of binding to PLP. Out of sixteen PLP binding residues, AZIs have major mutations in five positions whereas EhODC possesses a single mutation at position 99 with substitution of Ala to Ser (Figure 7 and Figure 8). The novel active site architecture revealed from the crystal structure of EhODC and previously reported low catalytic efficiency of the enzyme hints towards possible adaptive evolution which lead to DFMO insensitive. AZI is an inactive ODC homolog that possesses a broader active site due to unusual packing of AZI dimers [61]. The architecture of AZI active site does not favour the accommodation of substrate as well as the co- factor for enzyme catalysis, which makes it an inactive homolog of ODC. Recently, it has been proposed that several homologs of ODCs including putative antizyme inhibitors apparently arise independently through evolution [46]. Robust sequence analysis and active site structure comparisons were performed to explore the evolutionary relationship of EhODC with respect to ODC homologs including AZI and to uncover the possibility of additional EhODC functions. Multiple sequence alignment was done and phylogenetic tree was generated for ODC homologues including functional ODC and nonfunctional AZI (Figure 6). In HsODC, five residues Tyr323, Tyr331, Asp332, Cys360 and Asp361 are reported to be key active site residues which interact with L-ornithine and these residues are highly conserved in all functional ODCs (Figure 7). However, EhODC is an exception where only Cys334 is conserved while both Tyr and both Asp residues are substituted by His296, Phe305, Glu306 and Asn335 respectively. Crystal Structure of ODC from E. histolytica identified 27 residues from sequence alignment of ODCs from different organisms responsible for the formation of active site pocket and in ODC enzyme dimerization (Figure 7). Out of 27 residues, 16 residues contribute to the active site formation by interacting with cofactor PLP, 5 residues for substrate binding, 3 residues for salt bridge formation, 3 residues as critical interface residues and 1 residue for dimerization (Figure 7). enzymes are 0.24 mM and 0.09 mM respectively, whereas for DFMO resistant EhODC it is 1.5 mM [29,60]. Not only substituted residues, but also the displacement of loops (His’296 loop/Phe305 and Glu306 loop) away from EhODC active site seems to contribute towards DFMO insensitivity. Comparative analysis of active site architecture These residues are present at the same position in EhODC active site except Ala111 where it is substituted by Ser99 in EhODC. Overall, the architecture of EhODC for binding to PLP is similar to that of TbODC. DFMO, a substrate analogue makes a stable covalent bond with conserved Cys residue in the active site of ODC enzyme and inhibits its catalytic reaction. Binding of DFMO in the proper orientation for covalent bond formation with its active site is also supported by its interaction with other residues that are there in the substrate binding pocket. To extricate the intricate structural details of EhODC responsible for low substrate affinity and/or DMFO insensitivity, structural comparison of EhODC active site architecture for substrate/DFMO binding was done with the active site of DFMO bound TbODC crystal structure (Figure 5) (54, PDB ID: 2TOD). In TbODC, Cys’360 from counterpart Figure 5. Superimposition of active site of EhODC with TbODC bound to DFMO. Residues of active site at the dimer interface are represented in sticks. TbODC residues are colored with green, EhODC residues are colored with orange. PLP and DFMO are colored with blue and polar interactions were indicated by black dashes; water molecule in shown in red sphere. Residues with (9) symbol are of opposite monomer. doi:10.1371/journal.pone.0053397.g005 Figure 5. Superimposition of active site of EhODC with TbODC bound to DFMO. Residues of active site at the dimer interface are represented in sticks. TbODC residues are colored with green, EhODC residues are colored with orange. PLP and DFMO are colored with blue and polar interactions were indicated by black dashes; water molecule in shown in red sphere. Residues with (9) symbol are of opposite monomer. doi:10.1371/journal.pone.0053397.g005 January 2013 \| Volume 8 \| Issue 1 \| e53397 January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 7 Crystal Structure of ODC from E. histolytica EhODC sequence and structural comparison with ODC homologs Colour indication: Violet columns signifies the mutation in AZI; Orange columns signifies the mutated residues in E. histolytica ODC which are similar to AZI; Gray shows the unique mutations in EhODC which is neither conserved in ODC nor in AZI; Blue indicate the mutation in EhODC which are rarely found in AZI and functional ODC; Olive color point out the mutations in EhODC which are similar to some ODC. Sequence analysis and numbering has been done according to EhODC. Residues which are not conserved are shown by single letter, the conserved residues are indicted by – and D indicates the deleted amino acids. % identity indicates the identity of EhODC sequence with other homologous ODC sequences [46]. doi:10.1371/journal.pone.0053397.g007 Asn335 positions are unique to EhODC as these two residues are found to be conserved as Tyr323 and Asp361 in both AZI as well as in functional ODC. Interestingly, substitution at Glu306 instead of Asp332 is similar to AZIs, the inactive homologs of ODCs. However, in AZIs only Asp332 is substituted by Glu, whereas other four residues are mostly conserved (Figure 7). Asn335 positions are unique to EhODC as these two residues are found to be conserved as Tyr323 and Asp361 in both AZI as well as in functional ODC. Interestingly, substitution at Glu306 instead of Asp332 is similar to AZIs, the inactive homologs of ODCs. However, in AZIs only Asp332 is substituted by Glu, whereas other four residues are mostly conserved (Figure 7). of AZI as classified on the basis of conserved key amino acid residues was found to be a functional ODC [67]. In contrast to this, ODC from Aedes aegypti is found to be enzymatically non- functional [46]. Thus, mutations in and around active site, ranging from substitution of one residue to substitution of fourteen residues in single polypeptide may cause enzyme inactivation. In T. nigroviridis, 11 residues are altered in the active site whereas in mammals 4 residues are altered to convert a functional ODC to a nonfunctional homolog [46]. However, in Drosophila melanogaster, though all 18 key residues of active site are conserved, but a single mutation of Asp332Tyr hinders dimer formation in ODC in addition to cofactor and substrate binding, which makes it a nonfunctional ODC. Furthermore, in case of TbODC and other functional ODCs, Tyr331 contributes to form an aromatic zipper responsible for complementary packing in two monomers [53,54]. EhODC sequence and structural comparison with ODC homologs The substitution of active site residues at His296 and AZI is an inactive homolog of ODC which has lost decarbox- ylation activity due to mutation of critical residues in the active site [24,25,26,46,62]. However, they are important in mammals as they are responsible for antizyme down regulations, thus regulate the ODC activity in cell system [21]. In this study, we have Figure 6. Multiple sequence alignment of ornithine decarboxylase and its homologues antizyme inhibitor to determine the conservation of sequence and mutation of active site and substrate binding residues. Circles indicate the residues important for enzymatic activity. Numbering is according to EhODC. doi:10.1371/journal.pone.0053397.g006 Figure 6. Multiple sequence alignment of ornithine decarboxylase and its homologues antizyme inhibitor to determine the conservation of sequence and mutation of active site and substrate binding residues. Circles indicate the residues important for enzymatic activity. Numbering is according to EhODC. doi:10.1371/journal.pone.0053397.g006 January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 8 Crystal Structure of ODC from E. histolytica Figure 7. Sequence analysis of ODC and antizyme inhibitor, comparing the active site residues of ODC/AZI from various organisms. Abbreviation denoted: Cf for cofactor binding; Bs salt bridge formation; S substrate binding residues; If dimer interface residues; Di important for dimer formation. Species with the name of protein are shown on left side. Colour indication: Violet columns signifies the mutation in AZI; Orange columns signifies the mutated residues in E. histolytica ODC which are similar to AZI; Gray shows the unique mutations in EhODC which is neither conserved in ODC nor in AZI; Blue indicate the mutation in EhODC which are rarely found in AZI and functional ODC; Olive color point out the mutations in EhODC which are similar to some ODC. Sequence analysis and numbering has been done according to EhODC. Residues which are not conserved are shown by single letter, the conserved residues are indicted by – and D indicates the deleted amino acids. % identity indicates the identity of EhODC sequence with other homologous ODC sequences [46]. doi:10.1371/journal.pone.0053397.g007 Figure 7. Sequence analysis of ODC and antizyme inhibitor, comparing the active site residues of ODC/AZI from various organisms. Abbreviation denoted: Cf for cofactor binding; Bs salt bridge formation; S substrate binding residues; If dimer interface residues; Di important for dimer formation. Species with the name of protein are shown on left side. EhODC sequence and structural comparison with ODC homologs In AZI, this is mutated to Ser rendering a loose contact between monomers [61]. But in EhODC, same residue is substituted by aromatic amino acid Phe305 that is expected to perform same job in aromatic zipper. The mutation of Tyr to Phe is also reported in Plasmodium falciparum, Leishmania donovani and Glycine max ODCs (Figure 7, Figure 7) [46,66]. The evolutionary relationship of ODC and AZI can be evaluated by considering the root of phylogenic tree which connects the branch of both homologs. Evidences indicated that both the homologs are from same subfamily and have evolved and AZI genes have accumulated mutations in key residues that are important for ODC activity. In Petromyzon marinus, the homologue January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org January 2013 \| Volume 8 \| Issue 1 \| e53397 9 Crystal Structure of ODC from E. histolytica Figure 8. Active sites comparison of functional ODC, antizyme inhibitor and EhODC. A) Human ODC active site residues colored in blue. B) EhODC active site residues identical to human ODC colored blue, residues identical to AZI colored green and unique to EhODC colored red. C) AZI interface region showing residues identical to human ODC in blue and those are mutated colored green. doi:10.1371/journal.pone.0053397.g008 Figure 8. Active sites comparison of functional ODC, antizyme inhibitor and EhODC. A) Human ODC active site residues colored in blue. B) EhODC active site residues identical to human ODC colored blue, residues identical to AZI colored green and unique to EhODC colored red. C) AZI interface region showing residues identical to human ODC in blue and those are mutated colored green. doi:10.1371/journal.pone.0053397.g008 that contributes to dimerization. Though, same residues i.e. Lys169, Asp364, Asp134, and Lys294 are conserved in AZI (mouse), still residues do not approach to form the salt bridge [61]. Furthermore, AZI is inefficient to bind to PLP consequently unable to carry out decarboxylation reaction. The structure of AZI (mouse) reveals that the active site is too wide to make suitable pocket for substrate and PLP binding. However, EhODC binds to PLP and catalyzes decarboxylation of L-ornithine and relatively less active as compared to other active ODCs. It is interesting to note that though EhODC possesses similar property with other ODC on the basis of structure and function, it shares some similarity with AZI based on amino acid sequence. EhODC sequence and structural comparison with ODC homologs Firstly, the substrate binding residue Asp332 (HsODC) is conserved in functional ODCs where in EhODC same residue is altered to Glu306 and Glu is well conserved in AZI. Secondly, PLP binding residue Ala is altered to Ser in EhODC and such alternation is reported in AZI of Danio rerio, Tetraodon nigroviridis and Anolis crolinensis. Thirdly, EhODC possesses unique mutations at His296 and Asn335 those are neither reported in any functional ODC or AZI. Such alternation of critical residues particularly in protozoa provides the evidences of adaptive evolution of ODC. AZI dependent ODC regulation is only reported in higher organisms and absent from lower organisms. Even such regulation is not reported in protozoa till date. However, it can be hypothesized that ODC in protozoa takes the modification towards AZI though it functions less efficently as an active ODC and its function as AZI needs to be investigated. diverged according to their function. In phylogenetic tree, the group of AZI and ODC make different clusters according to the sequence alignment. Interestingly, EhODC is clustering to the ODC group just beneath the Aedes aegypti which is a nonfunctional ODC due to His197Asn and Asp332Arg substitutions as shown in Figure 7 and Figure 9. ODC of Aedes aezypti, being a non- functional ODC, represents the border line of functional ODCs and nonfunctional AZI. EhODC, the enzyme with low catalytic efficiency is found to be more evolutionarily related to nonfunc- tional ODC of Aedes aezypti and AZIs. These evidences from sequence alignment and phylogeny profile of EhODC allow us to establish the fact that during the course of evolution it gained DFMO resistance by acquiring critical alternation in its sequence similar to both functional ODCs and non-functional AZI. Though, the evolutionary changes in the sequence also influenced its catalytic efficiency. However, the possibility of additional biological role of EhODC such as antizyme inhibitory activity needs to be investigated. References 9. Pegg AE (2006) Regulation of ornithine decarboxylase. J Biol Chem 281: 14529–14532. 1. Rosas-Arreguı´n P, Arteaga-Nieto P, Reynoso-Orozco R, Villago´mez-Castro JC, Sabanero-Lo´pez M, et al. (2008) Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica. Exp Parasitol 119: 398–402. 10. Evageliou NF, Hogarty MD (2009) Disrupting polyamine homeostasis as a therapeutic strategy for neuroblastoma. Clin Cancer Res 15: 5956–5961. 2. Lo´pez-Vallejo F, Castillo R, Ye´pez-Mulia L, Medina-Franco JL (2011) Benzotriazoles and indazoles are scaffolds with biological activity against Entamoeba histolytica. J Biomol Screen 16: 862–868. 2. Lo´pez-Vallejo F, Castillo R, Ye´pez-Mulia L, Medina-Franco JL (2011) Benzotriazoles and indazoles are scaffolds with biological activity against Entamoeba histolytica. J Biomol Screen 16: 862–868. 11. Oredsson SM (2003) Polyamine dependence of normal cell-cycle progression. Biochem Soc Trans 31: 366–370. 12. Fleidervish IA, Libman L, Katz E, Gutnick MJ (2008) Endogenous polyamines regulate cortical neuronal excitability by blocking voltage-gated Na+ channels. Proc Natl Acad Sci U S A 105: 18994–18999. y J 3. Tanyuksel M, Petri WA Jr (2003) Laboratory diagnosis of amebiasis. Clin Microbiol Rev 16: 713–729. y 3. Tanyuksel M, Petri WA Jr (2003) Laboratory diagnosis of amebiasis. Clin Microbiol Rev 16: 713–729. 13. Krause T, Lu¨ersen K, Wrenger C, Gilberger TW, Mu¨ller S, et al. (2000) The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S- adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expres- sion and catalytic properties of two different constructs. Biochem J 352: 287– 292. 4. Bansal D, Sehgal R, Chawla Y, Mahajan RC, Malla N (2004) In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar. Ann Clin Microbiol Antimicrob 3: 27. 4. Bansal D, Sehgal R, Chawla Y, Mahajan RC, Malla N (2004) In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar. Ann Clin Microbiol Antimicrob 3: 27. p 5. Heby O, Roberts SC, Ullman B (2003) Polyamine biosynthetic enzymes as drug targets in parasitic protozoa. Biochem Soc Trans 31: 415–419. 14. Palanimurugan R, Scheel H, Hofmann K, Dohmen RJ (2004) Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme. EMBO J 23: 4857–4867. 6. Gillin FD, Reiner DS, McCann PP (1984) Inhibition of growth of Giardia lamblia by a-difluoromethylornithine, a specific inhibitor of polyamine biosynthesis. J Protozool 31: 161–163. 15. Conclusion In the present report, we successfully determined the 3D structure of EhODC to elucidate its intricate active site architec- ture that made it DFMO insensitive. Further on the basis of sequence analysis, we unveiled many unique characteristics of EhODC that show similarity with both functional ODC and non- functional AZI. EhODC exists as a dimer like other functional ODCs and in contrast AZI is monomer in solution due to weaker interaction between two monomers. Interface of EhODC shows 45 contacts and 16 hydrogen bonds in addition to salt bridges which stabilize the dimer. As studied in AZI structure (mouse AZI), only 43 contacts and 15 hydrogen bonds are reported which lack salt bridge formation and make interface less interactive as compared to ODC [61]. Structure of EhODC at 2.8 A˚ revealed two salt bridges between Lys157-Asp238 at a distance 2.9 A˚ and Asp122- Arg277 at 3.2 A˚ . Same salt bridges are also reported in HsODC Our study will facilitate to investigate the molecular evolution of ODCs and AZI. It also suggests additional functional properties for EhODC such as it may also play a role similar to that of AZI in E. histolytica. Additionally, availability of EhODC crystal structure will be helpful in development of structure based anti-amoebiasis drugs. January 2013 \| Volume 8 \| Issue 1 \| e53397 PLOS ONE \| www.plosone.org 10 Crystal Structure of ODC from E. histolytica Figure 9. Phylogenetic relationship of EhODC with antizyme inhibitor and ODC. Sequence of ODC and their evolutionary related homologous were retrieved from various sources. Antizyme inhibitor of Homo sapiens (BAA23593.1), Nomascus leucogenys (XP_003256127.1), Macaca mulatta (XP_002805501.1), Mus musculus (AAB87464.1), Rattus norvegicus (BAA23594.1), Monodelphis domestica (XP_001369332.1), Xenopus laevis (NP_001087584.1), Danio rerio (BAB84695.1), Tetraodon nigroviridis (ENSTNIT00000008148.1), Anolis carolinensis (XP_003219500.1), Gallus gallus (NP_001008729.1), Ornithorhynchus anatinus (XP_001506230.1), Canis familiaris (XP_849306.1), Bos Taurus (NP_001076080.1), Loxodonta africana (XP_003408472.1). Ornithine decarboxylase sequence from Aedes aegypti (EAT48998.1), Entamoeba histolytica (AAX35675.1), Plasmodium falciparum (AAF14518.1), Leishmania donovani (AAA29259.1), Datura stramonium (CAA61121.1), Solanum lycopersicum (NP_001234616.1), Glycine max (CAD91349.1), Chlamydomonas reinhardtii (CAE46409.1), Monodelphis domestica (XP_001371947.1), Bos Taurus (AAA92339.1), Macaca mulatta (NP_001185615.1), Homo sapiens (NP_002530.1), Mus musculus (NP_038642.2), Anolis carolinensis (XP_003215471.1), Trypanosoma brucei (AAA30219.1), Xenopus laevis (CAA39760.1). doi:10.1371/journal.pone.0053397.g009 Figure 9. Phylogenetic relationship of EhODC with antizyme inhibitor and ODC. Sequence of ODC and their evolutionary related homologous were retrieved from various sources. Acknowledgments The authors thank Macromolecular Crystallographic Unit (MCU), IIC, IIT Roorkee for providing protein expression, purification, data collection Accession number Structure factors and final refined atomic coordinates for EhODC have been deposited in the Protein Data Bank (http:// www.rcsb.org) with accession number 4AIB. Author Contributions Conceived and designed the experiments: Preeti S. Tomar RM. Performed the experiments: Preeti S. Tapas PK S. Tomar. Analyzed the data: Preeti S. Tapas RM PK S. Tomar. Contributed reagents/materials/analysis tools: S. Tomar RM PK. Wrote the paper: Preeti S. Tapas S. Tomar. Conclusion Antizyme inhibitor of Homo sapiens (BAA23593.1), Nomascus leucogenys (XP_003256127.1), Macaca mulatta (XP_002805501.1), Mus musculus (AAB87464.1), Rattus norvegicus (BAA23594.1), Monodelphis domestica (XP_001369332.1), Xenopus laevis (NP_001087584.1), Danio rerio (BAB84695.1), Tetraodon nigroviridis (ENSTNIT00000008148.1), Anolis carolinensis (XP_003219500.1), Gallus gallus (NP_001008729.1), Ornithorhynchus anatinus (XP_001506230.1), Canis familiaris (XP_849306.1), Bos Taurus (NP_001076080.1), Loxodonta africana (XP_003408472.1). Ornithine decarboxylase sequence from Aedes aegypti (EAT48998.1), Entamoeba histolytica (AAX35675.1), Plasmodium falciparum (AAF14518.1), Leishmania donovani (AAA29259.1), Datura stramonium (CAA61121.1), Solanum lycopersicum (NP_001234616.1), Glycine max (CAD91349.1), Chlamydomonas reinhardtii (CAE46409.1), Monodelphis domestica (XP_001371947.1), Bos Taurus (AAA92339.1), Macaca mulatta (NP_001185615.1), Homo sapiens (NP_002530.1), Mus musculus (NP_038642.2), Anolis carolinensis (XP_003215471.1), Trypanosoma brucei (AAA30219.1), Xenopus laevis (CAA39760.1). doi:10 1371/journal pone 0053397 g009 and computational facilities. We also thank Sonali Dhindwal for her help in data collection and refinement. and computational facilities. We also thank Sonali Dhindwal for her help in data collection and refinement. and computational facilities. We also thank Sonali Dhindwal for her help in data collection and refinement. 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https://openalex.org/W2077212425	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-11-39	English	null	Gene expression profiles in Atlantic salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes	BMC genomics	2,010	cc-by	12,215	Abstract Background: Excessive fat deposition is one of the largest problems faced by salmon aquaculture industries, leading to production losses due to high volume of adipose tissue offal. In addition, increased lipid accumulation may impose considerable stress on adipocytes leading to adipocyte activation and production and secretion of inflammatory mediators, as observed in mammals. Results: Microarray and qPCR analyses were performed to follow transcriptome changes during adipogenesis in the primary culture of adipose stromo-vascular fraction (aSVF) of Atlantic salmon. Cellular heterogeneity decreased by confluence as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature lineages. Transgelin (TAGLN), a marker of the multipotent pericyte, was prominently expressed around confluence while adipogenic PPARg was up-regulated already in subconfluent cells. Proliferative activity and subsequent cell cycle arrest were reflected in the fluctuations of pro- and anti-mitotic regulators. Marked regulation of genes involved in lipid and glucose metabolism and pathways producing NADPH and glycerol-3-phosphate (G3P) was seen during the terminal differentiation, also characterised by diverse stress responses. Activation of the glutathione and thioredoxin antioxidant systems and changes in the iron metabolism suggested the need for protection against oxidative stress. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occured in parallel with the increased lipid droplet (LD) formation and production of secretory proteins (adipsin, visfatin). The UPR markers XBP1 and ATF6 were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Concurrently, translation was suppressed as evidenced by the down-regulation of genes encoding elongation factors and components of the ribosomal machinery. Notably, expression changes of a panel of genes that belong to different immune pathways were seen throughout adipogenesis. The induction of AP1 (Jun, Fos), which is a master regulator of stress responses, culminated by the end of adipogenesis, concurrent with the maximal observed lipid deposition. Conclusions: Our data point to an intimate relationship between metabolic regulation and immune responses in white adipocytes of a cold-blooded vertebrate. Stress imposed on adipocytes by LD formation and expansion is prominently reflected in the ER compartment and the activated UPR response could have an important role at visceral obesity in fish. however, known about the factors regulating develop- ment and functions of adipose tissue in fish, and whether increased fat deposition may lead to health pro- blems. RESEARCH ARTICLE Open Access Open Access * Correspondence: marijana.todorcevic@nofima.no † Contributed equally 1Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, P. O. Box 5010, Ås NO-1430, Norway Abstract Previously regarded principally as energy storage, white adipose tissue (WAT) in mammals is now recog- nized as a highly active endocrine tissue producing numerous secretory proteins, including adipokines, a suite of small signalling proteins specifically produced in WAT [2,3]. Under normal conditions, adipocytes are Marijana Todorčević1,2†, Stanko Škugor1,2†, Aleksei Krasnov1, Bente Ruyter1 Marijana Todorčević1,2†, Stanko Škugor1,2†, Aleksei Krasnov1, Bente Ruyter1 © 2010 Todorčević 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. Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 © 2010 Todorčević et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creati 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. © 2010 Todorčević 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. Cell culture characterization A short summary of adipocyte development and cultiva- tion conditions is presented in Fig. 1. In the subconflu- ent stage during the first seven days of culture, aSVF cells had the highest proliferative activity (Fig. 2A). Approximately at day 7, cells reached the confluent stage. Two-day adipogenic hormonal induction was applied at that time point. This was followed by the terminal differentiation stage during which cells acquired a more rounded shape. Morphological changes observed by electron microscopy images at days 15 and 30 were characteristic of the terminally differentiating phenotype of adipocytes, including a relatively low mito- chondrial number, large size of LD and the nucleus located between LDs and cell membrane (Fig. 3A, B). Oil red O staining also showed a high degree of lipid accumulation in mature adipocytes at day 30 (Fig. 4A). A steady decrease in the extracellular superoxide dismu- tase (SOD) activity was observed from day 1 to day 30 (Fig. 4B). In mammals, adipogenesis includes three distinct stages. Following active proliferation and the phase of determination, cells reach confluence, followed by hor- monal induction and terminal differentiation. Re-entry into the cell cycle of growth-arrested cells at confluence involves several rounds of proliferation, referred to as mitotic clonal expansion. Secondary growth arrest and induction of the transcription factors CCAAT-enhancer- binding protein (C/EBP) a and peroxisome proliferator- activated receptor (PPAR) g mark the end of the mitotic clonal expansion phase and entry into terminal differen- tiation with transcriptional activation of genes defining the mature adipocyte phenotype. Background Feeds used in modern Atlantic salmon aquaculture con- tain large amounts of lipids, which provide rapid growth, reduce environmental load from farms, but increase visceral fat deposition [1]. Very little is, Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 2 of 17 The aSVF primary culture enabled investigation of early events involved in the adipogenic determination as well as processes characteristic of the later terminal dif- ferentiation phase. Particular emphasis was placed on genes governing nutrient metabolism and stress and inflammatory responses. involved in the regulation of a broad range of physiolo- gical processes but at obesity increased production of cytokines and adipokines lead to the chronic low-grade inflammatory state. The extent of conservation of endo- crine WAT functions in the cold-blooded vertebrate, including its capacity to influence systemic inflammatory responses and development of life style related disorders has not been studied until now. Results In salmonid fish, precursor cells differentiate into adi- pocytes continuously [4], however, the exact origin of preadipocytes is not known. In mammals, development of white adipocytes is thought to begin with progenitor cells from the perivascular compartment (pericytes) in WAT, characterised by expression of smooth muscle actin 22a, also known as transgelin (TAGLN), and other vascular markers. Isolated pericytes differentiate into mesenchymal stem cells (MSCs), which in turn, give rise to various cell types including osteoblasts, chondrocytes, smooth muscle cells, fibroblasts, macrophages and adi- pocytes [5-9]. Mammalian adipose stromal-vascular frac- tion (aSVF) harbours a population of progenitor cells that is also capable to differentiate ex vivo into cells and tissues of mesodermal origin, thus suggesting their peri- vascular origin. Atlantic salmon WAT contains a large number of fibroblast-like precursor cells that can differ- entiate to mature adipocytes in vitro [10,11], however, their stem cell features have not been characterized yet. Subconfluence (days 1 and 4) Microarray analyses of aSVF in the first days of culture, revealed concurrent expression of genes characteristic of MSCs, immune cells and cells of perivascular origin (Fig. 5). Platelet-derived growth factor A (PDGFA) is a major regulator of proliferation and migration of mam- malian adipose-derived MSCs [13]. Glomulin is essential for normal development of the vasculature while the lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) is a marker of lymph vessels and endothelial cells in mammals. Chemokine receptor 4 (CXCR4) and other chemokine/cytokine receptors are involved in cell mobilisation and retention in several populations of MSCs and in immune cells [14-16]. We have selected eight co-expressed genes encoding proteins involved in nucleotide metabolism, DNA replication and regulation of cell cycle and their averaged profile, due to their involvement in the similar cellular functions, was shown in Fig. 2B. The lowest expression of genes from this category was at day 1 (also see Fig. 5). The subsequent increase was in concordance with the results of prolifer- ating cell nuclear antigen (PCNA) assay that revealed the highest proliferation rate at day 4 (Fig. 2A). In paral- lel, microarray analyses showed a decreased expression of non-adipogenic cell markers. These genes had high levels at day 1 (Fig. 5). Tumour necrosis factor (TNF) a, The few performed studies indicate that adipogenesis in fish bears many resemblances to that of terrestrial vertebrates [10-12], though there certainly exist as yet undiscovered species-specific differences. Atlantic sal- mon precursor cells take longer than mammalian to acquire the mature adipocyte phenotype in culture, due to low incubating temperature, typical of salmonid fish habitat. Further, mammalian cells are able to produce lipid droplets (LDs) from glucose alone, while salmon preadipocytes require lipids in order to achieve the mature phenotype [10]. Hence it is to be expected that white adipocytes and their precursors in a cold-blooded vertebrate may have both evolutionarily conserved and specific features. Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 3 of 17 D1 D4 D5 D6 D7 D9 D10 D11 D12 D15 D20 D23 D30 Medium= Medium Medium FBS (10%) L-15 +Insulin +lipid mix Antibiotics L-glutamin +Dex +IBMX Subconfluent Confluent Terminal differentiation Hormonal induction +Biotin +Pantoten +T3 FBS..........Fetal Bovine Serum Dex.......... Dexametasone IBMX....... Isobutylmethylxanthine T3.............Triiodothyronine D1,D4,D7,D9,D15,D30 = Microarray & qPCR D5,D6,D10,D11,D12,D20,D23,D30 = qPCR Figure 1 Summary of procedures and the key stages. Figure 1 Summary of procedures and the key stages. Subconfluence (days 1 and 4) Confluence (day 7) and hormonal induction (day 9) Cells reached confluence at day 7. Up-regulation of reti- noblastoma-like protein 1 (RBL1) and p53-like protein (Fig. 5) was in concordance with the temporary cell cycle arrest at G0/G1, which is commonly observed at this stage in most mammalian cell lines. After stimula- tion with hormones, cells re-enter the cell cycle and undergo mitotic clonal expansion before ultimate exit from the cell cycle. At day 9, expression of RBL1 and p53-like protein decreased while cyclins E1 and B2 were stimulated. Two days after addition of the differentiation media, cells changed to a more rounded shape. This coincided with the up-regulation of a suite of motor contractile proteins (Fig. 5). Interestingly, microarray showed elevated expression of TAGLN at day 7 and day 9. TAGLN is a marker of pericytes, smooth muscle-like cells surrounding vasculature, that are closely related to the MSCs and fibroblasts [19]. Positive and negative reg- ulators of myogenic differentiation showed opposite changes: decrease in myogenic factor D (MyoD) and increase in bone morphogenic protein 4 (BMP4) (Fig.7). We also observed up-regulation of several genes for Ca2 + binding proteins (Fig. 5). Calcium is involved in the control of the whole adipogenic process, from multipo- tent stem cells to adipocytes [20,21]. Increases in cyto- plasmic calcium during this phase inhibit adipogenesis [21-24]. Days 7 and 9 were marked by the expression changes of C/EBPs. The early adipogenic marker C/ EBPb, was up-regulated until confluence, whereas tran- sient induction of C/EBPδ and consistent increase of C/ EBPa were observed after the addition of hormones at day 9 (Fig. 7). Overall, C/EBPb and C/EBPδ work sequentially and predate the expression of C/EBPa [12]. We observed different expression changes in a number of genes encoding chemokines and cytokines. The leu- kocyte cell-derived chemotaxin 2 (LECT2) was re-acti- vated at day 9 and remained up-regulated until the end of the study period (Fig. 6). LECT2 is a potent neutro- phil chemoattractant, which also affects development of a proinflammatory cytokine and a potent negative regu- lator of adipocyte differentiation in terrestrial verte- brates, was up-regulated before confluence as well as a panel of TNF-related genes and receptors (Fig. 5 and 6). We observed up-regulation of PPARg already at day 4 (Fig. 7). This is the master regulator that co-ordinately activates transcription of adipocyte-specific genes [17,18]. Figure 2 Cell division, DNA replication and nucleotide metabolism. Subconfluence (days 1 and 4) A: cell proliferation was assessed by immunocytochemical detection of proliferating cell nuclear antigen (PCNA). Data (labeling index) are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤0.05). B: expression changes (mean log-ER ± SE) of eight genes (Cdc45, cell division control protein 42 homolog, anti-silencing function 1B, ribonucleoside-diphosphate reductase large subunit, cold inducible RNA binding protein-1 and 2, chromosome segregation 1-like protein, chromosome-associated kinesin KIF4A) analyzed with microarrays. Figure 2 Cell division, DNA replication and nucleotide metabolism. A: cell proliferation was assessed by immunocytochemical detection of proliferating cell nuclear antigen (PCNA). Data (labeling index) are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤0.05). B: expression changes (mean log-ER ± SE) of eight genes (Cdc45, cell division control protein 42 homolog, anti-silencing function 1B, ribonucleoside-diphosphate reductase large subunit, cold inducible RNA binding protein-1 and 2, chromosome segregation 1-like protein, chromosome-associated kinesin KIF4A) analyzed with microarrays. Figure 2 Cell division, DNA replication and nucleotide metabolism. A: cell proliferation was assessed by Figure 2 Cell division, DNA replication and nucleotide metabolism. A: cell proliferation was assessed by immunocytochemical detection of proliferating cell nuclear antigen (PCNA). Data (labeling index) are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤0.05). B: expression changes (mean log-ER ± SE) of eight genes (Cdc45, cell division control protein 42 homolog, anti-silencing function 1B, ribonucleoside-diphosphate reductase large subunit, cold inducible RNA binding protein-1 and 2, chromosome segregation 1-like protein, chromosome-associated kinesin KIF4A) analyzed with microarrays. Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 4 of 17 Figure 3 Atlantic salmon aSVF primary cell culture. A, B: Electron micrographs of representative salmon adipocytes day 15 (A) and day 30 (B). Three weeks after induction of differentiation (day 30), a large portion of cytoplasmic space was filled with LDs, the number mitochondria was reduced and the nuclei were located between LDs and cell membranes. Bars: 5 μm. Arrows points to mitochondria and arrowheads to lipid droplets Figure 3 Atlantic salmon aSVF primary cell culture. A, B: Electron micrographs of representative salmon adipocytes day 15 (A) and day 30 (B). Three weeks after induction of differentiation (day 30), a large portion of cytoplasmic space was filled with LDs, the number mitochondria was reduced and the nuclei were located between LDs and cell membranes. Subconfluence (days 1 and 4) Bars: 5 μm. Arrows points to mitochondria and arrowheads to lipid droplets Figure 3 Atlantic salmon aSVF primary cell culture. A, B: Electron micrographs of representative salmon adipocytes day 15 (A) and day 30 (B). Three weeks after induction of differentiation (day 30), a large portion of cytoplasmic space was filled with LDs, the number mitochondria was reduced and the nuclei were located between LDs and cell membranes. Bars: 5 μm. Arrows points to mitochondria and arrowheads to lipid droplets chondrocytes and osteoblasts [25,26]. In contrast, TNFa and related genes were down-regulated (Fig. 5 and 6). Terminal differentiation and the late phase of white adipocyte maturation (days 12-30) phosphogluconate dehydrogenase (PGD) from the oxi- dative pentose phosphate pathway (PPP) were up-regu- lated from day 12 to the end of adipogenic differentiation indicating increased need for NADPH that is required for TAG biosynthesis. To synthesise nucleotides, cells need large amounts of ribose-5-phos- phate (R5P). The PPP uses glucose to provide R5P and NADPH, however, the output can be modified accord- ing to the cells’ current needs, towards the production of either one of the products. The increase of phospho- fructokinase (PFK) and pyruvate kinase (PK) expression from day 15 could be an evidence for the importance of glycolysis in TAG-accumulating adipocytes. In addition to glycolysis and contrary to what was earlier believed, adipocyte maturation (days 12-30) The glutathione peroxidases (GPXs) (Fig. 5 and 9), which are sensitive to lipid mediated peroxidation [36], were similarly induced and a suite of genes coding for proteins of glutathione metabolism also had high levels, at the time when cells engaged in increased LD forma- tion and expansion. The thioredoxin (TXN) antioxidant system is also involved in the regulation of intracellular ROS [37]. The production of ROS is catalyzed with iron and hem, therefore, the up-regulation of ferritin (the intracellular iron storage protein), and two key enzymes in the metabolism of hem, aminolevulinate δ synthetase and heme oxygenase 2 (HMOX2) suggest the induction of mechanisms that protect against oxidative stress at day 30. Figure 4 A: lipid content in adipocytes was quantified with Oil Red O. Absorbance of extracted dye was measured at 500 nm. Data are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤0.05). B: superoxide dismutase (SOD) activity in culture media. Different letters indicate significant differences (p ≤0.05). Data shown are mean ± SEM (n = 5). Figure 4 A: lipid content in adipocytes was quantified with Oil Red O. Absorbance of extracted dye was measured at 500 nm. Data are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤0.05). B: superoxide dismutase (SOD) activity in culture media. Different letters indicate significant differences (p ≤0.05). Data shown are mean ± SEM (n = 5). adipocytes seem to be able to use alternative means (glyceroneogenesis) to produce glycerol-3-phosphate (G3P) needed for TAG synthesis [28,29]. Two key gly- ceroneogenic enzymes, cytoplasmic malate dehydrogen- ase (MDHC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKC) were also up-regulated during terminal differentiation. These changes did not necessa- rily mean a switch to anaerobic metabolism. Microarray analyses revealed a decrease of hypoxia inducible factor (HIF1a) while the negative regulator of HIF1a (HIF prolyl hydroxylase) was induced (Fig. 5). Day 15 was marked with the expression changes of genes involved in differentiation and cell cycle. Increase was observed in the anti-osteogenic [30] growth arrest specific protein 6 (GAS6) and adipogenic markers adip- sin and visfatin (Fig. 5). The up-regulations of RBL1, p53-like protein and growth arrest and DNA-damage- inducible (GADD45) g were in concordance with the marked attenuation of cell proliferation. Interestingly, many immune-related genes exhibited sustained up-reg- ulations, which began shortly before or at the onset of terminal differentiation (Fig. 6). adipocyte maturation (days 12-30) The increase in intracellular lipid levels measured by Oil Red O (Fig. 4A) was in line with the up-regulation of genes for several proteins of lipid metabolism (Fig. 5), including lipoprotein lipase (LPL) (Fig. 7). The expres- sion of fatty acid synthase (FAS) was highest from day 15 to day 30 (Fig. 7) in agreement with the profile of FAS observed in murine adipocytes [27]. Metabolic alterations were also supported by the regulation of key genes from major carbohydrate pathways (Fig. 8). Glu- cose-6-phosphate dehydrogenase (G6PD) and 6- Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 5 of 17 Figure 4 A: lipid content in adipocytes was quantified with Oil Red O. Absorbance of extracted dye was measured at 500 nm. Data are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤0.05). B: superoxide dismutase (SOD) activity in culture media. Different letters indicate significant differences (p ≤0.05). Data shown are mean ± SEM (n = 5). receptor signalling and biosynthesis of eicosanoids (lipid mediators of inflammation). Galectins (Fig. 5) are carbo- hydrate binding proteins involved in various immune processes [33]. Notably, expression of a panel of immune genes decreased by day 30. The most noticeable aspect of the last stage of adipo- genesis was co-ordinated activation of genes involved in various stress responses. Several components of the acti- vator protein complex 1 (AP1) (c-Fos, c-Jun, JunB1, JunB2, and JunD), which co-ordinates responses to pathogens and stressors, showed highest expression levels at day 30 (Fig. 5). The observed gene expression changes clearly revealed the endoplasmic reticulum (ER) stress in our culture at this stage. The up-regulated oxi- dant stress-activated serine/threonine kinase 25 (YSK1, Fig. 5) is a Golgi complex-associated regulator of trans- port of proteins and lipids to plasma membrane [34]. The A-kinase anchor protein 9 (AKAP9) is an essential mediator in lipolytic pathways and is necessary for the maintenance of Golgi structure through interactions with signalling proteins, including the protein kinase N (PKN1); both AKAP9 and PKN1 showed strong down- regulation at day 15 (Fig. 5). The ER stress response involves a set of mechanisms referred to as unfolded protein response (UPR) [35]. The classical UPR markers X-box-binding protein 1 (XBP1) and activating tran- scription factor 6 (ATF6) were up-regulated during the lipid-loading phase of adipogenesis (Fig. 9). Our findings point to the increased responses to oxidative stress with time. adipocyte maturation (days 12-30) Programmed death ligand 1 (PD-L1) is involved in the blockage of cell cycle in T-cells [31]. The up-regulation of decoy receptor interleukin (IL) 13 receptor alpha 2 (IL13Ra2) implies suppression of the anti-inflammatory IL4/IL13 axis [32]. Janus kinase 1 (JAK1) and 5-lipoxygenase activating pro- tein (FLAP) play pivotal roles in respectively, cytokine The massive down-regulation of components of the translational machinery and translation initiation factors suggested reduction of ribosomal biogenesis and attenuation of protein translation while the highest expression level of 18S at day 15 suggested profound changes in the composition of ribosomes during term- inal differentiation (Fig. 5, 9 and 10). Averaged expres- sion profiles of thirty five highly co-expressed ribosomal proteins, components of the 40S and 60S ribosomal sub- units, were shown in Fig. 10. Interestingly, eukaryotic initiation translation factor 5 (eIF5) involved in the Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 6 of 17 Figure 5 Results of microarray analyses, selected genes with expression changes. Samples from days 1, 4, 7, 9, 15 and 30 (5 pooled flasks per day) were compared to an equalised mixture from all time-points. Data are log-ER (expression ratios). Significantly up- and down-regulated genes (p < 0.01, t-test, 12 spot replicates per gene) are highlighted with red and green scales. NS - not significant, ND - not detected. Figure 5 Results of microarray analyses, selected genes with expression changes. Samples from days 1, 4, 7, 9, 15 and 30 (5 pooled flasks per day) were compared to an equalised mixture from all time-points. Data are log-ER (expression ratios). Significantly up- and down-regulated genes (p < 0.01, t-test, 12 spot replicates per gene) are highlighted with red and green scales. NS - not significant, ND - not detected. observed at day 30 while up-regulation of lysosomal proteases (cathepsins) was seen from day 15 (Fig. 5). joining of ribosomal subunits resulting in the formation of a functional 80S initiation complex was up-regulated at the last time point (Fig. 5). Discussion TNFa - tumour necrosis factor alpha, TNF decoy - tumour necrosis factor decoy receptor, FLAP - 5-lipoxygenase-activating protein, JAK1 - janus kinase 1, PD-L1 - programmed cell death ligand 1, IL-13 a2 - interleukin 13, LECT2 - leukocyte cell- derived chemotaxin a2, C5 - complement component 5. Figure 6 Immune genes analyzed with real-time qPCR. In this and other panels data are shown as -ΔΔCt ± SE (n = 5). Significant difference from day 1 (t-test, p < 0.05) is indicated with . TNFa - tumour necrosis factor alpha, TNF decoy - tumour necrosis factor decoy receptor, FLAP - 5-lipoxygenase-activating protein, JAK1 - janus kinase 1, PD-L1 - programmed cell death ligand 1, IL-13 a2 - interleukin 13, LECT2 - leukocyte cell- derived chemotaxin a2, C5 - complement component 5. Figure 6 Immune genes analyzed with real-time qPCR. In this and other panels data are shown as -ΔΔCt ± SE (n = 5). Significant difference from day 1 (t-test, p < 0.05) is indicated with . TNFa - tumour necrosis factor alpha, TNF decoy - tumour necrosis factor decoy receptor, FLAP - 5-lipoxygenase-activating protein, JAK1 - janus kinase 1, PD-L1 - programmed cell death ligand 1, IL-13 a2 - interleukin 13, LECT2 - leukocyte cell- derived chemotaxin a2, C5 - complement component 5. stages of the culture. Decrease in gene expression of markers of osteo/chondrogenic, myogenic, immune and vasculature cell lineages (Fig. 5) and increase in adipo- genic markers indicate that the preadipocyte precursors, probably due to their active proliferation, become the preponderant cell type at confluence. WAT population of stem cells and early events in the determination phase are much less studied than the terminal differentiation phase of adipogenesis. The breakthrough in the understanding of elusive origins of the adipogenic MSCs was made only recently in a study of Tang et al. [9], which demonstrated that pericytes surrounding blood vessel walls in WAT are precursor cells of preadipocytes. Gradual attenuation of the vascu- lature specific genes glomulin and LIVE1 and increase of the pericytic marker TAGLN at days 7 and 9 support perivascular origin of salmon preadipocytes as well. Early PPARg expression could represent an additional Recent studies in mammals showed that tissue-resi- dent MSCs originate from the smooth-muscle-like peri- cytes [39,40], which are laid over junctions of endothelial cells in blood vessel walls [41]. Discussion The increase of several protein folding heat shock pro- teins and genes involved in protein degradation was evi- dent during this period (Fig. 5 and 10). The components of the 26S proteasome, ubiquitin and several enzymes involved in the ubiquitin conjugation to target proteins were up-regulated at day 15 and even more markedly at the end of the study period. High levels of 26S protea- some non-ATPase regulatory subunit 14 (POH1), were Our transcriptomic analyses suggest, as expected, that early salmon aSVF culture contains a number of cell types, including vascular cells, macrophages and lym- phocytes, in addition to preadipocytes. Similar cell com- position of aSVF was reported in mammals [38]. The gene expression profiles at days 1 and 4 implied reten- tion of multipotency of aSVF cells during the early Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 7 of 17 Figure 6 Immune genes analyzed with real-time qPCR. In this and other panels data are shown as -ΔΔCt ± SE (n = 5). Significant difference from day 1 (t-test, p < 0.05) is indicated with . TNFa - tumour necrosis factor alpha, TNF decoy - tumour necrosis factor decoy receptor, FLAP - 5-lipoxygenase-activating protein, JAK1 - janus kinase 1, PD-L1 - programmed cell death ligand 1, IL-13 a2 - interleukin 13, LECT2 - leukocyte cell- derived chemotaxin a2, C5 - complement component 5. stages of the culture. Decrease in gene expression of markers of osteo/chondrogenic myogenic immune and WAT population of stem cells and early events in th determination phase are much less studied than th Figure 6 Immune genes analyzed with real-time qPCR. In this and other panels data are shown as -ΔΔCt ± SE (n = 5). Significant difference from day 1 (t-test, p < 0.05) is indicated with . TNFa - tumour necrosis factor alpha, TNF decoy - tumour necrosis factor decoy receptor, FLAP - 5-lipoxygenase-activating protein, JAK1 - janus kinase 1, PD-L1 - programmed cell death ligand 1, IL-13 a2 - interleukin 13, LECT2 - leukocyte cell- derived chemotaxin a2, C5 - complement component 5. Figure 6 Immune genes analyzed with real-time qPCR. In this and other panels data are shown as -ΔΔCt ± SE (n = 5). Significant difference from day 1 (t-test, p < 0.05) is indicated with *. Discussion Once liber- ated from the endothelial cell, pericyte is activated and can be considered a stem cell [42]. Characterisics of the Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 8 of 17 Figure 7 Cell lineage markers analyzed with real-time qPCR. PPARg - peroxisome proliferator-activated receptor g, MyoD - myogenic factor D, BMP4 - bone morphogenic protein 4, 1C. C/EBP - CCAAT/enhancer-binding proteins a, b and δ, LPL - lipoprotein lipase, FAS - fatty acid synthase. Figure 7 Cell lineage markers analyzed with real-time qPCR. PPARg - peroxisome proliferator-activated receptor g, MyoD - myogenic factor D, BMP4 - bone morphogenic protein 4, 1C. C/EBP - CCAAT/enhancer-binding proteins a, b and δ, LPL - lipoprotein lipase, FAS - fatty acid synthase. Figure 7 Cell lineage markers analyzed with real-time qPCR. PPARg - peroxisome proliferator-activated receptor g, MyoD - myogenic factor D, BMP4 - bone morphogenic protein 4, 1C. C/EBP - CCAAT/enhancer-binding proteins a, b and δ, LPL - lipoprotein lipase, FAS - fatty acid synthase. mesenchymal lineages. Starting from day 6 we observed consistently decreased expression of MyoD, an early marker of the myogenic lineage. MyoD regulates the cell cycle in terminally differentiated myocytes by inducing CDK inhibitor p21, which irreversibly arrests prolifera- tion [43]. MyoD may play a similar role in mature adi- pocytes and in theory this could account for the increased expression at day 30. Activation of BMP4 from day 9 was an additional evidence for the inhibition of myogenesis. This growth factor equally promotes dif- ferentiation of the adipose, cartilage or bone lineages depending on the culture conditions [44,45]. Therefore, activation of BMP4 is necessary but not sufficient for the commitment of preadipocytes. PPARg is a master evidence of the pericytic identity of adipocytes’ precur- sors in our model since Tang et al. confirmed PPARg as a specific marker of the perivascular fraction in subcon- fluent aSVF [9]. Indeed, our finding of high levels of PPARg mRNA well before confluence contradicts the cell culture studies reviewed in [18] as the expression of C/EBPb and C/EBPδ predate the expression of PPARg and C/EBPa [17,18], which then activate adipocyte-spe- cific genes during terminal differentiation. This illus- trates the advantage of primary cell cultures, which offer possibilities to gain valuable insight into the early mole- cular events of adipogenic differentiation. Commitment of multipotent MSCs to the adipogenic lineage involves simultaneous inhibition of other Todorčević et al. Discussion BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 9 of 17 Figure 8 Genes for enzymes from different pathways of carbohydrate and lipid metabolism analyzed with real-time qPCR. HK hexokinase, G6PD - glucose-6-phosphate dehydrogenase, PFK - phosphofructokinase, PK - pyruvate kinase, MDHC - malate dehydrogenase, PEPCKC - phosphoenolpyruvate carboxykinase C, PGD - 6- phosphogluconate dehydrogenase, TKT - transketolase, MTP - microsomal triglycerid transfer protein, CD36 - cluster of differentiation 36, ATP binding - ATP-binding cassette transporter 2. Figure 8 Genes for enzymes from different pathways of carbohydrate and lipid metabolism analyzed with real-time qPCR. HK - hexokinase, G6PD - glucose-6-phosphate dehydrogenase, PFK - phosphofructokinase, PK - pyruvate kinase, MDHC - malate dehydrogenase, PEPCKC - phosphoenolpyruvate carboxykinase C, PGD - 6- phosphogluconate dehydrogenase, TKT - transketolase, MTP - microsomal triglyceride transfer protein, CD36 - cluster of differentiation 36, ATP binding - ATP-binding cassette transporter 2. Figure 8 Genes for enzymes from different pathways of carbohydrate and lipid metabolism analyzed with real-time qPCR. HK - hexokinase, G6PD - glucose-6-phosphate dehydrogenase, PFK - phosphofructokinase, PK - pyruvate kinase, MDHC - malate dehydrogenase, PEPCKC - phosphoenolpyruvate carboxykinase C, PGD - 6- phosphogluconate dehydrogenase, TKT - transketolase, MTP - microsomal triglyceride transfer protein, CD36 - cluster of differentiation 36, ATP binding - ATP-binding cassette transporter 2. Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 10 of 17 Figure 9 Genes for antioxidants and proteins involved in unfolded protein responses analyzed with real-time qPCR. PRDX1 - peroxiredoxin 1, GPx2 - glutathione peroxidase 2, ATF6 - activating transcription factor 6, XBP1 - x-box binding protein 1, GRP78 - glucose- regulated protein 78, 18S. Figure 9 Genes for antioxidants and proteins involved in unfolded protein responses analyzed with real-time qPCR. PRDX1 - peroxiredoxin 1, GPx2 - glutathione peroxidase 2, ATF6 - activating transcription factor 6, XBP1 - x-box binding protein 1, GRP78 - glucose- regulated protein 78, 18S. Figure 9 Genes for antioxidants and proteins involved in unfolded protein responses analyzed with real-time qPCR. PRDX1 - peroxiredoxin 1, GPx2 - glutathione peroxidase 2, ATF6 - activating transcription factor 6, XBP1 - x-box binding protein 1, GRP78 - glucose- regulated protein 78, 18S. and earliest increased level of the anti-osteogenic GAS6 was observed at day 15. adipogenic regulator, which simultaneously inhibits myogenesis, osteogenesis and chondrogenesis [44,46,47]. Discussion Biosynthesis of carbohydrates in carnivourous fish species could be important due to the very low carbohydrate dietary level in their natural diets [51]. Indeed, we have seen regula- tions in all major glucose metabolic pathways in our model (Fig. 8); however, differentiation of adipocytes did not involve induction of HK, one of the key enzymes of glucose metabolism that only showed slight up-regula- tion at day 30. This indirectly suggested that the trans- formation of gluconeogenic amino acids into glucose is important in adipocytes of Atlantic salmon, at least in vitro. In addition, late activity of two key glyceroneo- genic genes, MDHC and PEPCKC implied that glycero- neogenesis, which can be fed with mitochondrial intermediates derived from lactate and gluconeogenic amino acids, in addition to pyruvate, was active in devel- oping fish adipocytes. To conclude, these findings illu- minate potentially novel aspects of nutrient metabolism in adipocytes, which might take advantage of the cou- pling of glycolysis, the PPP and glyceroneogenesis, in order to fulfil shifting demands for the three major pro- ducts of these pathways: G3P, R5P and NADPH. Accumulation of lipids in adipocytes coincided with the activation of genes coding for secreted proteins, such as adipokines adipsin and visfatin. Together with the previous finding of leptin secretion by Atlantic sal- mon adipocytes [10], this supports the notion of WAT as an active endocrine organ in fish. Enhanced secretion may impose an additional load on the ER actively engaged in lipid droplet synthesis [55]. The ER compart- ment is suspected to exert a great deal of control over adipogenesis and association of obesity with ER-stress is firmly established [56]. Up-regulation of XBP1 and ATF6 at day 15 in salmon adipocytes implied activation of UPR in response to perturbations in the ER homeos- tasis [57]. Moreover, endoplasmic GRP78, which is located downstream from ATF6 in the UPR signalling cascade was down-regulated throughout the whole stu- died period but increased in expression at day 30. A number of changes that are typical of UPR [35] was a hallmark of the latest stages of adipogenesis in our model. UPR as a collection of pathways aimed at restor- ing ER function may serve different adaptive roles and is observed both under pathological and normal physiolo- gical situations [58]; for example, it is part of the devel- opmental program in highly specialized secretory B and T cells [59] and ß-pancreatic cells [60]. Discussion Thus, high expression of PPARg observed long before confluence with the peak at day 6 probably posed a bar- rier to the induction of non-adipogenic mesenchymal cell lineages. Non-adipogenic developmental pathways are expected to be strongly suppressed by the addition of hormones. Yet, we did not find any noticeable changes in the markers of different cell types after day 9 Our PCNA results suggested the highest proliferative activity during the first days of culture followed by a moderate reduction of mitotic activity at day 6 and much greater decrease by day 30. Overall, the expres- sion patterns of genes involved in cell cycle were in concordance with these changes. Highest levels of the key negative cell cycle regulators (e.g. RBL1, p53-like, GADD45g) were observed during days 15-30. An addi- tional evidence for the anti-proliferative status was the up-regulation of several pro-apoptotic genes, including retinoic acid-regulated apoptosis-related protein 3 (APR3). APR3 arrests cell cycle by suppressing the activity of cyclin D1 [48], which was also induced at the latest stage. Differentiation of adipocytes in mam- mals is associated with a reduction in proliferation at confluence followed by subsequent activation or clonal expansion and eventually with cell cycle arrest at term- inal differentiation [49]. Our results revealed the diver- sity of mechanisms potentially employed in the execution of the similar stage of events that occur dur- ing Atlantic salmon adipogenesis. Given that WAT is a highly specialized organ that functions to regulate Figure 10 Ribosomal proteins analyzed with microarrays. Data are mean log-(ER) ± SEM of twenty and ten genes involved in 60S and 40S subunit assembly respectively. Figure 10 Ribosomal proteins analyzed with microarrays. Data are mean log-(ER) ± SEM of twenty and ten genes involved in 60S and 40S subunit assembly respectively. Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 11 of 17 Page 11 of 17 NADPH produced by PPP is also crucially important for the cellular antioxidant defence since it is required for the regeneration of oxidized glutathione by glu- tathione reductase (GSR). Up-regulation of GSR and a suite of genes involved in metabolism of glutathione, thioredoxin and iron was a prominent feature of late salmon adipocyte differentiation (days 15-30). Indeed, a recently introduced concept [52-54,4] clearly demon- strated ROS as anti-adipogenic molecules that inhibit preadipocyte proliferation and differentiation. Discussion A reduced redox state in WAT is now recognised as a distinct characteristic of visceral obesity in mammals, with over- expression of GPX peroxidases and high content of hydrophilic antioxidant gluthathione associated with pro-adipogenic processes. Induction of the number of GPXs, known to be sensitive to lipid peroxidation, implied ROS-provoked response in our culture and the need to maintain highly reduced state of the intracellu- lar environment. However, contrary to what could be expected in situations of high ROS production, neither gene expression nor activity of SOD increased. In fact, the activity of this important ROS scavenger progres- sively declined during adipocyte differentiation. Thus, our data corroborated the indespensible role of glu- tathione-based antioxidant system in the maintenance of the reduced intracellular state in white adipocytes of fish. metabolic homeostasis and energy balance, nutrient sensing mechanisms could be opted for a role in the control of proliferation. Growth cycle arrest of salmon adipocytes was in parallel with the up-regulation of the fatty acid binding protein 3 (FABP3) that participates in the uptake, intracellular metabolism and transport of long chain fatty acids. FABP3 seems like a good candidate that could be involved in this type of regula- tion since its growth arrest activity was previously demonstrated in mammalian epithelial cells [50]. Further, minimizing protein synthesis may also control proliferation as cell cycle arrest is a well established consequence of the general translational arrest, implied from day 15 onwards. Microarray analyses suggested changes in nutrient metabolism in the late phase of adipogenesis (day 30). As a consequence of the up-regulation of pyruvate dehy- drogenase kinase that inactivates pyruvate dehydrogen- ase, the Krebbs cycle most likely relied on the preferential use of acetyl-CoA from FA oxidation rather than pyruvate to cover the cellular energy requirements. Also, the expression of genes involved in FA b-oxida- tion, acyl-Coenzyme A dehydrogenase and acyl-Coen- zyme A binding protein, steadily increased towards the end of adipogenesis. The up-regulation of G6PD and PGD pointed to the importance of the PPP, which gen- erates NADPH required for accumulation of lipids. The PPP can be seen as an alternative to glycolysis, because in addition to generating NADPH and R5P, it can also provide cells with G3P, the phosphorylated glycerol backbone suitable for TAG synthesis. Induction of gly- colytic PFK was also in line with the increased produc- tion of G3P and TAG biosynthesis. Discussion The revealed UPR in developing fish adipocytes appears as a highly tailored homeostatic mechanism that is activated when Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 12 of 17 Page 12 of 17 receptors, TNFa and a panel of TNF-related genes could be explained by the changes in relative abundance of non-adipogenic cells that possess pronounced immune properties. In mammals, TNFa has high anti- adipogenic activity [64], which was recently shown to be conserved in fish [65]. Therefore, down-regulation of the TNF axis at days 7-9 was probably important for the onset of adipocyte differentiation. However, a number of immune genes showed either stable increase since day 4 (complement component C5) or biphasic regulation with the second activation after hormone induction (LECT2). Highest expression levels of many genes with diverse roles in different inflammatory pathways were observed during terminal adipocyte differentiation. Numerous independent studies have provided evidence that a set of inflammatory and stress-response genes is activated in obesity in mammals (reviewed in [66]). Some of these genes likely have important roles in the coordination of homeostasis in WAT, similar to the established roles of TNFa in modulating proliferative abilities in preadipocytes or metabolic activities in mature adipocytes. increased number and size of LDs meat the limited ER capacity. Although ER is mainly considered to be a pro- tein-folding factory, all proposed models of LD forma- tion emphasize the engagement of the ER compartment in the process [61]. UPR utilises two broad strategies to relieve stress in the ER: increased clearance of misfolded proteins from the ER and reduction of new protein influx into the ER. We found ample evidence for the activation of the 26S proteasome and autophagy-lysosomal proteolytic path- ways, which degrade misfolded proteins. Components of the membrane protein complex, derlin 1 and p97-inter- acting cofactor p47, which mediate transport from the ER lumen into the cytosol were induced at day 30, while other proteolytic pathways were up-regulated even ear- lier. UPR-induced proteolysis may be employed to initi- ate the breakdown of superfluous proteins and organelles that must be replaced by more highly specia- lised cellular components during the adipocyte develop- ment. Without a doubt, considerable remodelling of the cellular architecture occurs during terminal differentia- tion of Atlantic salmon adipocytes. Discussion An intriguing example is a group of virus responsive genes including TLR3, a receptor of double stranded RNA and a group of galectins, previously not reported to have roles in any aspect of adipocyte biology. The lat- ter includes galectin 9, which showed highly specific responses to viruses in previous studies from our group [67,68]. However, a large portion of immune genes, including complement component C5, JAK1, the key actor of interferon signaling and FLAP, the regulator of eicosanoid metabolism, decreased expression by the time of AP1 establishment. Finally, our results do not permit for major conclusions as to whether immune genes are important for adipogenesis or change expres- sion as a consequence of differentiation and/or stress responses. However, it is clear that Atlantic salmon WAT possesses high potential for immune activity. UPR also activates genes involved in protein folding. Hsp40s and other J-domain-containing proteins act as co-chaperones for Hsp70s helping to restore homeosta- sis in the ER and in addition may have pro-degradation roles. Down-regulation of translational machinery is commonly observed during UPR [35], and in our study, it involved a dramatic reduction in expression of riboso- mal proteins. Alteration of the ribosomal composition is most cer- tainly involved in the multiple regulation of the ER- stress response. The curious up-regulation of the 40S subunit 18S rRNA through most of the terminal differ- entiation against the majority of down-regulated riboso- mal proteins is bound to have important consequences given the high level of 18S constitutive expression. This could signify a switch to a less efficient protein transla- tion as increase in 18S negatively affects the ribosomal subunit ratio that favours protein production [62]. On the other side, reduced expression of 18S at the very end of differentiation could improve the effectiveness of protein synthesis in adipocytes that acquire secretory phenotype. In conclusion, fully matured adipocytes seem to reprogram the pattern of gene expression to sustain a certain level of protein production, congruent with the now recognised endocrine functions of WAT. It is tempting to explain the activation of immune genes in differentiating adipocytes as a side effect of adaptation to the oxidative and ER stress, since stress and immune responses share common regulatory path- ways. Ozcan et al. [56] showed that ER-stress-induced JNK-AP1 axis is the central link between TAG overload in liver and diabetes. Discussion Most interestingly, this is in agree- ment with our study, which shows that the same players are activated in fish adipocytes in response to increased lipid deposition. Continuous administration of lipids led to the maximal observed TAG loading at the end of the studied period, when the collective and highest expres- sion of the AP1 complex components, including all c- Jun and JunB members was also observed. This addi- tionally coincided with the induction of POH1, a regula- tory subunit of the 26S proteasome that has a specific Secreted adipsin and visfatin have multiple immune functions as most other adipokines [63]. Overall, expres- sion changes of a large number of immune genes were characteristic of Atlantic salmon aSVF culture. Dissimi- lar temporary profiles suggest different regulatory and physiological roles of these genes. Additionaly, high initial levels and subsequent decrease of the chemokine Page 13 of 17 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 13 of 17 laminin coated cell-culture flasks at a density of approximately 10 g tissue/25 cm2. The cells were kept at 13°C and media were changed every 3 days. Cells reached confluence after approximately 1 week. Conflu- ent cells were cultivated for 48 h in an differentiation inducing medium, i.e. growth medium supplemented with 1 μM dexamethasone, 33 μM biotin, 10 nM triio- dothyronine, 17 μM panthothenate and 25 μM isobutyl- methylxanthine, 20 μg/ml insulin and a lipid mixture (1 μl/ml; corresponding to 45 mg/ml cholesterol, 100 mg/ ml cod liver oil FA (methyl esters). After that cells were transferred to a maintenance differentiation media con- taining growth media only supplemented with 2 μl/ml of lipid mixture. Media was changed every 3 days until the cells reached the final differentiation stage with mor- phology of mature adipocytes (day 30). de-ubiquitinase activity towards c-Jun leading to its accumulation and subsequent increase in AP1-mediated gene expression [66]. A suite of highly expressed immune genes at this time supported an inflammatory type reaction in adipocytes. Hence, an unrelieved ER- stress could likely be the major cause of chronic inflam- matory responses in lipid overloaded white adipocytes in fish. Electron microscopy Cells for electron microscopy studies were taken from cultures at days 15 and 30. Cells were washed in 0.1 M PBS (pH 7.4), then fixed in 2% glutar aldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 24 h. The cells were the harvested, rinsed in 0.1 M cacodylate buffer and post-fixed for 60 min in 2% OsO4 containing 1.5% potassium ferrocyanide, followed by en bloc staining with 1.5% uranyl acetate. Cells were dehydrated in a ser- ies of ethanol solutions (70%, 90%, 96%, and 100%) and propylene oxide, and then embedded in epon resin, which was polymerized at 60°C for 24 h. Ultrathin sec- tions (approximately 50 nm) were cut on a Reichert Ultracut E ultramicrotome using a diamond knife. The sections were placed onto formvar/carbon-coated 75- mesh copper grids, post-stained for 2 min with 0.2% lead citrate solution in 0.1 M NaOH, and examined in a Philips CM 100 transmission electron microscope at an accelerating voltage of 80 kV. Preadipocyte isolation and culture conditions Preadipocyte isolation and culture conditions Atlantic salmon was reared at Nofima Marin station at Averøy, Norway on a commercial diet to weight of 2-3 kg. Fish were anaesthetized with metacain (MS-222), bled by cut of arch bows of the gills and killed by a blow to the head. The experiment was conducted according to the National Guidelines for Animal Care and Welfare of the Norwegian Ministry of Research. Visceral adipose tissue was excised and salmon preadi- pocytes were isolated as described in Vegusdal et al. [10]. Briefly, the dissected fat tissue was washed with phosphate buffered saline (PBS, pH 7.4) (unless other- wise stated, all chemicals were obtained from Sigma- Aldrich Chemical Co., St. Louis, MO, USA), minced and digested in 0.1% collagenase (type I) in HBSS (1 g tis- sue/5 mL HBSS) at 13°C for 1 h under shaking and fil- tered through 250 and 100 μm nylon. The resulting cell suspension was then centrifuged at 700 × g for 10 min at 10°C. The buoyant fat layer with mature adipocytes was removed by aspiration, while the preadipocytes were pelleted on the bottom. After washing twice, the cells were resuspended in growth medium containing L- 15, 10% fetal bovine serum (FBS), 2 mM L glutamine, 10 mM HEPES, and antibiotics (mixture of penicillin, streptomycin and amphotericin B) and seeded on Conclusions Our study revealed concordance between the gene expression profiles and the key events during adipogenic development of the primary culture of aSVF cells, including fluctuations of proliferative activity, induction of adipogenic differentiation and suppression of other mesenchymal cell lineages and final tuning of metabo- lism towards production and accumulation of lipids. For the first time in a fish species we show that the estab- lishment of mature adipocyte phenotype is associated with high activity of immune genes, activated UPR and responses to oxidative stress. These changes are likely to be part of the normal adipocyte development but may be accentuated when cells are overloaded with lipids. High expression of pro-inflammatory mediators imply that excessive growth of WAT in fish may cause dis- turbed endocrine function with possible negative effects on health, as seen in mammals. Microarray analyses h l f BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Table 1 Primers used for real-time qPCR an Gene Tumor necrosis factor alpha (TNFa) Tumor necrosis factor (TNF) decoy Arachidonate 5-lipoxygenase-activating protein (FLAP) Tyrosine-protein kinase 1 (Jak1) Programmed death ligand 1 (PD-L1) Interleukin 13 receptor alpha 2 (IL13Ra2) Leukocyte cell-derived chemotaxin 2 (LECT2) Complement component 5 (C5) Proliferator-activated receptor gamma (PPARg) Bone morphogenic protein 4 (BMP4) Myogenic factor D (MyoD) CCAAT-enhancer-binding protein beta (C/EBPb) CCAAT-enhancer-binding protein delta (C/EBPδ) CCAAT-enhancer-binding protein alpha (C/EBPa) Hexokinase (HK) Glucose-6-phosphate dehydrogenase (G6PD) Phosphofructokinase (PFK) Pyruvate kinase (PK) Malate dehydrogenase cytoplasmic (MDHC) Cytosolic phosphoenolpyruvate carboxykinase (PEPCKC) 6-phosphogluconate dehydrogenase (PGD) Transketolase (TKT) Peroxiredoxin 1 (PRDX1) Glutathione peroxidase 2 (GPX2) Activating transcription factor 6 (ATF6) X-box binding protein 1 (XBP1) Microsomal triglyceride transfer protein (MTP) 78 kDa glucose-regulated protein (GRP78) Cluster of differentiation 36 (CD36) ATP-binding cassette transporter 2 Lipoprotein lipase (LPL) Fatty acid(FAS) 18S Elongation factor 1A (EF1A) Eukaryotic translation initiation factor 3 subunit 6 Table 1 Primers used for real-time qPCR Gene Tumor necrosis factor alpha (TNFa) Tumor necrosis factor (TNF) decoy Arachidonate 5-lipoxygenase-activating protein (FLAP) Tyrosine-protein kinase 1 (Jak1) Programmed death ligand 1 (PD-L1) Interleukin 13 receptor alpha 2 (IL13Ra2) Leukocyte cell-derived chemotaxin 2 (LECT2) Complement component 5 (C5) Proliferator-activated receptor gamma (PPARg) Bone morphogenic protein 4 (BMP4) Myogenic factor D (MyoD) CCAAT-enhancer-binding protein beta (C/EBPb) CCAAT-enhancer-binding protein delta (C/EBPδ) CCAAT-enhancer-binding protein alpha (C/EBPa) Hexokinase (HK) Glucose-6-phosphate dehydrogenase (G6PD) Phosphofructokinase (PFK) Pyruvate kinase (PK) Malate dehydrogenase cytoplasmic (MDHC) Cytosolic phosphoenolpyruvate carboxykinase (PEPCKC) 6-phosphogluconate dehydrogenase (PGD) Transketolase (TKT) Peroxiredoxin 1 (PRDX1) Glutathione peroxidase 2 (GPX2) Activating transcription factor 6 (ATF6) X-box binding protein 1 (XBP1) Microsomal triglyceride transfer protein (MTP) 78 kDa glucose-regulated protein (GRP78) Cluster of differentiation 36 (CD36) ATP-binding cassette transporter 2 Lipoprotein lipase (LPL) Fatty acid(FAS) 18S Elongation factor 1A (EF1A) Eukaryotic translation initiation factor 3 subunit 6 Table 1 Primers used for real-time qPCR analyses Gene Forward primer (5’-3’) Reverse primer (5’-3’) Accession number Tumor necrosis factor alpha (TNFa) AGGTTGGCTATGGAGGCTGT TCTGCTTCAATGTATGGTGGG AF321836 Tumor necrosis factor (TNF) decoy TCTCCTGGTATTTGCGCTCTGTGGT TATAAGTCGGTGTGTGAGCGCCTGA CA351440 Arachidonate 5-lipoxygenase-activating protein (FLAP) TCTGAGTCATGCTGTCCGTAGTGGT CCTCCCTCTCTACCTTCGTTGCAAA CA369467 Tyrosine-protein kinase 1 (Jak1) GAGGAGTTTGTCCAGTTCGGTCCGT CATGCACCAGCTTCTTATCCTCCAG CA368994 Programmed death ligand 1 (PD-L1) TCAACGACTCTGGGGTGTACCGATG TCCACCTCATCTCCACCACGTCTC CA366631 Interleukin 13 receptor alpha 2 (IL13Ra2) TCTCTGAGCCGCTCAACCTGTCAT CGTTCCACGACAGCTTTATACGGA CA348044 Leukocyte cell-derived chemotaxin 2 (LECT2) CTGTGTTGTCAGAGTGCGAGATGGT TACACACAATGTCCAGGCCCTGA EXOB2 Complement component 5 (C5) AGAACTCTTCCGAGTTGGCATGGT AGTGATGCTGGGATCCATCTCTGA CA364804 Proliferator-activated receptor gamma (PPARg) CGTGTATCAAGACGCCAGCT TTGCAGCCCTCACAGACATG EU655708 Bone morphogenic protein 4 (BMP4) TCAAGTTGCCCATAGTCAGT CACCTGAACTCTACCAACCA FJ195610 Myogenic factor D (MyoD) CCGCAACACGAAGCAACTATTACAGC GGAACCCTCCTGGCCTGATAACAC AJ557150 CCAAT-enhancer-binding protein beta (C/EBPb) CAAACTACATTACCAGGC GTTATGTGTTGCCAGTTG EU668996 CCAAT-enhancer-binding protein delta (C/EBPδ) TTGGGCGGTGGAGCCTAT TTTCCTCGCCCGTGTCAT EU668997 CCAAT-enhancer-binding protein alpha (C/EBPa) AGACCTCGGCGAGATTTGT TGTGGAATAGATCAGCCAGGAA EU668995 Hexokinase (HK) GCTGAAGACCAGAGGCATCTTTGA GCTGCATACCTCCTTGACGATGAT AY864082 Glucose-6-phosphate dehydrogenase (G6PD) TGGTGCAGAACCTCATGGTCCTCA ATCCCGGATGATTCCAAAGTCGTC BT044902 Phosphofructokinase (PFK) AATCCATCGGCGTTCTGACAAGC GCCCGTACAGCAGCATTCATACCTT BT059256 Pyruvate kinase (PK) TGCCTTCATTCAGACGCAGCA CAGATGATTCCGGTGTTGCGA BT043851 Malate dehydrogenase cytoplasmic (MDHC) AGACGTCCACCACTGCAAGGTCAA TTAACAGCGTCGAAGCAGGCCA BT043497 Cytosolic phosphoenolpyruvate carboxykinase (PEPCKC) AGGGCATGGACCAGGAACTCC GGGCTCTCCATCCTGGGATGT BT072418 6-phosphogluconate dehydrogenase (PGD) CCAATGAGGCTAAAGGCACCAAGA CCAGCTTGTCGATGAAGTCATCCA BT050391 Transketolase (TKT) TGCCATCTCCGAGAGCAACATC CCGTGGGAATGGCTCTGAACAT BT059642 Peroxiredoxin 1 (PRDX1) CACTGCTGTGGTGGATGGACAGTT CCAGCGGGTAGAAGAAGAACACCA est02b08 Glutathione peroxidase 2 (GPX2) TGTACCTCAAGGAGAAGCTGCCGT ATTAAGGCCATGGGATCGTCGC est04e05 Activating transcription factor 6 (ATF6) CTCACACCATCAAAGCTACAGCGA GTGTCGCCTCGTCGATTTAACTCA CA367172 X-box binding protein 1 (XBP1) CGACTCAAATTCGACAACTGGGC TTTCTGTCTCTGGCTGTCTGGGCT CA385697 Microsomal triglyceride transfer protein (MTP) CAAAGACCAGCGTCAACAACAA CGCCTCTGTCTCAAAGCTCACT CA042356 78 kDa glucose-regulated protein (GRP78) GTGCAGCATGACATCAAGTA CTCTTCTTCTCGATAACCTT CA368961 Cluster of differentiation 36 (CD36) GGATGAACTCCCTGCATGTGA TGAGGCCAAAGTACTCGTCGA AY606034 ATP-binding cassette transporter 2 AGCGGGGGAAACAGTAGCAGGA GCCTGGTCTTGAGATTGTGGGTGT CA343913 Lipoprotein lipase (LPL) TGCTGGTAGCGGAGAAAGACAT CTGACCACCAGGAAGACACCAT BI468076 Fatty acid(FAS) TGCCTCAGCACCCTACTCTG GCTTTACAACCTCAGGATTGGC BT048827 18S TGTGCCGCTAGAGGTGAAATT GCAAATGCTTTCGCTTTCG AJ427629 Elongation factor 1A (EF1A) TGCCCCTCCAGGATGTCTAC CACGGCCCACAGGTACTG BG933853 Eukaryotic translation initiation factor 3 subunit 6 GTCGCCGTACCAGCAGGTGATT CGTGGGCCATCTTCTTCTCGA CX040383 Table 1 Primers used for real-time qPCR analyses Gene Forward CCAATGAGGCTAAAGGCACCAAGA TGCCATCTCCGAGAGCAACATC CACTGCTGTGGTGGATGGACAGTT TGTACCTCAAGGAGAAGCTGCCGT CTCACACCATCAAAGCTACAGCGA CGACTCAAATTCGACAACTGGGC CAAAGACCAGCGTCAACAACAA GTGCAGCATGACATCAAGTA GGATGAACTCCCTGCATGTGA AGCGGGGGAAACAGTAGCAGGA TGCTGGTAGCGGAGAAAGACAT TGCCTCAGCACCCTACTCTG TGTGCCGCTAGAGGTGAAATT TGCCCCTCCAGGATGTCTAC GTCGCCGTACCAGCAGGTGATT hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), and twice in 0.06 × SSC (2 and 1 min, respectively). Microarray analyses h l f The samples for microarrays analyses were taken at days 1, 4, 7, 9, 12 and 30. The salmonid fish microarray (SFA2, immunochip) includes 1800 unique clones printed each in six spot replicates. Total RNA was extracted by using RNeasy® Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instruction. RNA was treated with RNase-free DNase I (Qiagen, Valencia, CA, USA), to remove any contaminating DNA. All RNA samples used in our experiments had A260/280 ratios between 1.80 and 2.30. The total RNA concentration was determined at 260 nm using spectro- photometry. Equal inputs from 5 cell flask replicates were pooled for each time point. Equalised control was made by mixing RNA from each time point. The test and control samples (15 μg RNA in each) were labelled with respectively Cy5-dUTP and Cy3-dUTP (Amersham Pharmacia, Little Chalfont, UK). The fluorescent dyes were incorporated in cDNA using the SuperScript™ Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 14 of 17 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 14 of 17 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. Microarray analyses h l f [70] on days 1, 4, 7, 9, 15 and 30. Briefly, cells were washed twice with PBS, fixed with 10% cold formalin for 30 min, rinsed in water and stained for 2 h with fil- tered oil red O in isopropanol at room temperature. For relative quantitative measurements of TAGs accumula- tion, bottles were washed with PBS to remove excess of stain solution, dried, the colour was dissolved in 100% isopropanol and the absorbance was measured spectro- photometrically in a Victor 3 microplate reader Perki- nElmer (Wellesley, MA, USA) at 500 nm. Author details 1 1Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, P. O. Box 5010, Ås NO-1430, Norway. 2Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, Ås NO-1432, Norway. Acknowledgements W h k I Ø K i g We thank Inger Ø. Kristiansen for her skilful technical assistance and Turku Centre of Biotechnology for the preparation of microarrays. This study was carried out with support from Norwegian Research Council (NRC). y Superoxide dismutase assay SOD activity was assayed with a kit (Cayman Chemicals, Ann Arbor, MI, USA), which utilizes a tetrazolium salt to detect superoxide radicals generated by xanthine oxi- dase and hypoxanthine. Colour was measured at 405 nm in a Titertek Multiskan PLUS MKII (Labsystems, Helsinki, Finland) plate reader. One unit of SOD was defined as the amount of enzyme needed to achieve 50% dismutation of the superoxide radical. Cell proliferation Cell proliferation was assessed by the immunocytochem- ical detection of PCNA)(Zymed Laboratories, South San Francisco, CA, USA). Cells were washed in PBS and fixed in 70% ethanol for 30 minutes at 4°C. Endogenous peroxidise activity was blocked with with 3% hydrogen peroxide in methanol for 10 min. The cells were washed three times with in PBS, then incubated with a mouse anti PCNA monoclonal antibody (clone PC10) using a PCNA immuno detection kit, following the manufac- turer’s instructions. The cells were counterstained with Mayer’s haematoxylin for 2 min, washed in water, dehy- drated in a graded series of alcohol solutions, cleared with xylene, and mounted with Histomount. PCNA-con- taining nuclei were stained dark brown. Two hundred cells were observed and the percentage of proliferating cell was calculated. Received: 31 July 2009 Accepted: 17 January 2010 Published: 17 January 2010 Authors’ contributions MT and BR contributed to the overall experimental design. SS and AK carried out microarray analyses. MT carried out cell culture work, and assay analyses. MT and SS carried out qPCR analyses and produced the first manuscript draft. All authors read, contributed to, and approved the final manuscript. Microarray analyses h l f Scanning was performed with Axon GenePix 4100A and images were processed with Gene- Pix 6.0 (Molecular Devices, Sunnyvale, CA, USA). The spots were filtered by criterion (I-B)/(SI+SB) ≥0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analysis and genes presented with less than three high quality spots on a slide were discarded. After subtraction of median Indirect cDNA Labelling System (Invitrogen, Carlsbad, CA, USA). The cDNA synthesis was performed at 46°C for 3 h in a 20 μl reaction volume, following RNA degradation with 0.2 M NaOH at 37°C for 15 min and alkaline neutralization with 0.6 M Hepes. Labelled cDNA was purified with Microcon YM30 (Millipore, Bedford, MA, USA). The slides were pretreated with 1% BSA fraction V, 5 × SSC, 0.1% SDS (30 min at 50°C) and washed with 2 × SSC (3 min) and 0.2 × SSC (3 min) and hybridized overnight at 60°C in a cocktail con- taining 1.3 × Denhardt’s, 3 × SSC 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After Page 15 of 17 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Lipid accumulation in adipocytes Lipid accumulation in adipocytes Lipid accumulation in adipocytes background from median signal intensities, the expres- sion ratios were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. The differential expression was assessed by difference of the mean log- expression ratios between the slides with reverse label- ling (6 spot replicates per gene on each slide, Student’s t-test, p < 0.01). Complete microarray results are sub- mitted to NCBI GEO Omnibus (GSE18389). background from median signal intensities, the expres- sion ratios were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. The differential expression was assessed by difference of the mean log- expression ratios between the slides with reverse label- ling (6 spot replicates per gene on each slide, Student’s t-test, p < 0.01). Complete microarray results are sub- mitted to NCBI GEO Omnibus (GSE18389). p p y The quantity of lipids in cytoplasm were estimated by oil-red O staining according to Ramirez-Zacarias et al. Quantitative real-time RT-PCR RNA isolated at days 1, 4, 5, 6, 7, 9, 10, 11, 12, 15, 20, 23 and 30 was used for qPCR. Approximately 200 ng of total RNA was reverse-transcribed into cDNA using TaqMan® Gold RT-PCR Kit (Applied Biosystems, Foster City, CA, USA), a 25 μl reaction system according to the manufacturer’s protocol. The PCR primers (Table 1) were designed using the Vector NTI (Invitrogen, Carls- bad, CA, USA) and synthesized by Invitrogen. Efficiency was checked from tenfold serial dilutions of cDNA for each primer pair. A 2 × SYBR® Green PCR Mastermix (Roche Diagnostics, Mannheim, Germany), 0.4 μM of each primer, and the cDNA template were mixed in 12 μl volumes. PCR was performed in duplicates in 96-well optical plates on Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). Different controls were used for microarray and qPCR. All time points were compared relative to day 1 in qPCR analyses. Relative expression of mRNA was calculated using the ΔΔCt method. Three commonly used genes (18S, elongation factor 1A and eukaryotic translation initiation factor 3, subunit 6) were tested for stability using the GeNorm and NormFinder. Finally, elongation factor 1A (EF1A) met criteria of sta- bility in the analyzed material. Differences between con- trol and cells at different developmental stages were assessed with Student’s t-test (p < 0.05). References Rabinovich GA, Riera CM, Landa CA, Sotomayor CE: Galectins: a key intersection between glycobiology and immunology. Brazilian Journal of Medical and Biological Research 1999, 32:383-393. 11. Todorcevic M, Vegusdal A, Gjoen T, Sundvold H, Torstensen BE, Kjaer MA, et al: Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; Effects of n-3 and n-9 fatty acids. Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids 2008, 1781:326-335. 34. 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J Biol Chem 2004, 279:40462-40469. doi:10.1186/1471-2164-11-39 Cite this article as: Todorčević et al.: Gene expression profiles in Atlantic salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes. BMC Genomics 2010 11:39. References 70. Ramirez-Zacarias JL, Castro-Munozledo F, Kuri-Harcuch W: Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 1992, 97:493-497. doi:10.1186/1471-2164-11-39 Cite this article as: Todorčević et al.: Gene expression profiles in Atlantic salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes. BMC Genomics 2010 11:39. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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https://openalex.org/W2617832597	https://europepmc.org/articles/pmc5442327?pdf=render	English	null	Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice	Frontiers in cellular and infection microbiology	2,017	cc-by	8,612	Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Beneﬁts in C. gattii Infected Mice Jannyson J. Jandú 1, Marliete C. Costa 2, Julliana R. A. Santos 3, Fernanda M. Andrade 1, Thais F. Magalhães 2, Márcia V. Silva 1, Maria C. A. B. Castro 4, 5, Luanna C. B. B. Coelho 1, Aline G. Gomes 6, Tatiane A. Paixão 6, Daniel A. Santos 2 and Maria T. S. Correia 1* Jannyson J. Jandú 1, Marliete C. Costa 2, Julliana R. A. Santos 3, Fernanda M. Andrade 1, Thais F. Magalhães 2, Márcia V. Silva 1, Maria C. A. B. Castro 4, 5, Luanna C. B. B. Coelho 1, Aline G. Gomes 6, Tatiane A. Paixão 6, Daniel A. Santos 2 and Maria T. S. Correia 1* 1 Departamento de Bioquímica, Universidade Federal de Pernambuco, Recife, Brazil, 2 Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 3 Laboratório de Micologia, Universidade CEUMA, São Luís, Brazil, 4 Núcleo de Enfermagem, Universidade Federal de Pernambuco, Vitória de Santo Antão, Brazil, 5 Laboratório de Imunogenética, Centro de Pesquisas Aggeu Magalhães, Recife, Brazil, 6 Departamento de Patologia Geral, Instituto Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins puriﬁed from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with ﬂuconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with ﬂuconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reﬂex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inﬂammatory inﬁltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These ﬁndings point toward the use of pCramoll in combination with ﬂuconazole as a viable, alternative therapy for cryptococcosis management. ORIGINAL RESEARCH published: 24 May 2017 doi: 10.3389/fcimb.2017.00211 Citation: Keywords: cryptococcosis, immunomodulation, lectins, Cratylia mollis lectin, ﬂuconazole, survival Jandú JJ, Costa MC, Santos JRA, Andrade FM, Magalhães TF, Silva MV, Castro MCAB, Coelho LCBB, Gomes AG, Paixão TA, Santos DA and Correia MTS (2017) Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Beneﬁts in C. gattii Infected Mice. Front. Cell. Infect. Microbiol. 7:211. doi: 10.3389/fcimb.2017.00211 Edited by: Joao Santana Silva, University of São Paulo, Brazil Reviewed by: Karen L. Wozniak, University of Texas at San Antonio, United States Carlos Pelleschi Taborda, University of São Paulo, Brazil Correspondence: Maria T. S. Correia mtscorreia@gmail.com Edited by: Joao Santana Silva, University of São Paulo, Brazil Reviewed by: Karen L. Wozniak, University of Texas at San Antonio, United States Carlos Pelleschi Taborda, University of São Paulo, Brazil Correspondence: Maria T. S. Correia mtscorreia@gmail.com Received: 09 August 2016 Accepted: 09 May 2017 Published: 24 May 2017 Keywords: cryptococcosis, immunomodulation, lectins, Cratylia mollis lectin, ﬂuconazole, survival INTRODUCTION Cryptococcus gattii is a pathogenic fungus that aﬀects mainly immunocompetent individuals. The desiccated yeasts and spores are inhaled and enter the body via the respiratory system, ﬁnally infecting the central nervous system (CNS) and causing meningo-encephalitis (Thompson et al., 2012), which frequently has a poor prognosis. Meningitis and meningo-encephalitis are May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org pCramoll-Fluconazole Beneﬁts to Cryptococcosis Jandú et al. typical in HIV/AIDS and transplant-recipient patients, but also in apparently healthy individuals (Sharon et al., 2012). The main therapies for cryptococcosis treatment caused by C. gatti are ﬂuconazole (antifungal azole) and amphotericin B (antifungal polyene; Santos et al., 2012; Reichert-Lima et al., 2016). Fluconazole is used in cases of pulmonary diseases with mild to moderate symptoms. In severe infections, amphotericin B is recommended (associated or not with 5-Flucytosine) followed by a prolonged therapy with ﬂuconazole. Itraconazole, voriconazole, and other azoles are recommended when the use of ﬂuconazole is contraindicated or ineﬀective (Perfect et al., 2010). Resistance against antifungals (Zhai et al., 2013) enhances clinical failures and increases morbidity and mortality (Ghannoum and Rice, 1999). Furthermore, side eﬀects due to the use of amphotericin B lead to dose-dependent nephrotoxicity frequently associated with increased mortality, requiring monitoring of the renal function of patients. Altogether, these ﬁndings increase the demand for new therapies against cryptococcosis. in 70% ethanol and the ends of each bone were cut. Both bones were ﬂushed with 5 mL of cold RPMI 1640 (HyClone, LGC Biotecnologia) using a 5-mL syringe and a 25-gauge needle. The cell suspension was centrifuged for 5 min at 1,200 rpm at 4◦C and washed once with cold RPMI. Then, bone marrow cells were counted using a hemocytometer and the concentration was adjusted to 2 × 106 cells/mL for plating on tissue culture–treated petri dishes in BMM medium (RPMI supplemented with 30% L929 growth conditioning media, 20% bovine fetal serum [Gibco], 2 mM glutamine [Sigma], 25 mM HEPES pH 72, 100 units/mL of penicillin-streptomycin [Life Technologies]). Fresh media were added every 48 h. Bone marrow–derived macrophages (BMDMs) were collected on day 7 and used for subsequent experiments. Under these conditions, macrophages/monocytes progenitors will proliferate and diﬀerentiate into a homogenous population of mature BMDMs, obtained >90% pure live macrophages (Zhang et al., 2008; Bouwman et al., 2014; Bhattacharya et al., 2015; Heung and Hohl, 2016). INTRODUCTION For all analyses, the cells were infected with L27/01 yeasts cells (growth on Sabouraud Dextrose Agar (SDA) medium, for 48 h at 35◦C) opsonized with 10% murine serum in RMPI suspension (0.4 × 106 cells/mL), at the proportion of 5 macrophages: 1 C. gattii, under cell treatment with 1 or 5 µM pCramoll and incubated for 3 or 24 h at 37◦C under 5% CO2. Previous studies have shown that lectins from plants may have immunomodulatory eﬀects: augmented recruitment and activation of mononuclear and polymorphonuclear leukocytes, development of Th1, Th2, and Th17 response and stimulation of phagocytosis (da Silva and Correia, 2014). Cratylia mollis is a native forage plant endemic to the Semiarid Region of Brazil (Caatinga biome), and popularly known as camaratu bean. Four isolectins (Cramoll 1, 2, 3, and 4) can be puriﬁed from seeds of the plant. pCramoll (preparation containing isolectins 1 and 4) has binding sites for the speciﬁc recognition of glucose/mannose receptors and presents anthelmintic, antiprotozoal, antitumoral, healing, and immunomodulatory eﬀects (Maciel et al., 2004; Melo et al., 2011; da Silva et al., 2014). Focusing on immunoregulatory properties, the aim of this study was to evaluate the immunomodulatory eﬀect of pCramoll in a murine model of infection by C. gattii. Our results revealed that this lectin is able to increase survival, decrease fungal burden in organs and reduce the morbidity of cryptococcosis. The phagocytic index was performed using 24-well plates and a single coverslip 13 mm in diameter. After 3 h, these coverslips containing adherent cells were removed, stained with Giemsa and the index was calculated as the percentage of cells with internalized C. gattii 24 h post-infection (Santos et al., 2014). Images from the phagocytosis assay were obtain using a Nikon COOLPIX 4500 camera coupled to a Nikon ECLIPSE E200 microscope with 100 x of magniﬁcation and analyzed using the ImageJ Protocol (http://rsbweb.nih.gov/ij/); the cell area (average of 100 cells per coverslip) was measuring as described by Baviskar (2011). The intracellular proliferation rate (IPR) assay was performed as previously described (Ma et al., 2009) with modiﬁcations. Non- internalized yeast cells in the supernatant were taken from the wells and the adherent phagocytes were washed with 200 µL PBS. INTRODUCTION These macrophages were lysed at 3 and 24 h with 200 µL of cold, sterile, distilled water and incubated for 30 min, then 100 µL was collected and plated on SDA medium, and the viable yeast cells were counted. The IPR was calculated as the quotient of the intracellular yeast cell numbers at 24 h (the point in time which featured the maximum intracellular yeast number) and 3 h. pCramoll C. mollis seed extract (10% w/v prepared in 0.15 M NaCl) was fractionated using ammonium sulfate (40–60% w/v) and the fraction obtained was submitted to aﬃnity chromatography in a Sephadex G-75. The pCramoll preparation was bioselectively eluted with 0.3 M of D-glucose in 0.15 M NaCl, dialyzed against 0.15 M NaCl for 24 h and subsequently lyophilized (Correia and Coelho, 1995) and diluted in PBS to analyze protein concentration, as determined by the BCA kit (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA). The supernatant of phagocytosis assay was used for measurement of NO production using the Griess assay. The nitrite concentration was quantiﬁed by extrapolation from a sodium nitrite standard curve, determined at 540 nm with a microplate reader (Benchmark Plus, Bio-Rad, CA, US). Phagocytosis Assay, Intracellular Proliferation Rate (IPR), Measurement of ROS, and NO Production by Macrophages Bone marrow cells were isolated as described previously (Weischenfeldt and Porse, 2008; de Souza et al., 2016). Femurs and tibias from mice were removed, disinfected by immersion Macrophages/well in RPMI-1640 without phenol red (Sigma- Aldrich) were also infected with C. gatti (same conditions that phagocytosis assay), and the supernatant was used for ROS quantiﬁcation using 2-7-dichloroﬂuorescein diacetate (DCFH- DA; Invitrogen, Life Technologies, Carlsbad, CA, USA) was used and the ﬂuorescence was measured with a ﬂuorometer (Synergy 2 May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 2 pCramoll-Fluconazole Beneﬁts to Cryptococcosis Jandú et al. SL Luminescence Microplate Reader; Biotek) the positive control was hydrogen peroxide at 10 µM (Ferreira et al., 2013). SL Luminescence Microplate Reader; Biotek) the positive control was hydrogen peroxide at 10 µM (Ferreira et al., 2013). to the manufacturer’s instructions (R&B Systems, Minneapolis, MN, USA) and the levels of IFN-γ and IL-17A were measured by cytometric bead array (BD Biosciences, San Jose, CA, USA). Also, the bronchoalveolar lavage ﬂuid (BAL) was obtained and centrifuged at 1,200 rpm for 5 min at 4◦C. The cell pellet was suspended in 3% albumin solution (100 µl). A 10 ul aliquot of albumin solution containing the cells washed was diluted in 10 uL of Türck for cell count totals in a Neubauer chamber with an optical microscope at 40x magniﬁcation. pCramoll For diﬀerential counts, smears were made in cellspin (CT-2000, CIENTEC), stained using the Giemsa method and cells were quantiﬁed by morphological criteria for the distinction of cellular types (mononuclear and polymorphonuclear) and the results were grouped according to the percent content of these two cells type (Maxeiner et al., 2007). Myeloperoxidase (MPO) and Myeloperoxidase (MPO) and N-Acetylglucosaminidase (NAG) Activities The inﬁltration of neutrophil in the lungs was indirectly measured by the assay of myeloperoxidase activity (MPO) according to Costa et al. (2016), by measuring the change in optical density (OD) using 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich). The absorbance reading was taken at 450 nm in a spectrophotometer. For N-acetylglucosaminidase activity (NAG), 2,24mMp-nitrophenyl-N-acetyl-β-D- glicosaminide (Sigma-Aldrich) was used and absorbance was determined at 400 nm (Baltazar et al., 2014). Initially, infected mice were intraperitoneally (i.p.) treated every 10 days with 1, 250, or 500 µg of lectin alone (the ﬁrst dose was administered 1 day before intratracheal infection). For the group treated with the combination ﬂuconazole (20 mg.kg−1) and pCramoll at 1, 250, or 500 µg (the ﬂuconazole was daily administered and every 10 days the association was used), or the mice were treated daily with ﬂuconazole alone (20 mg.kg−1) for survival monitoring. The untreated group (NT), and not infected group (NI) were inoculated with PBS. All animals were monitored twice daily for survival and behavior parameter analysis (SHIRPA protocol). The SHIRPA protocol analyzes the behavioral and functional assessment of neurological diseases (Rogers et al., 1997). The tasks are grouped into ﬁve functional categories: neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength, and reﬂex and sensory function (Santos et al., 2014; Costa et al., 2016). All mice were examined daily and the score for each functional category was calculated as the total of the evaluated parameters according to Lackner et al. (2006) and Pedroso et al. (2010) using the EpiData 3.1 software. The Table S1 describes all the parameters analyzed in the SHIRPA protocol. Histopathology The lungs were removed during necropsy and immediately ﬁxed in buﬀered 10% formalin (v/v). The tissue was embedded in paraﬃn, and the sections were stained with hematoxylin and eosin (Sigma) and examined under light microscopy. Statistical Analysis Statistical analysis of all data were performed using GraphPad Prism version 5.0 with p < 0.05 considered signiﬁcant. The survival curve was plotted by Kaplan-Meier analysis and the results were analyzed using the log rank test, for behavior parameters the area under the curve was analyzed. Also, the results of the phagocytosis assay, IPR, measurement of ROS and NO production by macrophages, MPO and NAG activity, and quantiﬁcation of cytokines were analyzed by analysis of variance (ANOVA) followed by Dunn’s Multiple Comparison Test and the Student’s t-test. Furthermore, other groups of mice were i.t. infected and treated with pCramoll at 1 µg per mouse (the concentration that provided the best results in the survival curve), combined or not with ﬂuconazole to obtain lungs and brain at 15 or 35 days post-infection (d.p.i). The animals were euthanized by cervical dislocation under anesthesia. The lungs and brain were removed for determining the colony-forming units (CFU) as described previously (Santos et al., 2014). For this, the organs were homogenized with sterile PBS and plated in SDA medium. Speciﬁcally, 100 or 50 mg of lung tissue was homogenized with 1 or 0.5 mL of extraction solution of protein, containing PBS (pH 7.0) and anti-proteases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, 20 Kallikrein inhibitor units of aprotinin A, all purchased from Sigma-Aldrich and 0.05% tween 20). The samples were centrifuged for 10 min at 3,000 × g, at 4◦C and the supernatant was frozen at −20◦C and utilized for cytokine analysis. The levels of IL-10 and IL-6 were determined using commercially available antibodies according Effect of pCramoll and Fluconazole in the Cryptococcus gattii Infection Model The protocol of the animal studies was approved by the Comissão de Ética no Uso de Animais (CEUA) at the Universidade Federal de Minas Gerais (Protocol 310/2014). All mice were housed in clean cages, with food and water ad libitum. The controlled environment was set to a 12 h light/dark cycle at 23◦C. Mice C57BL/6, 5–6 weeks old (n = 6/group) were anesthetized by intraperitoneal (i.p.) injection of ketamine hydrochloride (60 mg.kg−1) and xylazine (10 mg.kg−1) in PBS, and then inoculated by an intratracheal infection with 30 µL of 104 CFU/animal of C. gattii, L27/01 strain. Intratracheal infection with the C. gattii L27/01 strain (GVII molecular type) has been previously shown in other works (Santos et al., 2014; Ferreira et al., 2015). The Combination pCramoll + Fluconazole Reduces Fungal Burden and Increases Inﬂammatory Response All the treatments reduced the fungal burden in the lungs after 15 dpi. At 35 days post infection, ﬂuconazole combined with pCramoll was better than ﬂuconazole alone in reducing pulmonary fungal burden (p < 0.05; Figure 3A). In brain tissue, only the use of ﬂuconazole reduced the fungal burden after 15 dpi (Figure 3B), however, the combination (ﬂuconazole + pCramoll) was able to reduce cerebral fungal burden signiﬁcantly when compared with ﬂuconazole alone after 35 dpi (p < 0.05). Histological analysis of lung tissue from untreated infected mice at 15 dpi showed numerous yeasts in the pulmonary parenchyma, mild perivascular inﬁltrate and alveolar thickening. Moderate to intense perivascular inﬂammation was observed in lung tissue from pCramoll, and pCramoll + ﬂuconazole groups. Lungs from treated groups showed less yeast in alveolar space than the untreated group (Figure 3C). pCramoll and Fluconazole Increase the Survival and Improve the Behavior of Animals Infected with C. gattii g An increase in survival of animals treated with pCramoll was observed. The untreated group (NT) showed a median survival (MS) of 20 days (Figure 2A), while the MS for groups treated with the lectin at 500, 250, and 1 µg, were 30.5 (p < 0.05; data not shown), 30 (p < 0.01; data not shown) and 29.5 (p < 0.01) days (Figure 2A), respectively, an increase of 50% (approximately) for all doses tested, and an independent dose response. pCramoll at 500, 250, and 1 µg was also tested in uninfected animals and no change in the survival/behavior of animals compared to NI mice was found (data not shown). When combined with ﬂuconazole, the inﬂuence of lectins on survival was dose dependent. The group treated with ﬂuconazole alone showed an MS of 46 days, while the groups where pCramoll was combined with the antifungal demonstrated a MS of 51 days for 500 µg of pCramoll (data not shown), 58 days for 250 µg of pCramoll (data not shown) and 62 days for 1 µg of pCramoll (p < 0.05; Figure 2A). Based on these results, the dose of 1 µg of pCramoll combined or not with ﬂuconazole was used in the further experiments. Indeed, the cellular polysaccharide extracted from strain L27/01 (PSC) of C. gattii was associated with pCramoll and ﬂuconazole, and were tested in our model. The administration of PSC only hurried the death of infected animals (MS of 17 days), compared to the NT group (20 days). The same was observed where PSC was associated with pCramoll and ﬂuconazole (MS of 43 days), compared to the FCZ-treated group (MS of 46 days), where the animals had previously succumbed (data not shown). Regarding the inﬂammatory inﬁltrate in the bronchoalveolar lavage ﬂuid (Figure 4A), we veriﬁed a predominance of mononuclear cells for all groups except the one treated with the combination, for which there was a balance between mononuclear and polymorphonuclear cells, at both 15 and 35 dpi (Figure 4B). Additionally, MPO (Figure 4C) and NAG (Figure 4D) activities were performed in order to conﬁrm the presence of neutrophils and macrophages, respectively, where the combination demonstrated higher activities of both enzymes at 15 dpi. The levels of IFN-γ (Figure 4E) were higher for the group treated with the combination at 35 dpi. The levels of IL-6 (Figure 4F) and IL-10 (Figure 4G) were augmented in the NT group. pCramoll Increases Phagocytosis and Fungicidal Activity of Bone Marrow-Derived Macrophages (BMDM) pCramoll at 1 µM increased the phagocytic index after 3 h of incubation (Figure 1A), compared with the untreated group. At the same time, the levels of ROS were increased in the presence of pCramoll at 1 and 5 µM, both for infected and uninfected macrophages (Figure 1B), especially with pCramoll at 1 µM with signiﬁcant increase in the production of nitrosative species (Figure 1C). This increase in ROS levels was associated with a reduced IPR (Figure 1D). Another interesting observation was the presence of macrophage aggregates, vacuoles and increased expansion of the cells (area) in the presence of increasing concentration of pCramoll (Figure 1E). Bone marrow-derived macrophages stimulated with pCramoll at 1 and 5 µM showed, 3 h post-incubation, 0.080 and 0.197 µm2 of area, respectively, while at 24 h: 0.214 and 0.483 µm2, respectively. There was an increase of up to 5× in cell area compared with untreated macrophages infected with C. gattii after 3 h and to 7× after 24 h of incubation (Figure 1F). pCramoll and Fluconazole Increase the Survival and Improve the Behavior of Animals Infected with C. gattii An increase in IL-17A (Figure 4H) was veriﬁed for the combination and for ﬂuconazole alone at 15 dpi. Preliminary Tests of Toxicity and Antifungal Activity of pCramoll Previously, the pCramoll toxicity analysis in Caenorhabditis elegans model was performed complementarily. Non-toxicity was veriﬁed in these worms (data not shown) and conﬁrms the non- cytotoxicity of the pCramoll concentration used in this study and published previously by da Silva et al. (2014). Subsequently, we investigated the antifungal activity of pCramoll against C. gattii by screening and determining the minimum inhibitory May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Jandú et al. pCramoll-Fluconazole Beneﬁts to Cryptococcosis The behavior analysis of basic functions such as sensory- reﬂex (Figure 2B), muscle tone and strength (Figure 2C) demonstrated improved behavior by day 20 post-infection when animals were treated with pCramoll, ﬂuconazole and the combination compared to the untreated group (NT). Moreover, the combination is signiﬁcantly more eﬃcient than ﬂuconazole alone (p < 0.05). A similar behavioral proﬁle was observed for parameters involved in the neuropsychiatric state (Figure 2D) and body weight (Figure 2E), which improved by day 22 day post-infection (p < 0.05) for all the treated groups, but with better performance for groups treated with the combination (p < 0.05). There was no diﬀerence between the treated and untreated groups for motor behavior (Figure 2F). Finally, as shown in Figure 2G, autonomic function was only improved when the combination was used (p < 0.05). concentration (MIC). Neither of the concentrations tested (0.93– 120 µg/mL) were able to inhibit fungal growth (data not shown). Indeed, no interaction between pCramoll and ﬂuconazole was obtained by the checkerboard test (data not shown). concentration (MIC). Neither of the concentrations tested (0.93– 120 µg/mL) were able to inhibit fungal growth (data not shown). Indeed, no interaction between pCramoll and ﬂuconazole was obtained by the checkerboard test (data not shown). DISCUSSION We observed complementarily that pCramoll did not show toxicity in the C. elegans model, corroborating previously published in vitro cytotoxicity data in mammalian cells (Melo et al., 2010, 2011; da Silva et al., 2015). In addition, pCramoll May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 4 pCramoll-Fluconazole Beneﬁts to Cryptococcosis Immunomodulatory and fungicidal effect of pCramoll in murine bone marrow macrophages infected with Cryptococcus gattii. (A) After 3 h , the phagocytic index increased under pCramoll treatment to 1 µM; (B) After 3 h of incubation, treatment with pCramoll enhances ROS production in s infected with C. gattii, in a concentration-dependent manner. (C) Oxide Nitric production after 3 h after incubation; (D) Intracellular Proliferation Rate ncubation; (E) Macrophage morphology after 24 h of incubation with C. gattii, showing C. gattii phagocytosis (arrows). Cell cultures were observed under al zoom and 10 ﬁelds per coverslip. (F) Area of macrophages stimulated with lectins and subsequently infected with C. gatti. p < 0.05; p < 0.01; *p < ence between treatment groups and infected control); 8, Uninfected macrophages, 8Cg, C. gattii-infected macrophages; 8pC, Uninfected macrophages pCramoll; C+ 10 µM Hydrogen peroxide. Data represent the means ± SE from two independent experiments consisting of ten replicate assays. Jandú et al. pCramoll-Fluconazole Beneﬁts to Cryptococcosis FIGURE 1 \| Immunomodulatory and fungicidal effect of pCramoll in murine bone marrow macrophages infected with Cryptococcus gattii. (A) After 3 h of incubation, the phagocytic index increased under pCramoll treatment to 1 µM; (B) After 3 h of incubation, treatment with pCramoll enhances ROS production in macrophages infected with C. gattii, in a concentration-dependent manner. (C) Oxide Nitric production after 3 h after incubation; (D) Intracellular Proliferation Rate after 24 h of incubation; (E) Macrophage morphology after 24 h of incubation with C. gattii, showing C. gattii phagocytosis (arrows). Cell cultures were observed under 100 × optical zoom and 10 ﬁelds per coverslip. (F) Area of macrophages stimulated with lectins and subsequently infected with C. gatti. p < 0.05; p < 0.01; p < 0.005 (Difference between treatment groups and infected control); 8, Uninfected macrophages, 8Cg, C. gattii-infected macrophages; 8pC, Uninfected macrophages treated with pCramoll; C+ 10 µM Hydrogen peroxide. Data represent the means ± SE from two independent experiments consisting of ten replicate assays. DISCUSSION May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 5 Jandú et al. pCramoll-Fluconazole Beneﬁts to Cryptococcosis IGURE 2 \| Survival curve and behavioral proﬁle evaluation of mice infected with Crytococcus gattii and subjected to different treatments. (A) Six mice er group were inoculated with 104 cells of L27/01 strain by intratracheal line inoculation and treated with ﬂuconazole at 20 mg.kg−1 (FCZ), pCramoll at 1 ug (pC1), or he combination (pC1 + FCZ). Animals treated by pC1 + FCZ had an average increase of over 34.8% in survival compared to those treated with FCZ alone p < 0.05. B–G) Five animals per group were submitted to the SHIRPA Protocol. There was no behavioral difference between uninfected mice (NI) and those treated with pC1 lone, which did also not inﬂuence the survival of mice. Untreated mice (NT); p <0.05; p < 0.001 (difference when compared to NI); +p < 0.5; ++p < 0.001 difference when compared to NT); #p < 0.05 (difference between FCZ and combination). Data represent the means ± SE from three independent experiments. FIGURE 2 \| Survival curve and behavioral proﬁle evaluation of mice infected with Crytococcus gattii and subjected to different treatments. (A) Six mice per group were inoculated with 104 cells of L27/01 strain by intratracheal line inoculation and treated with ﬂuconazole at 20 mg.kg−1 (FCZ), pCramoll at 1 ug (pC1), or the combination (pC1 + FCZ). Animals treated by pC1 + FCZ had an average increase of over 34.8% in survival compared to those treated with FCZ alone p < 0.05. (B–G) Five animals per group were submitted to the SHIRPA Protocol. There was no behavioral difference between uninfected mice (NI) and those treated with pC1 alone, which did also not inﬂuence the survival of mice. Untreated mice (NT); p <0.05; p < 0.001 (difference when compared to NI); +p < 0.5; ++p < 0.001 (difference when compared to NT); #p < 0.05 (difference between FCZ and combination). Data represent the means ± SE from three independent experiments. pCramoll increased the phagocytosis of C. gattii and the production of ROS and NO, augmenting the fungicidal activity of macrophages. Furthermore, this eﬀect was observed in the cellular morphology of infected macrophages. Previously, the immunoregulatory inﬂuence of pCramoll has been portrayed, showing increased phagocytosis of S. DISCUSSION Mice treated with pC1 showed more prominent perivascular inﬂammatory inﬁltrate (thin arrow) and less yeasts (thick arrow) in alveolar space than untreated, infected mice. Mice from FLZ or pC1+ FLZ showed moderate inﬂammation. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 µg (pC1), Fluconazole (FCZ), ND (not recovered CFU). p < 0.05; *p < 0.01 (difference between treated groups and NT); #p < 0.05 (difference between FCZ alone and combination, at 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays. FIGURE 3 \| pCramoll in combination with ﬂuconazole shows increased survival and reduced fungal burden in the lungs and brain of Cryptococcus gattii-infected mice. Six mice per group were inoculated with 104 cells of the L27/01 strain by intratracheal line inoculation and given the combination treatment, analyzed at 15 and 35 dpi: (A,B) Colony-forming Units (CFU) recovered from brain and lungs, respectively, at 15 and 35 days post infection; (C) Histological sections of lung tissue stained with H&E at 200 ×, 15 days post inoculation. Non-infected mice showed normal histology. Mice treated with pC1 showed more prominent perivascular inﬂammatory inﬁltrate (thin arrow) and less yeasts (thick arrow) in alveolar space than untreated, infected mice. Mice from FLZ or pC1+ FLZ showed moderate inﬂammation. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 µg (pC1), Fluconazole (FCZ), ND (not recovered CFU). p < 0.05; *p < 0.01 (difference between treated groups and NT); #p < 0.05 (difference between FCZ alone and combination, at 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays. the blood-brain barrier (Patricio et al., 2011) and not directly acting on the CNS. Meanwhile, ﬂuconazole was present at higher concentration in the CNS after 14 days of use, reducing CFU in the brain (Mendes et al., 2010). Furthermore, pCramoll also stimulates C. gattii phagocytosis, increasing the early combat of the pathogen in the lungs. However, some yeasts have the capacity to survive in phagolysosomes, enabling transmigration of the fungus (Charlier et al., 2009). This phenomenon reduces fungal permanence in lung tissue, inhibiting strategies of fungal adaptation, avoiding cellular modiﬁcation which inhibit the phagocytosis, and more virulent growth, like giant, ﬂuconazole- resistant cells (Kronstad et al., 2011). (da Silva et al., 2014). DISCUSSION aureus, as well as enhancement of NO, ROS, and pro-inﬂammatory cytokines did not present antimicrobial action against C. gatti, an activity rarely found in the legume lectin domain (Dias et al., 2015). However, a few studies have shown the immunomodulatory potential of plant lectins to combat bacterial and fungal infections (Alegre-Maller et al., 2014), in this context pCramoll inﬂuences the inﬂammatory responses against C. gatti. May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Jandú et al. pCramoll-Fluconazole Beneﬁts to Cryptococcosis FIGURE 3 \| pCramoll in combination with ﬂuconazole shows increased survival and reduced fungal burden in the lungs and brain of Cryptococcus gattii-infected mice. Six mice per group were inoculated with 104 cells of the L27/01 strain by intratracheal line inoculation and given the combination treatment, analyzed at 15 and 35 dpi: (A,B) Colony-forming Units (CFU) recovered from brain and lungs, respectively, at 15 and 35 days post infection; (C) Histological sections of lung tissue stained with H&E at 200 ×, 15 days post inoculation. Non-infected mice showed normal histology. Mice treated with pC1 showed more prominent perivascular inﬂammatory inﬁltrate (thin arrow) and less yeasts (thick arrow) in alveolar space than untreated, infected mice. Mice from FLZ or pC1+ FLZ showed moderate inﬂammation. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 µg (pC1), Fluconazole (FCZ), ND (not recovered CFU). p < 0.05; *p < 0.01 (difference between treated groups and NT); #p < 0.05 (difference between FCZ alone and combination, at 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays. nation with ﬂuconazole shows increased survival and reduced fungal burden in the lungs and brain of Cryptococcus 4 FIGURE 3 \| pCramoll in combination with ﬂuconazole shows increased survival and reduced fungal burden in the lungs and brain of Cryptococcus gattii-infected mice. Six mice per group were inoculated with 104 cells of the L27/01 strain by intratracheal line inoculation and given the combination treatment, analyzed at 15 and 35 dpi: (A,B) Colony-forming Units (CFU) recovered from brain and lungs, respectively, at 15 and 35 days post infection; (C) Histological sections of lung tissue stained with H&E at 200 ×, 15 days post inoculation. Non-infected mice showed normal histology. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org DISCUSSION pCramoll-Fluconazole Beneﬁts to Cryptococcosis FIGURE 4 \| Inﬂammatory response of pCramoll in combination with ﬂuconazole in Cryptococcus gattii-infected mice, analyzed at 15 and 35 dpi: (A,B) Total and differential count of leukocytes (neutrophils and mononuclear cells) in the bronchoalveolar lavage, respectively; at 15 and 35 days after infection; (C,D) Myeloperoxidase and N-acetylglucosaminidase activities in lungs of infected animals; (E–H) Levels of cytokines IFN-γ, IL-6, IL-10, and IL-17A in lungs. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 µg (pC1), Fluconazole (FCZ), p < 0.05; *p < 0.01 (difference between treated groups and NT); #p < 0.05 (difference when comparing treated groups); ###p < 0.005 (difference between FCZ alone and combination, at 35 dpi); +p < 0.5 (difference between 15 dpi and 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays. FIGURE 4 \| Inﬂammatory response of pCramoll in combination with ﬂuconazole in Cryptococcus gattii-infected mice, analyzed at 15 and 35 dpi: (A,B) Total and differential count of leukocytes (neutrophils and mononuclear cells) in the bronchoalveolar lavage, respectively; at 15 and 35 days after infection; (C,D) Myeloperoxidase and N-acetylglucosaminidase activities in lungs of infected animals; (E–H) Levels of cytokines IFN-γ, IL-6, IL-10, and IL-17A in lungs. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 µg (pC1), Fluconazole (FCZ), p < 0.05; **p < 0.01 (difference between treated groups and NT); #p < 0.05 (difference when comparing treated groups); ###p < 0.005 (difference between FCZ alone and combination, at 35 dpi); +p < 0.5 (difference between 15 dpi and 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays. and pro-inﬂammatory cytokine production (Wright et al., 2002; Leopold et al., 2016). On the other hand, the therapy tested in this work has shown that, at 15 dpi, the animals treated with pCramoll alone increase leukocyte migration with a predominance of monocytes to macrophages contained in the BAL, although with less expression of inﬂammatory cytokines IFN-γ, IL-4, and TNF- α in the lungs (data not shown). The recruitment of these cells and their presence in the inﬂammatory response are crucial for immunological responses and pathogen depletion, mainly by phagocytosis (Melo et al., 2010). DISCUSSION Phagocytosis is the main mechanism for fungal elimination (Leopold et al., 2016), and is associated with increased survival of infected animals. Artin M (another mannose-speciﬁc lectin, like pCramoll) demonstred that phagocytosis was also the mechanism responsible for Candida albicans elimination, reducing the fungal burden in target organs and increasing animal survival (Loyola et al., 2012). The murine model used in this study mimics the natural route of infection by C. gattii, as conﬁrmed by the results of fungal burden and alterations in histopathology and behavior. pCramoll alone, as well as when combined with ﬂuconazole, enhanced the survival of animals and improved the behavioral alterations caused by neuro-cryptococcosis. pCramoll in association with ﬂuconazole decreased distinct alterations in related parameters, such as sensory-reﬂex function, the neuropsychiatric state, muscle tone, and strength, as well as body weight. In the course of the disease, these clinical manifestations are determinants for neurological sequelae and death (Chen et al., 2012). Interestingly, 35 days post-infection, there was a signiﬁcant decrease in CFU in the lungs and brain in animals treated with the combination compared to the use of ﬂuconazole alone. Ongoing use of the combination resulted in better neurological and behavioral conditions. This therapeutic characteristic is very important because previous studies have demonstrated that most patients infected with C. gattii presented CNS cryptococcosis, especially with meningitis 64–76% (Chen et al., 2000, 2012). The reduced number of yeasts in the brain improves the general conditions of mice, as conﬁrmed by the results shown in the survival curve. The pCramoll and ﬂuconazole treatments decreased CFU in the lungs, at 15 dpi. Not only does this association enable a signiﬁcant reduction in CFU, it is also more eﬀective than the use of ﬂuconazole alone in treating pulmonary cryptococcosis. Although yeasts were recovered from the brain of animals treated with the combination, they were not found in animals treated only with ﬂuconazole. This may be due to pCramoll not crossing Infections with C. gattii leads to suppression of the host’s immune responses, including decreased leukocyte recruitment May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 Jandú et al. REFERENCES Charlier, C., Nielsen, K., Daou, S., Brigitte, M., Chretien, F., and Dromer, F. (2009). Evidence the role of monocytes in dissemination and brain invasion by Cryptococcus neoformans. Infect. Immun. 77, 120–127. doi: 10.1128/IAI.01065-08 Alegre-Maller, A. C., Oliveira, A. F., Dos Reis Almeida, F. B., Roque-Barreira, M. C., and Hanna, E. S. (2014). Recombinant paracoccin reproduces the biological properties of the native protein and induces protective Th1 immunity against Paracoccidioides brasiliensis infection. PLoS. Negl. Trop. 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Inﬂammasome activation by Campylobacter jejuni. J. Immunol. 193, 4548–4557. doi: 10.4049/jimmunol.1400648 da Silva, L. C. , Alves, N. M., de Castro, M. C., Pereira, V. R., da Paz, N. V., Coelho, L. C., et al. (2015). SUPPLEMENTARY MATERIAL Therefore, pCramoll is an immunomodulatory lectin without antifungal activity but its combination with ﬂuconazole increases the survival of animals with cryptococosis, improving aspects of morbidity present in disease progression. The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fcimb. 2017.00211/full#supplementary-material AUTHOR CONTRIBUTIONS JJ: performed plant collection, biochemical, antimicrobial, and immunologycal assays, analysis and wrote the paper; JS: contributed in the experimental developing with animals; MC: permed experimental procedures in vivo, in vitro, as well as theoretical aspects of the paper. FA: participated plant collection, and puriﬁcation process of the lectin. TF: contributed in experimental procedures with animals. AG: Contributed in the histological analysis. MS, LC: Scientiﬁc and structural support for research development. MB: Participated in the analysis of inﬂammatory factors by ﬂow cytometry. TP: collection, and puriﬁcation process of the lectin.Contributed in the histological analysis and theoretcal aspects. DS: Coordinated the project, supervised all development of this work, such theoretical and experimental aspects this research. MC: Coordinated the project. Scientiﬁc and structural support for research development. DISCUSSION At 15 dpi, when pCramoll was combined with ﬂuconazole, despite the large inﬂammatory inﬁltrate, the leukocyte content was balanced (neutrophils and macrophages), with lower Th1 cytokine expression (IFN-γ) as well as IL-10 auto-regulator. May 2017 \| Volume 7 \| Article 211 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 8 pCramoll-Fluconazole Beneﬁts to Cryptococcosis Jandú et al. CONCLUSION This is very important because the highly detrimental eﬀect of IL-10 in cryptococcal infection models, as shown in NT mice, plays a major role in downregulating cryptococcal clearance (Olszewski et al., 2014). The same way as at 15 dpi, the high IL-17A production was observed for the combination. This inﬂammatory proﬁle is not limited to the Th1/Th2 balance polarization against the infection, this response has been credited to IL-17A cytokine, which is known to have a role in cellular recruitment (Kolls and Lindén, 2004; Steinman, 2007) and contribute to anti-cryptococcal protection (Voelz et al., 2009). Immunotherapy with lectins in the treatment of cryptococcosis has not been reported, making this work pioneering. The immunoregulatory eﬀect of pCramoll increases the survival of animals and improves clinical signs. Combination with ﬂuconazole was able to reduce fungal burden in the lungs and brain of infected animals, increase C. gattii phagocytosis and produce higher levels of ROS. As such, the combination thereby represents a promising alternative in the treatment of cryptococcosis. Up to 35 dpi, an increase of IFN-γ was observed in the lungs of mice treated with the combination, followed by IL- 17A production, however with lower inﬂammatory inﬁltrate compared to the ﬂuconazole group. The early decrease of IL- 10 as seen in the groups treated with pC and their combination enhances IFN-γ and IL-17A eﬀector responses and promotes fungal clearance in mice with cryptococcal lung infection (Murdock et al., 2014a). Previous studies have shown that pCramoll lectin stimulates Th17- and modulate the Th1-type cytokines production (Oliveira et al., 2013). 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https://openalex.org/W1982566777	http://hal.in2p3.fr/in2p3-01093962/document	English	null	Single-neutron orbits near 78 Ni: Spectroscopy of the<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:mi>N</mml:mi><mml:mo>=</mml:mo><mml:mn>49</mml:mn></mml:math>isotope 79 Zn	Physics letters. B	2,015	cc-by	5,974	Single-neutron orbits near 78Ni: Spectroscopy of the N = 49 isotope 79Zn Box 35, FIN-40014 Jyväskylä, Finland q Institut für Kernphysik, Technische Universität Darmstadt, D-64289 Darmstadt, Germany r AB Department, CERN 1211, Geneva 23, Switzerland s IPHC, CNRS/IN2P3, Université de Strasbourg, F-67037 Strasbourg, France t Università degli studi di Padova and INFN Sezione di Padova, Padova I-35131, Italy u Institut für Kernphysik, Universität zu Köln, D-50937 Köln, Germany v GANIL, CEA/DSM-CNRS/IN2P3, F-14076 Caen, France Instituto de Estructura de la Materia, IEM-CSIC, Madrid, E-28006, Spain a Instituto de Estructura de la Materia, IEM-CSIC, Madrid, E-28006, Spain c School of Engineering, University of the West of Scotland, Paisley, P d d Scottish Universities Physics Alliance (SUPA), United Kingdom Contents lists available at ScienceDirect Contents lists available at ScienceDirect Physics Letters B 740 (2015) 298–302 Physics Letters B 740 (2015) 298–302 Single-neutron orbits near 78Ni: Spectroscopy of the N = 49 isotope 79Zn R. Orlandi a,b,c,d,e,∗, D. Mücher f, R. Raabe b, A. Jungclaus a, S.D. Pain g, V. Bildstein f, R. Chapman c,d, G. de Angelis h, J.G. Johansen i, P. Van Duppen b, A.N. Andreyev c,d,j,e, S. Bottoni b,k, T.E. Cocolios m, H. De Witte b, J. Diriken b, J. Elseviers b, F. Flavigny b, L.P. Gaffney n,b, R. Gernhäuser f, A. Gottardo h, M. Huyse b, A. Illana a, J. Konki l,o,p, T. Kröll q, R. Krücken f, J.F.W. Lane c,d, V. Liberati c,d, B. Marsh r, K. Nowak f, F. Nowacki s, J. Pakarinen l,o,p, E. Rapisarda b, F. Recchia t, P. Reiter u, T. Roger b,v, E. Sahin h, M. Seidlitz u, K. Sieja s, J.F. Smith c,d, J.J. Valiente Dobón h, M. von Schmid q, D. Voulot r, N. Warr u, F.K. Wenander r, K. Wimmer f a Instituto de Estructura de la Materia, IEM-CSIC, Madrid, E-28006, Spain b KU Leuven, Instituut voor Kern- en Stralingsfysica, B-3001 Heverlee, Belgium c School of Engineering, University of the West of Scotland, Paisley, PA1 2BE, United Kingdom d Scottish Universities Physics Alliance (SUPA), United Kingdom e Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki, 319-1195, Japan f Physik Department E12, Technische Universität München, D-85748 Garching, Germany g Physics Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA h Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Legnaro, Legnaro, I-35020, Italy i Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus, Denmark j Department of Physics, University of York, Heslington, YO10 5DD, United Kingdom k Dipartimento di Fisica, Università di Milano and INFN Sezione di Milano, I-20133, Italy l PH Department, CERN 1211, Geneva 23, Switzerland m School of Physics and Astronomy, University of Manchester, Manchester, M13 9PL, United Kingdom n Oliver Lodge Laboratory, University of Liverpool, Liverpool L69 9ZE, United Kingdom o Helsinki Institute of Physics, University of Helsinki, P.O. Box 64, FIN-00014 Helsinki, Finland p Department of Physics, University of Jyväskylä, P.O. a r t i c l e i n f o Additional data (ap- proximately 20 hours), collected using a thick (∼1.7 mg/cm2) DPE target, permitted to conﬁrm weak coincidences observed in the thin-target data. The region of isotopes near 78Ni is the focus of intense exper- imental and theoretical research (cf., for example, [7–12] and ref- erences therein). Whether 78Ni can be considered a doubly-magic spherical nucleus depends ultimately on the size of the Z = 28 and N = 50 shell gaps. To date, however, scarce information is available on 78Ni and on its immediate neighbors, and contrasting predic- tions have been made [12,13] about its magicity. The properties of nuclei lying close to 78Ni also impact strongly on astrophysical models of stellar nucleosynthesis and evolution. A recent example is related to the measurement of the 82Zn bind- ing energy and its implications on the composition of neutron- star crust [14]. Furthermore, a sensitivity study on the effect of neutron-capture rates on the A ∼80 and A ∼130 r-process peaks [15] revealed that 78Zn and 79Zn are among the few isotopes which can cause the largest change (>15%) in the overall abun- dance pattern, affecting the abundances of masses as high as A ∼195. The reaction was studied using the segmented T-REX array of Si telescopes [23], and eight triple-cluster HPGe detectors of Miniball [24], which surrounded the T-REX scattering chamber. The coin- cident detection of light charged particles and γ rays led to the identiﬁcation of states which could not be resolved using only the proton data. Furthermore, the charged-particle data constrained the placement of states in the level scheme which would have been ambiguous from the γ -ray data alone. Single-nucleon transfer reactions are a very sensitive technique to populate single-particle states and to investigate the structure of the isotopes produced [16–19]. Performing such reactions on 78Ni will reveal the energies of the single-particle orbits governing the properties of 78Ni and its neighbors. The necessary experiments however still require years of developments in radioactive-ion- beam production. Revealing insights about the structure of 78Ni can nonetheless be gained by studying close-lying isotopes. More- over, an accurate description of the evolution of nuclear structure across neighboring nuclei is an implicit test for theoretical predic- tions of the properties of 78Ni. γ In Fig. 1 (a), the 79Zn excitation energy deduced from reaction kinematics is shown for proton singles and protons in coincidence with all detected γ rays. a r t i c l e i n f o Article history: Received 5 October 2014 Received in revised form 18 November 2014 Accepted 3 December 2014 Available online 9 December 2014 Editor: D.F. Geesaman Keywords: Nuclear structure γ -Ray transitions Transfer reactions N = 50 shell closure Single-neutron states in the Z = 30, N = 49 isotope 79Zn have been populated using the 78Zn(d, p)79Zn transfer reaction at REX-ISOLDE, CERN. The experimental setup allowed the combined detection of protons ejected in the reaction, and of γ rays emitted by 79Zn. The analysis reveals that the lowest excited states populated in the reaction lie at approximately 1 MeV of excitation, and involve neutron orbits above the N = 50 shell gap. From the analysis of γ -ray data and of proton angular distributions, characteristic of the amount of angular momentum transferred, a 5/2+ conﬁguration was assigned to a state at 983 keV. Comparison with large-scale-shell-model calculations supports a robust neutron N = 50 shell-closure for 78Ni. These data constitute an important step towards the understanding of the magicity of 78Ni and of the structure of nuclei in the region. g © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. * Corresponding author at: Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki, 319-1195, Japan. E-mail address: orlandi.riccardo@jaea.go.jp (R. Orlandi). g/ /j p y 014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licen p // g/ /j p y 0370-2693/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativec SCOAP3. R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 299 Fig. 1. (Color online.) a) 79Zn excitation energy deduced from proton kinematics for all the transfer protons (black solid line) and from the protons in coincidence with any γ ray (green, solid ﬁll). b) 79Zn excitation energy in coincidence with the 983-keV γ ray, corrected for γ -ray eﬃciency. c) Same as b), but in coincidence either with the 236-keV (blue) or the 1859-keV γ transitions (red). Shell structure characterizes several many-body systems of fermions moving in a common potential, such as atomic elec- trons, metal clusters and nuclei. Angular momentum quantization induces a bunching of the single-particle states, resulting in shells separated by energy gaps. a r t i c l e i n f o Three main peaks can be seen, cen- tered respectively around 1.2, 2.5 and 3.3 MeV. Due to kinematic compression and to the detection threshold, only transfer protons corresponding to the lowest-energy peak could be detected both at forward and backward angles, and meaningfully compared to DWBA calculations. At this beam energy, the transferred neutrons should populate mainly states or groups of states corresponding to low-ℓorbits, mostly above the N = 50 gap, namely d5/2, s1/2 and d3/2. The g7/2 orbit above the gap (ℓ = 4) and the neutron–hole states based on p or f conﬁguration are likely to be populated very weakly. As an illustration, the 79Zn case can be compared to the study of the N = 81 isotone 131Sn via the 130Sn(d, p)131Sn re- action [17], in which only neutron orbits above the N = 82 gap were populated. In this Letter, the ﬁrst spectroscopic study of the Z = 30, N = 49 isotope 79Zn is presented. In this nucleus, neutrons can oc- cupy orbits which lie both below and above the N = 50 shell gap. Prior to this work, the available information about 79Zn was lim- ited to its ground-state half life, 0.995(19) s [20]. The beta decay of 79Zn [8] supports a Jπ = 9/2+ ground-state conﬁguration, in line with the shell-model expectation that the odd neutron (hole) oc- cupies the g9/2 orbit, and with N = 49 systematics. In the present work, the 9/2+ assignment for the ground state has been adopted. 79 In this Letter, the ﬁrst spectroscopic study of the Z = 30, N = 49 isotope 79Zn is presented. In this nucleus, neutrons can oc- cupy orbits which lie both below and above the N = 50 shell gap. Prior to this work, the available information about 79Zn was lim- ited to its ground-state half life, 0.995(19) s [20]. The beta decay of 79Zn [8] supports a Jπ = 9/2+ ground-state conﬁguration, in line with the shell-model expectation that the odd neutron (hole) oc- cupies the g9/2 orbit, and with N = 49 systematics. In the present work, the 9/2+ assignment for the ground state has been adopted. In this work, excited states in 79Zn have been populated using the 78Zn(d, p)79Zn reaction in inverse kinematics at REX-ISOLDE, CERN (Q value = 1.796 MeV [21]). a r t i c l e i n f o In the nuclear medium, such shell gaps are revealed by nuclei with neutron and proton numbers corre- sponding to closed-shell conﬁgurations. The properties of these so-called magic nuclei and of their neighbors, which were cardi- nal to the development of the nuclear shell model, could only be reproduced when the role played by the nuclear spin–orbit inter- action was recognized [1]. In recent years, experiments with radioactive ion beams have shown that in some neutron-rich nuclei well-established shell clo- sures can vanish, and new magic numbers appear [2,3]. The chal- lenge to explain and predict the size of shell gaps away from beta stability has led to considerable progress in nuclear physics, both experimentally and theoretically. Despite some remarkable steps forward in describing the evolution of shell structure, e.g. the in- clusion of the tensor interaction [4] and three-body forces [5,6], rare-isotope data are still essential to test and guide theoretical advances. Nuclei away from the valley of beta stability with magic numbers of neutrons and protons, and isotopes in their vicinity, have become new cornerstones for the development of a reliable theoretical picture of all nuclei. Fig. 1. (Color online.) a) 79Zn excitation energy deduced from proton kinematics for all the transfer protons (black solid line) and from the protons in coincidence with any γ ray (green, solid ﬁll). b) 79Zn excitation energy in coincidence with the 983-keV γ ray, corrected for γ -ray eﬃciency. c) Same as b), but in coincidence either with the 236-keV (blue) or the 1859-keV γ transitions (red). with a UCx target. 78Zn atoms were laser ionized using the RILIS set up [22], mass separated, and post-accelerated by the REX- LINAC to 2.83 MeV per nucleon. The 78Zn beam impinged on a thin (105(10) μg/cm2) deuterated polyethylene (DPE) target. In ad- dition to 78Zn, which made up ∼75% of the total intensity, the beam also contained 78Rb (∼20%) and 78Ga (∼5%). Exploiting the fact that without laser ionization only 78Zn disappeared from the beam cocktail, the contribution from the contaminants could be identiﬁed and subtracted oﬄine by collecting data with the laser periodically turned on and off (in total, approximately 100 hours with and 35 hours without laser ionization). From the analysis of elastically scattered deuterons, the estimated average 78Zn beam intensity was 7.8(7) · 105 particles per second. a r t i c l e i n f o Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. discussed below, the analysis of proton angular distributions are indeed consistent with transfer of neutrons to the d5/2 and s1/2 or- bits. The green curve in the same ﬁgure shows that, in coincidence with γ -rays, the intensity of the ﬁrst peak decreases signiﬁcantly more than that of the two higher-lying peaks. This observation suggests the presence, in the excitation-energy range of the ﬁrst peak, of one or more states which are too long lived (more than few nanoseconds) for their decay(s) to be detected in ﬂight. The analysis of the 236- and 1859-keV single-γ -ray-gated exci- tation energy spectra, shown in Fig. 1 (c), is key to positioning the 236-keV transition in the level scheme. A peak at ∼3.20(15) MeV can be seen in both spectra (blue and red in the ﬁgure), but only the 236-keV gate shows also a peak at 2.65(15) MeV, where the large uncertainty is due to the Si-detector resolution (the thin red peak appearing at 2.6 MeV in the 1859-keV gated spectrum is not statistically signiﬁcant). Their observed coincidence implies that the 236-keV lies below the 1859-keV γ ray (otherwise no peak could appear also at 2.65 MeV excitation energy). Hence, the 236-keV transition must be the decay of a state with energy at least as low as the difference between the 3.2 MeV excitation energy and the 1859-keV γ ray, i.e. 1.34(15) MeV. In Fig. 1 (c), however, no peak appears around or below 1.3 MeV, which means that the state at ∼1.34 MeV is not (or too weakly) populated in the direct reaction and only fed from higher-lying states. The prompt character of the 236-keV transition implies that it has E1 or M1 multipolarity. The position of the 1185-keV transition was deduced from its γ -gated excitation energy and the coincidence with the 236-keV γ ray. The γ rays emitted by 79Zn were identiﬁed by requiring a coincidence with the protons ejected in the neutron-transfer re- actions. The Doppler-corrected γ -ray energy spectrum is shown in Fig. 2 (a). Fig. 1 (b) and (c) are examples of excitation-energy spec- tra of 79Zn gated by individual γ rays. Similarly, Fig. a r t i c l e i n f o The strongest peaks are labeled by their energy. The peak labeled “c” is due to a small amount of 78Ga in the beam, due to in-ﬂight 78Zn β decay. b) Same spectrum gated on 79Zn excitation energy in the 0.8–1.7 MeV range, where only the 441- and 983-keV transitions can be seen. See text for details. Fig. 3. Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. Fig. 2. a) Doppler-corrected 79Zn γ -ray spectrum, gated by all transfer protons. The strongest peaks are labeled by their energy. The peak labeled “c” is due to a small amount of 78Ga in the beam, due to in-ﬂight 78Zn β decay. b) Same spectrum gated on 79Zn excitation energy in the 0.8–1.7 MeV range, where only the 441- and 983-keV transitions can be seen. See text for details. Fig. 4. Partial level scheme of 79Zn deduced in this work. The width of the arrows is proportional to the relative transition intensities. With the exception of the isomeric state ‘X’ at 1.10(15) MeV, all energies are in keV, with the error given in brackets. See text for details. Fig. 3. Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. ing to a neutron–hole in the f5/2 orbit. Interestingly, comparable B(E2; 5/2+ →9/2+) strengths have been measured in neighbor- ing N = 49 isotones 81Ge and 83Se (respectively, 0.0383(20) and ≈0.13 W.u. [25]). The direct and indirect feeding to the 983-keV state, which makes its ground-state decay the most intense transition in the spectrum, supports a 5/2+ assignment. The DWBA analysis dis- cussed below conﬁrms the 5/2+ assignment deduced from the γ -ray analysis. In the case of the 1424-keV state, the prompt character of the 441-keV transition, the coincidence between the 983- and 441-keV γ rays and the unobserved crossover ground-state transition favor a spin 3/2+ or 5/2+. Few additional weak transitions, of 888, 1774 and 2321 keV, were tentatively placed in the level scheme: their γ -ray-gated excitation energy spectra in fact exhibit a peak respec- tively at 2.35(15), 3.30(15) and 3.45(15) MeV, compatible with a direct feeding to either the 1424- or the 983-keV states. For the 888-keV transition, this placement is also supported by the ob- served coincidence with the 441-keV transition. Fig. 3. a r t i c l e i n f o 78Zn (T1/2 = 1.47(15) s) was produced in collisions of 1.4 GeV protons from the CERN PS Booster In this work, excited states in 79Zn have been populated using the 78Zn(d, p)79Zn reaction in inverse kinematics at REX-ISOLDE, CERN (Q value = 1.796 MeV [21]). 78Zn (T1/2 = 1.47(15) s) was produced in collisions of 1.4 GeV protons from the CERN PS Booster Fig. 1 (a) reveals that the lowest-lying states which are strongly populated via transfer are found near 1 MeV. This observation is important, since the position of the excited states based on neutron orbits above the N = 50 shell reﬂects the gap size. As R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 300 Fig. 4. Partial level scheme of 79Zn deduced in this work. The width of the arrows is proportional to the relative transition intensities. With the exception of the isomeric state ‘X’ at 1.10(15) MeV, all energies are in keV, with the error given in brackets. See text for details. Fig. 2. a) Doppler-corrected 79Zn γ -ray spectrum, gated by all transfer protons. The strongest peaks are labeled by their energy. The peak labeled “c” is due to a small amount of 78Ga in the beam, due to in-ﬂight 78Zn β decay. b) Same spectrum gated on 79Zn excitation energy in the 0.8–1.7 MeV range, where only the 441- and 983-keV transitions can be seen. See text for details. Fig. 3. Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. Fig. 4. Partial level scheme of 79Zn deduced in this proportional to the relative transition intensities. W state ‘X’ at 1.10(15) MeV, all energies are in keV, w See text for details. ing to a neutron–hole in the f5/2 orbit. B(E2; 5/2+ →9/2+) strengths have bee ing N = 49 isotones 81Ge and 83Se (res ≈0.13 W.u. [25]). The direct and indirect feeding to t makes its ground-state decay the most spectrum, supports a 5/2+ assignment. cussed below conﬁrms the 5/2+ assign γ -ray analysis. In the case of the 1424-keV state, the 441-keV transition, the coincidence betw γ rays and the unobserved crossover gro a spin 3/2+ or 5/2+. Few additional wea and 2321 keV, were tentatively placed i d Fig. 2. a) Doppler-corrected 79Zn γ -ray spectrum, gated by all transfer protons. a r t i c l e i n f o The large amount of feeding from higher lying states favors a positive parity, but a negative parity cannot be excluded. Illustrative angular distributions are shown in Fig. 5 (a) and (b). In both cases, the excitation energy of 79Zn was restricted to the lowest peak of Fig. 1 (a). The data in Fig. 5 (a) were not gated on any γ -ray line, and are poorly ﬁtted by any single ℓtrans- fer. These data are instead well described by a sum of ℓ = 0 and ℓ = 2 distributions. The simultaneous ﬁt of two distribu- tions yields a signiﬁcantly smaller reduced χ 2 (2.1) than obtained by ﬁtting a single ℓ(χ 2 = 17.8 and 18, respectively). The scal- ing factors were left as free parameters in the ﬁt and found to be, respectively, 0.41(3)(10) and 0.51(4)(12) (where the ﬁrst error is statistical, and the second systematic). If, in addition to ℓ = 0 and 2, also a third, ℓ = 1 distribution is added to the simultane- ous ﬁt, the latter has a scaling factor compatible with zero while the former are substantially unchanged, indicating that mostly d and s orbits were populated in this energy range. It is instructive to compare the 5/2+-state energy, 983 keV, with state-of-the-art shell-model calculations [12]. The large shell- model space includes the proton orbits p1/2, p3/2, f72 and f5/2, and the neutron orbits p1/2, p3/2, f5/2, g92 and d5/2, which lies above the N = 50 shell gap. Details about the interaction can be found in Refs. [12,34]. As it can be seen in Fig. 6, these calcu- lations reproduce very well the measured energies of the lowest lying 5/2+-state N = 49 isotones. For 79Zn they predict an energy of 1029 keV, strikingly close to the measured 983 keV. This lowest calculated 5/2+ state (SF = 0.53) is formed by the promotion of one neutron from the g9/2 to the d5/2 orbit. The calculated B(E2) transition strength to the ground state is only 4 e2 fm4 (0.2 W.u.). The equivalent lifetime, 240 ps, is compatible with the observed tail of the 983-keV transition. The angular distribution changes instead considerably by requir- ing the coincident detection of the 983-keV γ ray. This is shown in Fig. 5 (b), together with DWBA calculations for transfer to, re- spectively, pure ℓ = 0 or ℓ = 2 states. a r t i c l e i n f o For the neutron bound-state potential, the radius and diffuseness parameters were 1.25 and 0.65 fm, respectively. Exper- imental spectroscopic factors (SF) were determined from the ratio of experimental and DWBA cross sections, which were calculated for SF = 1. The low beam energy leads to appreciable differences in the DWBA distributions calculated using different optical-model- potential parameterizations. Additional ﬁts were therefore also per- formed using different parameters from Refs. [30–33], and varying the bound-state radius parameter from 1.20 to 1.30. The system- atic uncertainties amount to approximately 20–25% variation in the calculated SFs. It should be remarked however that the arguments based on the analysis of angular distributions do not depend on the chosen parameterization. Refs. [28,29]. For the neutron bound-state potential, the radius and diffuseness parameters were 1.25 and 0.65 fm, respectively. Exper- imental spectroscopic factors (SF) were determined from the ratio of experimental and DWBA cross sections, which were calculated for SF = 1. The low beam energy leads to appreciable differences in the DWBA distributions calculated using different optical-model- potential parameterizations. Additional ﬁts were therefore also per- formed using different parameters from Refs. [30–33], and varying the bound-state radius parameter from 1.20 to 1.30. The system- atic uncertainties amount to approximately 20–25% variation in the calculated SFs. It should be remarked however that the arguments based on the analysis of angular distributions do not depend on the chosen parameterization. The ℓ = 0 strength revealed by the proton data (not gated by any γ ray) of Fig. 5 (a) testiﬁes the presence of an isomeric 1/2+ state near 1 MeV, with SF = 0.41(3)(10). The proton-singles data were also split in three smaller ranges in excitation energy and compared to DWBA calculations similar to those in Fig. 5 (a). The weighted average of the ℓ = 0 strengths measured in the sub-sets indicates that the 1/2+ state lies at approximately 1.05(15) MeV. It seems likely that this s1/2 neutron state corresponds in fact to the 1.10(15)-MeV state deduced from gamma-ray gated excitation energy spectra (labeled X in Fig. 4). If the strongly populated 1/2+ state coincides indeed with the level at 1.1 MeV, then the prompt observation of the 236-keV γ -ray limits the possible spin of the 1.34(15)-MeV state to (1/2) or (3/2) (X +236 in Fig. 4). a r t i c l e i n f o 2 (b) shows that only the 441- and 983-keV lines are still clearly visible if the 79Zn excitation energy is restricted to the lowest peak of Fig. 1 (a) (from 0.8 to 1.7 MeV). Such spectra, together with observed γ coincidences, such as those presented in Fig. 3, proved essen- tial to place the main transitions in the level scheme. No other γ ray was observed below 0.8-MeV excitation energy. It should also be noted that the γ -ray detection eﬃciency drops drastically below 120 keV. Fig. 3 shows that both the 441–983-keV and the 236–1859-keV pairs were each found to be coincident. The partial level scheme of 79Zn deduced in this work is pre- sented in Fig. 4. According to measured intensities and coincidence relations, the 983-keV state decays directly to the ground state, and it is fed by the 441-keV transition. Since the 983-keV tran- sition is prompt, despite a low energy tail, its multipolarity can only be dipole or quadrupole. If the tail is due to the lifetime of the 983-keV state, and not to unobserved feeding, it suggests a lifetime of the order of few hundred picoseconds, with mul- tipolarity E2 or M2. A slow E2 (0.05 < B(E2) < 0.2 W.u.) seems much more likely. An M2 character would in fact require this low- lying and strongly populated state to be a 5/2−state, correspond- From the unobserved coincidence with the 983-keV transition it follows that the 236-keV γ ray feeds an isomeric state lying approximately at 1.10(15) MeV. Proton angular distributions, which are characteristic of the angular momentum transfer, ℓ, provide a strong argument to identify this 1.10(15)-keV state with a 1/2+ state. The measured proton angular distributions were compared to DWBA calculations performed with the codes FRESCO [26] and TWOFNR [27] using global optical model potentials from R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 301 Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy range 0.85–1.55 MeV and scaled DWBA calculations for ℓ = 0, 2 and a sum of the two. b) Same proton data, gated also on the 983-keV γ -ray line, and DWBA calculations for ℓ = 0 or 2. Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. a r t i c l e i n f o The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). and feed the 983-keV state. In summary, approximately 75% of the Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy range 0.85–1.55 MeV and scaled DWBA calculations for ℓ = 0, 2 and a sum of the two. b) Same proton data, gated also on the 983-keV γ -ray line, and DWBA calculations for ℓ = 0 or 2. Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy 0 85 155 M V d l d DWBA l l i f ℓ 0 2 d f Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy range 0.85–1.55 MeV and scaled DWBA calculations for ℓ = 0, 2 and a sum of the two. b) Same proton data, gated also on the 983-keV γ -ray line, and DWBA calculations for ℓ = 0 or 2. and feed the 983-keV state. In summary, approximately 75% of the measured ℓ = 2 strength in this energy range results in the direct or indirect feeding of the state at 983 keV, again consistent with a 5/2+ assignment. Refs. [28,29]. a r t i c l e i n f o A pure ℓ = 2 distribution (transfer to a d5/2 or d3/2 neutron state) yields a better ﬁt and conﬁrms the 5/2+ assignment of the 983-keV state deduced from γ -ray data. Jπ = 3/2+ can in fact be excluded since it would lead to an isomeric M3 ground-state transition. Fig. 6 shows that the calculations also reproduce reasonably well the evolution of the N = 50 gap deduced from 2-neutron sep- aration energies [14,21], with a minimum at Z = 32 and a larger gap in zinc than in germanium, although the experimental gap size in these two isotones is 450 keV smaller. The calculations predict a doubly-magic 78Ni with shell gaps of 4.7 MeV for neutrons and 5.0 MeV for protons, and a ﬁrst 2+-state lying at nearly 4 MeV. If at least the relative increase in gap size between zinc and nickel is correct, the N = 50 gap in 78Ni can be expected to be at least as large as 4.2 MeV. A similar distribution is observed when gating the same excita- tion energy region by the 441-keV transition: the data, not shown here, are best ﬁtted by ℓ = 2, and yield SF = 0.05(1)(2). The comparison between Fig. 1 (b) and the intensity strength of the 441-keV transition suggests that the 983-keV state is also fed by additional low-energy undetected γ -ray transition(s) from one or more states between 1.0 and 1.4 MeV. The measured angular dis- tributions imply however that these states have ℓ = 2 character In conclusion, this spectroscopic study of 79Zn has permitted the identiﬁcation of key states near 1 MeV of excitation energy, R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 302 based on the occupancy of single-particle states above the N = 50 shell gap. The agreement between the current measurement and recent large-scale shell-model calculations supports the picture of a robust N = 50 shell closure for 78Ni. This newly acquired knowl- edge about neutron single-particle states in 79Zn will also be im- portant to constraining neutron-capture rates on 78Zn, which have been shown to impact the ﬁnal r-process abundance pattern dur- ing freeze-out periods. [9] Z.Y. Xu, S. Nishimura, G. Lorusso, F. Browne, P. Doornenbal, et al., Phys. Rev. Lett. 113 (2014) 032505, http://dx.doi.org/10.1103/PhysRevLett.113.032505. [10] T. Marchi, G. de Angelis, T. Baugher, D. Bazin, J. Berryman, EPJ Web Conf. 66 (2014) 02066, http://dx.doi.org/10.1051/epjconf/20146602066. Acknowledgements [15] R.A. Surman, M.R. Mumpower, G.C. Mclaughlin, R. Sinclair, W.R. Hix, K.L. Jones, in: P.E. Garrett, B. Hadinia (Eds.), Proceedings of the 14th International Sympo- sium on Capture Gamma-Ray Spectroscopy and Related Topics, Guelph, 2013, World Scientiﬁc, 2013, p. 304. This work was supported by the European Commission through the Marie Curie Actions Contracts Nos. PIEFGA-2011-30096 (R.O.) and PIEFGA-2008-219175 (J.P.), by the Spanish Ministerio de Ciencia e Innovación under contracts FPA2009-13377-C02 and FPA2011-29854-C04, by the Spanish MEC Consolider – Ingenio 2010, Project No. CDS2007-00042 (CPAN), by FWO-Vlaanderen (Belgium), by GOA/2010/010 (BOF KU Leuven), by the Interuni- versity Attraction Poles Programme initiated by the Belgian Sci- ence Policy Oﬃce (BriX network P7/12), by the European Union Seventh Framework Programme through ENSAR, contract no. RII3-CT-2010-262010, and by the German BMBF under contracts 05P09PKCI5, 05P12PKFNE, 05P12RDCIA and 06DA9036I. R.O., R.C., J.F.W.L., V.L. and J.F.S. also acknowledge support from STFC, Grant Nos. PP/F000944/1, ST/F007590/1, and ST/J000183/2. The authors thank A.M. Moro for valuable discussions. [16] K.L. Jones, et al., Nature 465 (2010) 454–457, http://dx.doi.org/10.1038/ nature09048. [17] R.L. Kozub, et al., Phys. Rev. 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https://openalex.org/W4312801129	https://www.scielo.br/j/jatm/a/NCrnkt8mCLszRwpLnmVh57r/?lang=en&format=pdf	English	null	Modeling Resilience of Air Traffic Management Systems Based on Complex Networks	Journal of Aerospace Technology and Management	2,022	cc-by	12,010	ERROR: type should be string, got "https://doi.org/10.1590/jatm.v14.1273\nTHEMATIC SECTION \| AVIATION SAFETY AND CONTINU https://doi.org/10.1590/jatm.v14.1273\nTHEMATIC SECTION \| AVIATION SAFETY AND CONTINU ABSTRACT The air transport market has a strategic character, promotes economic and social development, and has a strong correlation \nwith the level of economic activity. The Air Traffic Management (ATM) system plays an essential role in air transportation and \ncan be characterized as a sociotechnical system that is too complex to research through classical approaches such as systems \nengineering. In this sense, the application of Complex Network Theory (CNT) analysis and modeling paradigms is driven by the \nneed to accommodate the growth of air traffic within an already saturated ATM infrastructure. The present work describes \nthe development of a resilience evaluation model of ATM systems based on CNT, its metrics and analysis tools. The model was \napplied to the Brazilian ATM system, demonstrating its usefulness in identifying the air traffic control (ATC) agencies that have the \ngreatest influence on the network. The results also showed that the Brazilian ATM network is resilient to random failures; however, \nit is particularly vulnerable to targeted attacks to the ATC agencies with the highest centrality. The research findings can be applied \nto prioritize the deployment of systems and equipment that enhance the resilience and operability of the Brazilian ATM system. Keywords: Air traffic management; ATM; Complex networks; CNT; Resilience. Modeling Resilience of Air Traffic \nManagement Systems Based on \nComplex Networks Gomes Sampaio1,* \n, Raimundo Nogueira Costa Filho2 \n, Marcelo Xavier Guterres1 Francisco Claudio Gomes Sampaio1,* \n, Raimundo Nogueira Costa Filho2 \n, Marcelo 1.Departamento de Ciência e Tecnologia Aeroespacial \n – Instituto Tecnológico de Aeronáutica – Divisão de Transporte Aéreo e \nAeroportos – São José dos Campos/SP, Brazil.2.Universidade Federal do Ceará \n – Departamento de Física – Fortaleza/CE, Brazil. *Correspondence author: claudiofcgs@hotmail.com J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 This is an open access article distributed under the terms of the Creative Commons license. Received: May 17, 2022 \| Accepted: Aug 16, 2022\nPeer Review History: Single-Blind Peer Review.\nSection editor: Donizeti de Andrade INTRODUCTION In this line of reasoning, the characterization of the ATM as a complex system highlights the relevance of the application \nf C\nl\nN t\nk Th\n(CNT)\ni l b\nit\nid\nd li\nt\nl\nd\nl\ni t\nh i\nth t ll\ndi\ni This management strategy combines, in a strongly integrated way, the work of people with technical subsystems and operational \nprocedures in a complex system characterized by numerous simultaneous interactions between its various components, not being \npossible to fully understand through the study of its components individually, then adding its characteristics (Cook et al. 2015). h\ng\ngy\n,\ng y\ng\ny,\np\np\ny\np\nprocedures in a complex system characterized by numerous simultaneous interactions between its various components, not being \npossible to fully understand through the study of its components individually, then adding its characteristics (Cook et al. 2015). In this line of reasoning, the characterization of the ATM as a complex system highlights the relevance of the application \nof Complex Network Theory (CNT), mainly because it provides modeling tools and analysis techniques that allow discovering \nfundamental properties and suggest design guidelines to support high-performance nonlinear systems (Strogatz 2001).hfi In this line of reasoning, the characterization of the ATM as a complex system highlights the relevance of the application \nof Complex Network Theory (CNT), mainly because it provides modeling tools and analysis techniques that allow discovering \nfundamental properties and suggest design guidelines to support high-performance nonlinear systems (Strogatz 2001). Thus, questions arise that instigate critical thinking on the subject: Why is it relevant to model an air traffic system? How to \nmodel? How does a system react to eventual or intentional disturbances? How to measure the resilience of an ATM system? To help reflect on these issues, the present work can be characterized as an investigative approach to the CNT and the process \nof modeling ATM systems, more specifically as a study on the systematic application of a set of metrics of the CNT to measure \nthe resilience of an ATM system. Assuming that an air traffic system can be modeled and has its resilience measured based on the CNT, this work has the main \nobjective to develop a model for evaluating the resilience of ATM systems based on the concepts, metrics and analysis techniques \nof CNT applied to the Brazilian ATM network. INTRODUCTION This analysis model, in essence, is a major contribution of this research. The studies on the subject found in the literature are mainly directed to the structural analysis of the implemented airport \nnetwork (Zanin and Lillo 2013). The annual reports published by the National Civil Aviation Secretariat, for example, portray the \nreality of the Brazilian air transport with a reference centered on the volume of passengers and the cargo handled at the airports. The research is, therefore, limited to the analysis of interactions between flight operations and the ATM system, due to the \nlevel of complexity of air operations and the volumes of data to be processed to produce the appropriate results. The studies on the subject found in the literature are mainly directed to the structural analysis of the implemented airport \nnetwork (Zanin and Lillo 2013). The annual reports published by the National Civil Aviation Secretariat, for example, portray the \nreality of the Brazilian air transport with a reference centered on the volume of passengers and the cargo handled at the airports.hl reality of the Brazilian air transport with a reference centered on the volume of passengers and the cargo handled at the airports. The research is, therefore, limited to the analysis of interactions between flight operations and the ATM system, due to the \nlevel of complexity of air operations and the volumes of data to be processed to produce the appropriate results.fii The research is, therefore, limited to the analysis of interactions between flight operations and the ATM system, due to the \nlevel of complexity of air operations and the volumes of data to be processed to produce the appropriate results.fii For the Academy, air traffic modeling according to CNT metrics fills a gap in this area of knowledge. It is expected that the \nanalysis of air traffic networks, based on the air traffic control (ATC) agencies responsible for conducting air operations, will allow \nthe identification of the most important and central of the network, as well as the robustness, the reliability and resilience of the \nentire ATM system. The establishment of an analysis model also makes it possible to compare the characteristics and structural \nproperties of air traffic systems in different countries. INTRODUCTION The air transport market has a strategic character for countries, promotes economic and social development and is correlated \nwith the level of economic activity. With regard to passenger traffic, demand for air transport continues at its own pace, with \npositive growth rates. International travel and tourism, in particular, remain unstoppable and supported by the dynamics of the \nindustry, according to the prospective analysis of the Airports Council International. In the Brazilian market, government actions—such as the airport concessions program, the release of 100% of foreign capital \nparticipation in Brazilian companies, tax incentives aimed at reducing costs with aviation kerosene and positive signs for the \napproval of the open skies policy—implemented a project a favorable prospective scenario. In the view of Stefan et al. (2012), to understand an air transport system it is necessary to examine the interactions between the components \nthat make it up, including airlines, airports and, with special attention, Air Traffic Management (ATM), that comprises a sociotechnical system \nthat is too complex to be researched and improved through the use of classical approaches such as systems engineering and human factors. Received: May 17, 2022 \| Accepted: Aug 16, 2022\nPeer Review History: Single-Blind Peer Review. Section editor: Donizeti de Andrade J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX\n2 Sampaio FCG, Costa Filho RN, Guterres MX The application of complexity science paradigms to the analysis and modeling of future operations is driven by the need to \naccommodate long-term air traffic growth within an already saturated ATM infrastructure (Blom and Bouarfa 2016).h The application of complexity science paradigms to the analysis and modeling of future operations is driven by the need to \naccommodate long-term air traffic growth within an already saturated ATM infrastructure (Blom and Bouarfa 2016).h This management strategy combines, in a strongly integrated way, the work of people with technical subsystems and operational \nprocedures in a complex system characterized by numerous simultaneous interactions between its various components, not being \npossible to fully understand through the study of its components individually, then adding its characteristics (Cook et al. 2015). INTRODUCTION The research results can be applied, eventually, in the prioritization process for the implementation of systems and equipment \nthat expand the operability of the ATC agencies, which would result, for example, in the expansion of the flight control capacity \nin the same portion of airspace; in the case of installation of radars; or increasing the probability of aircraft landing at an airport, \nby installing precision aids for aircraft approaches in adverse weather conditions. ATM RESILIENCE AND CNT This part of the work provides the technical basis, theoretical foundation, and a reflection on resilience in air traffic systems, \nall of them necessary to understand ATM systems, the CNT philosophy, and the real possibility of mapping non-linear systems \nthrough CNT metrics and their analysis techniques. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Air Traffic Management For Stefan et al. (2012), in a typical air transport system, air operations result from the dynamic interaction between airlines, \nairport operations and the ATM system, of which operational concept, according to the International Civil Aviation Organization \n(ICAO 2019), involves the dynamic and integrated management of traffic and airspace, including air traffic services (ATS), strategic \nmanagement of airspace and air traffic flow—with order, safety, regularity, economy and efficiency—by through the provision of \nfacilities and continuous services, involving air and ground facilities. In Brazil, activities involving the management and control of airspace are treated with strategic relevance and national security \ninterest, thus being entrusted to the Brazilian Air Force, with the Department of Airspace Control (DECEA) as the central J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Modeling Resilience of Air Traffic Management Systems Based on Complex Networks 3 organization of the Brazilian Airspace Control System. The Brazilian management model integrates civil ATC and military air \ndefense operations, which results in the rationalization of human and material resources, and optimizes the decision-making \ncycle in processes involving operational safety matters (DECEA 2019a). Airspaces can be understood as complex partitioned systems, primarily for purposes of organization, control, adequacy of \noperational capacity and protection against saturation of available ATC means. The classical ordering of airspace, according to the \nICAO (2005), involves the concept of Flight Information Region (FIR), which can be defined as a continuous volume of airspace, \ndelimited by duly established lateral and vertical limits, where ATS are provided. The FIRs are arranged contiguously and cover \nthe entire national territory. The Brazilian airspace is structured based on five FIRs associated with the respective control centers responsible for each of the \nregions (Fig. 1d). From this organization, it follows that the Amazonian (ACC-AZ), Recife (ACCRE), Brasília (ACC-BS), Curitiba \n(ACC-CW) and Atlantic (ACC-AO) control centers are responsible for the respective FIRs that bear their names: Amazonian FIR \n(SBAZ); Recife FIR (SBRE); Brasília FIR (SBBS); Curitiba FIR (SBCW); and, finally, the Atlantic FIR (SBAO).h The Brazilian airspace is structured based on five FIRs associated with the respective control centers responsible for each of the \nregions (Fig. 1d). Air Traffic Management From this organization, it follows that the Amazonian (ACC-AZ), Recife (ACCRE), Brasília (ACC-BS), Curitiba \n(ACC-CW) and Atlantic (ACC-AO) control centers are responsible for the respective FIRs that bear their names: Amazonian FIR \n(SBAZ); Recife FIR (SBRE); Brasília FIR (SBBS); Curitiba FIR (SBCW); and, finally, the Atlantic FIR (SBAO).h The airspace of an FIR is normally divided into smaller fractions called a sector, with the aim of facilitating management and \nexpanding its occupancy capacity for a greater number of flights. There are also other portions of airspace—smaller than an FIR \nand larger than a sector—destined for specific purposes and managed by specialized ATC agencies. Among these areas, some in \nwhich ATC services are provided are detailed below, characterizing them as controlled air spaces (DECEA 2013):fifi The airspace of an FIR is normally divided into smaller fractions called a sector, with the aim of facilitating management and \nexpanding its occupancy capacity for a greater number of flights. There are also other portions of airspace—smaller than an FIR \nand larger than a sector—destined for specific purposes and managed by specialized ATC agencies. Among these areas, some in \nwhich ATC services are provided are detailed below, characterizing them as controlled air spaces (DECEA 2013):fifi •\t ATZ – Aerodrome Traffic Zone (Fig. 1a) is an airspace established around an aerodrome for the protection of local air traffic \nof aircraft under visual flight rules. The Control Tower (TWR) is the ATC agency responsible for this type of airspace;tl •\t CTR – Control Zone (Fig. 1b) is a controlled airspace that aims to protect the trajectories of aircraft under instrument flight \nrules in departure or arrival procedures at one or more nearby aerodromes. The Approach Control (APP) is the ATC agency \nresponsible for this type of airspace; •\t TMA – Terminal Control Area (Fig. 1c) is a controlled airspace to which the air routes to the aerodromes contained in \nthis TMA converge. A TMA can be formed by several CTRs and ATZs, thus protecting the trajectory of the aircraft \nin procedures of arrival or departure from the aerodromes inserted in it. Air Traffic Management APP is also the agency responsible for this type \nof controlled airspace; •\t CTA – Control Area is a controlled airspace that contains the lower airways and the TMAs inserted in it, with the Area \nControl Center (ACC) as the control agency responsible for this type of airspace; •\t UTA – Superior Control Area is a controlled airspace similar to the CTA, differing in that it contains the superior airways and \nother parts of the superior airspace, defined as superior control areas. The ACC is the ATC agency responsible for this type \nof airspace. •\t UTA – Superior Control Area is a controlled airspace similar to the CTA, differing in that it contains the superior airways and \nother parts of the superior airspace, defined as superior control areas. The ACC is the ATC agency responsible for this type \nof airspace. Source: Adapted from DECEA (2019a) and Bastos and Baum (2009). Figure 1. Airspaces and ATC agencies: (a) Aerodrome Traffic Zone (ATZ); (b) Control Zone (CTR); (c) Terminal \nControl Area (TMA); and (d) Flight Information Regions (FIRs) and their respectively Area Control Centers (ACCs). Source: Adapted from DECEA (2019a) and Bastos and Baum (2009). Source: Adapted from DECEA (2019a) and Bastos and Baum (2009). Figure 1. Airspaces and ATC agencies: (a) Aerodrome Traffic Zone (ATZ); (b) Control Zone (CTR); (c) Terminal \nControl Area (TMA); and (d) Flight Information Regions (FIRs) and their respectively Area Control Centers (ACCs). J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX\n4 Sampaio FCG, Costa Filho RN, Guterres MX Complementing the set of ATC/ATS agencies, there is the Aerodrome Flight Information Service (AFIS), which is an air traffic \nservice provided to all aircraft operating in the movement area and in flight in the lower airspace, within a radius of 27 nm (50 km), \naround approved aerodromes that do not have a TWR. AFIS is carried out by an aeronautical telecommunications station, which \nprovides meteorological and coordination information for the safety and efficiency of air operations. Complex Network Theory The origins of the CNT, according to Anderson (1972), are related to studies in the field of statistical physics, the dynamics of \nnonlinear systems and information theory, and can be conceptualized as the multidisciplinary study of complex systems. In this \nsense, air transport, in general, and ATM can be analyzed and studied through these powerful tools available (Cook et al. 2015). Using CNT, a network can be modeled and represented by a set of nodes, connected to each other by a set of links. The connections \nbetween the nodes can also be defined as directed, nondirected, with nodes of the same type or of different types, weighted and, \nstill, of dynamic behavior, when there is variation of the structural elements of the network, nodes, and links, in time function \n(Zanin and Lillo 2013). As a practical example of this modeling system (that can be seen in Fig. 2), an air transport network can be formed by airports \n(nodes), linked by flights from one node to another (directed connection) and weighted based on the number of passengers. If the \nflights have information about the proposed departure time and duration, the network can be dynamically analyzed with respect \nto time. Another example of structuring could be observed in a representative network of airspace sectors or navigation points \nsuch as nodes, connected by flights (edges) that cross those sectors or fly over these navigation points. These types of network organization facilitate ATC modeling (Cai et al. 2012) and, by analyzing centrality metrics, allow \nidentifying potential traffic bottlenecks. The detection of communities in these types of networks has proved to be an effective \ninstrument to rationalize airspace restructuring based on traffic analysis (Gurtner et al. 2014). Source: Adapted from Newman (2003). Figure 2. Examples of complex networks: (a) network formed by 8 nodes and 10 edges; (b) directed network, \ni\nhi h\nh d\nh\nifi di\ni\n( )\ndi\nd\nk\ni h\nl\nd\nd Source: Adapted from Newman (2003). re 2. Examples of complex networks: (a) network formed by 8 nodes and 10 edges; (b) directed network\ni\nhi h\nh d\nh\nifi di\nti\n( )\ndi\nt d\nt\nk\nith\nl\nd t\nd Figure 2. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Complex Network Theory Examples of complex networks: (a) network formed by 8 nodes and 10 edges; (b) directed network, edge has a specific direction; (c) nondirected network with only one \none edge type; and (d) network with weighted nodes and edges. In the understanding of Cook et al. (2015) and Sallan and Lordan (2020), metrics are measured with properly established \ncalculation systems that allow understanding or characterizing a network; identify its organization; its critical nodes; and the \nbehaviors that emerge from the dynamic relationships and interactions between its components. A summary of the CNT metrics that characterize the structure of a network is detailed below, based on the works of Albert \nand Barabási (2002) and Newman (2003). J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Modeling Resilience of Air Traffic Management Systems Based on Complex Networks The shortest path between two nodes l (m, n) is defined as the path with the fewest intermediate nodes between the origin \nand the destination. Since L is the set of shortest paths and \|L\| is the number of shortest paths in the network, the average shortest \npath in the network R, l(R) can be defined through Eq. 1. \b\n(1) \b (1) (1) According to Freeman (1979), betweenness centrality represents the control capacity that intermediate nodes have over \nother nodes that locally depend on this intermediary to connect, that is, it is the relationship between the shortest paths that pass \nthrough the node n and the shortest paths between all pairs of nodes. Let N be the set of network nodes, σ(m,o) be the number of \nshortest paths between nodes m and o, and σ(m,o) (n) the number of these paths that cross the node n, the betweenness centrality \nof a node n, β(n), can be calculated by Eq. 2. This metric makes it possible to assess the proportion of a given node of a network \nas a necessary link between different groups belonging to the same network, based on the shortest possible path. \b\n(2) (2) Proximity centrality represents how close a node is to the other nodes in the network that it can reach. Being π(m, n) the length \nof the shortest path between a node m and any other reachable node n, the proximity centrality γ(m) can be defined by means \nof Eq. 3. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Complex Network Theory Broadly speaking, the analysis of this metric makes it possible to understand how a node is perceived as the center of \nthe network; to what extent does it employ the help of intermediate nodes to reach more distant ones; and how it positions itself \n(dependent/independent) regarding the structure of the network. \b\n(3) (3) The clustering coefficient can be conceptualized as the degree to which the nodes of a network tend to cluster. In practical \nterms, it represents a measure of the density of edges between the neighbors of a node, that is, if the neighbors are directly \nconnected—number of triangles in the network. It is also known as the network transitivity measure. Equation 4 proposes the \nformula for calculating the clustering coefficient C of a network N. Here, emo represents the edges that connect neighboring nodes \nto node n; vm and vo represent the vertices neighboring to node n; Rn defines the neighboring graph to node n; and kn represents \nthe number of neighbors of vertex n. \b\n(4) (4) Equation 5 defines the density D of a network as the ratio between the number of edges \|ϵ\| and the total number of possible \nedges of the complete network, where \|ν\| is the number of the nodes. The analysis of this metric allows an understanding of the \ncohesion of a network, because the higher the density, the more cohesive it will be (Nooy et al. 2005). Equation 5 defines the density D of a network as the ratio between the number of edges \|ϵ\| and the total number of possible \nedges of the complete network, where \|ν\| is the number of the nodes. The analysis of this metric allows an understanding of the \ncohesion of a network, because the higher the density, the more cohesive it will be (Nooy et al. 2005). \b\n(5) (5) The diameter d of a network R can be defined as the longest shortest path between any pair of nodes in the network and can \nbe calculated by Eq. 6. The analysis of this metric allows the understanding of the dispersion of the network. \b\n(6) (6) The degree of a node corresponds to the total number of links connected to it and can be divided into in-degree and out-\ndegree. The in-degree of a node n, kin(n), represents the total number of links that arrive at this node. The out-degree, kout(n), J. Aerosp. Complex Network Theory Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX describes the total number of connections that leave the node. Thus, the degree of a node n is equivalent to the total number of \nlinks that arrive or leave the node: k(n) = kin(n) + kout(n). In this reasoning, the average degree k of a network R (Eq. 7) is the result \nof the relation between the sum of the degrees of nodes in the network and the total number of nodes \|N\|, where N is the set of \nnodes in the network. In the case of weighted networks, the strength of a node is defined as the sum of the weights of the node \nconnections, whether input or output. describes the total number of connections that leave the node. Thus, the degree of a node n is equivalent to the total number of \nlinks that arrive or leave the node: k(n) = kin(n) + kout(n). In this reasoning, the average degree k of a network R (Eq. 7) is the result \nof the relation between the sum of the degrees of nodes in the network and the total number of nodes \|N\|, where N is the set of \nnodes in the network. In the case of weighted networks, the strength of a node is defined as the sum of the weights of the node \nconnections, whether input or output. \b\n(7) (7) (7) Based on several studies on this metric, Barabási (2009) identified a specific type of network that he conceptualized as \nscale-free network. This conception is mainly supported by studies by Barrat et al. (2004) and Guimerà et al. (2005), which \nshowed that the probability that the degree (or strength) of a node k is greater than x can be characterized by the power law \nP (k > x) = Nx−g e−ax, where 1 ≤ g ≤ 2 is the tail exponent, a is the parameter that controls the speed of exponential decay and \nN a normalization constant. As a network with random distribution increases in size, the relationship between nodes with \nhigh degrees and other nodes in the network decays. In a scale-free network, however, this relationship remains constant as a \nfunction of the size of the network. Resilience Air traffic management is an example of a complex sociotechnical system, which encompasses interactions between different \norganizations, technical systems, operational units, regulatory agencies, and consumers (DeLaurentis and Ayyalasomayajula 2009). In this extremely specialized field, technology plays a prominent role and the value of studying ATM resilience is perceived as \nrelevant due to the studies of several researchers, especially Hollnagel et al. (2006). Routinely, the air traffic network coexists with different types of internal and external disturbances, which daily test its resilience. These disturbances can interact with each other, enhancing their cascading effects, which can extend over different scales of time \nand space. According to Eurocontrol (2009), the management of disturbances in air traffic systems involves adjustments and \nadaptations in the processes of their subsystems, which result from their inherent complexities, enhanced by the finite human \nand material resources available in the operational control agencies. In the Brazilian ATM activities, minor disturbances are managed by control agencies distributed throughout the country, \nwhile disturbances that may have global reach in the network are handled by a collegiate decision-making process, with \nrepresentatives of airlines, airport administrators and ATM agencies. Flights are rerouted, or timed, to allow for more effective \naccommodation of system-wide disturbances. As an example, in the case of airspace congestion, supervisors of control agencies \ncan coordinate with airlines to redirect or adjust their flight schedules. In these cases, safety and fluidity are prioritized at \nthe expense of the economy, managing conflicting interests that need to be conveniently resolved without generating major \ninconveniences for passengers.fii Despite the coordinated work, there are times when the resilience of the air traffic system cannot contain significant delays in \nflights, which can even spread through the network and compromise several control areas and airports for an extended period. Effects caused by bad weather are classic examples of this type of behavior. In addition to more common cases with restricted \nconsequences, there are other atypical cases with very serious consequences. The two cases can be classified into two broad \ncategories, those that produce effects that strongly affect the performance of the system; and catastrophic accidents involving a \nsingle aircraft or a group of them. The examples described in the previous paragraphs highlight a wide range of events with profound consequences. However, due \nto the resilience of ATM systems, the vast majority of these potential disturbances generate effects that go unnoticed by the users \nof the system. Complex Network Theory The analysis of the average degree metric—strength for weighted networks—allows the identification of the most active and \ninfluential nodes in the network, since they have a higher number of links and, consequently, higher values of k(n). For weighted \nnetworks, the strength of a node can also be properly correlated with the number of flights and passengers passing through this \nnode (Barrat et al. 2004; Guimerà et al. 2005). J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Resilience Analysis Model We developed a model to assess the resilience of ATM systems based on complex networks, in which the system under analysis \nis represented by a directed graph G (A, F) with the ATC/ATS agencies (A, nodes) connected by fractions of the managed flights \n(F, links) in which ATM control and services are effectively performed. We simulated network operations under disturbances and assess resilience in direct and random attack scenarios, \nremoving nodes from the core and using a new metric created by us, the resilience index (Ire), which measures the level of \nresilience comparing interconnections between the ATC/ATS agencies of the initial ATM system and the resulting one after \nthe disturbance simulation. Resilience Index Real world networks represent systems that are constantly modified by the removal or addition of new components, involving \nthe establishment of new connections which are influenced by the degree distribution of the network nodes. In the case of the \nATC/ATS system, new agencies can be activated to support growing air activities in a region of operational interest, normally \nassociated with a significant increase in air movements or the entry into operation of a new airport. The resilience of a network is a property that is related to the distribution of degrees, to connectivity—the existence of \nlinks between vertices—and, mainly, to the removal of nodes from this network. Normally, the loss of nodes results in a \nprogressive reduction in the operational capacity of the network, so its resilience can be measured by removing its nodes, \nwith different criteria for the removal process, either randomly or directed to the nodes that have higher degree or centrality \n(Newman 2003). Considering that the coordination between ATC/ATS agencies for the management and transfer of flights control by adjacent \nagencies depends on the effective connection between them, then the vertices of the largest component formed after removing a \nnode n of the network still maintain, among themselves, a significant coordination capacity in relation to the original network.l In this line of thought, the resilience index reflects the reduction of the ATM network operational capacity with the unavailability \n(removal) of a specific ATC/ATS agency, that is, an index of 0.11 means the loss of 11% of the network’s operational capacity. In short, the lower the resilience index, the greater the resilience of the network. Resilience Despite this, according to Zanin and Lillo (2013), little effort has been devoted to understanding the relationships J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Modeling Resilience of Air Traffic Management Systems Based on Complex Networks between the topology of ATM networks and their vulnerabilities. As, in essence, complexity stems from the interaction between \nvarious subsystems, CNT modeling tools that focus on interactions are better suited to capture the phenomenon of resilience, \nwhich has greater adherence to the approach of sociotechnical systems rather than technical systems. The value of the CNT for investigating the impact of disturbances on a sociotechnical system, in general terms, can be \nestablished based on the capacities to absorb them, adapt to them, and restore its operational performance to pre-disturbance \nlevels (Francis and Bekera 2014).if Absorptive capacity is defined as the extent to which a system can absorb disturbances and minimize their effects. Incorporating \nan additional time in advance into the effective duration of flights is a proactive action to accommodate delays that increases \nabsorption capacity. The ability of a system to accommodate to disturbances by modifying its internal processes is defined as \nadaptive capacity, which differs from absorption capacity in that an adaptive system can change its reaction. Restorative capacity \nrefers to the power that a system has to recover from disturbances and restore or improve its operational capacity, based on the \nlevel of performance presented before the disturbing event. Based on these concepts, a system that has only the ability to absorb disturbances is characterized as robust, while a system that \nhas both absorption and restoration capabilities is said to be reliable. Therefore, resilience can be characterized as the attribute of \na system capable of absorbing, adapting, and recovering from disturbances. This highlights the need to prioritize the development \nof adaptive capacity to increase the resilience of a system. Resilience Analysis Model Thus, being N the set of nodes of an ATM network, we can define a resilience index, IRe(n), of the network in relation to the \nloss of a node, n, using the percentage number of nodes of the largest component that remains connected, that is the fraction \nbetween the number of vertices of the largest component of the network that remains connected after the removal of this node, \n\|SN−{n}\|, and the total number of vertices of the network before the start of the disturbance (\|N\|) (Eq. 8). Thus, J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX \b\n(8) (8) Model Development J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Model Development Manag., São José dos Campos, v14, e2222, 2022 Modeling Resilience of Air Traffic Management Systems Based on Complex Networks SBEG)—will result in five transfers of control records in the ATM flights file: (TWR-FZ, APP-WZ); (APP-WZ, ACC-RE); (ACC-RE, \nACC-AZ); (ACC-AZ, APP-WN); (APP-WN, TWR-EG). For the network modeling (M2 process) the dataset formed by the two files generated in P 1.1 and P 1.2 is used. It starts with \nimporting the dataset into the Gephi workbench, with special attention to the type of graph to be selected (directed) and the \nmerge strategy for edges (sum), therefore, for flights with similar origin and destination, the system will create only one origin/\ndestination edge with weight equal to the sum of the occurrences of parallel edges in the ATM file. The following CNT metrics used in the model, along with the resilience index (Ire, Eq. 8) have their use justified by the \nability to characterize a network and identify its organization, robustness, reliability, and resilience: average shortest path (Eq. 1); \nbetweenness centrality (Eq. 2); proximity centrality (Eq. 3); clustering coefficient (Eq. 4); density (Eq. 5); diameter (Eq. 6); and \naverage degree (Eq. 7). For the statistical treatment and graphic visualization of the dataset that serves as the basis for the modeling of the complex \nstructured network, the interactive free software platform Gephi version 0.9.2 (Bastian et al. 2009) is used. Metric calculations \n(P 2.1 process) are performed using the tools available in the Statistics Tab, selecting each of the metrics of interest. Calculation results \nare automatically entered into fields in the graph nodes file. In order to measure the resilience of the network (module M3), the results obtained through the statistical calculations, summarized \nin the metrics file (A 2.1), are analyzed regarding the maintenance of the minimum operational capacity of the network, which \nincludes the verification of the operability of the system in terms of absorption, accommodation, and response to disturbances, \nthrough the evaluation of updated metrics generated at each iteration. This step is terminated when the minimum operational \ncapacity of the network is verified, which occurs when the number of vertices of the largest component of the network that remains \ninterconnected after the removal of a node is less than 50% of the number of nodes in the undisturbed network (\|S\| < 0.5). RESULTS AND DISCUSSION This section details the results and describes the discussions carried out with the application of the resilience analysis model \nto the Brazilian ATM system. The subsections that compose it are structured with a focus on the results achieved, specifying \nthe general characteristics of the network (General Analysis), deepening research on the resilience of the Brazilian ATM system \n(Resilience Analysis), identifying the communities present in the structure (Community Analysis), and evaluating the weekly \ndistribution of flights in relation to days and times (Dynamics Analysis). Model Development As long as the network remains operational, the most central ATC/ATS agencies are removed from the network (process P 3.1), \none at a time, and the calculation of the statistics is iteratively reworked, up to the point where the network loses its operational \ncapacity (\|S\| < 0.5), when then, the process is terminated. During this process, the metrics file (A 2.1) is indexed based on the \nbetweenness centrality and the most central node in the network—the ATC/ATS agency with the highest centrality value—has all \nits links excluded, that is, all flights referring to that node are eliminated from the graph. The flow is directed to a new statistical \ncalculation (P 2.1) with generation of a new set of metrics. The process of discussing the results (module M4) involves, in a generic way, the identification of the general characteristics of \nthe ATM network based on the calculated metrics; the assessment of established communities, which makes it possible to understand \nthe dependencies between control agencies; understanding of dynamic behavior over time; and the analysis of resilience, which \nultimately reflects the system’s ability to maintain its operability in situations of degradation. Model Development p\nThe flowchart shown in Fig. 3 consolidates the proposed resilience analysis model. The modules and processes used in the \nmodel, which appear in the flowchart, are detailed below. The flowchart shown in Fig. 3 consolidates the proposed resilience analysis model. The modules and processes used in the \nmodel, which appear in the flowchart, are detailed below. Source: Elaborated by the authors. Figure 3. Representative flowchart of the Resilience Analysis Model of ATM Systems Based on Complex Networks. Source: Elaborated by the authors. y\nFigure 3. Representative flowchart of the Resilience Analysis Model of ATM Systems Based on Complex Networks. y\nFigure 3. Representative flowchart of the Resilience Analysis Model of ATM Systems Based on Comp y\nFigure 3. Representative flowchart of the Resilience Analysis Model of ATM Systems Based on Complex Networks. presentative flowchart of the Resilience Analysis Model of ATM Systems Based on Complex Networks. The M1 module is responsible for generating the dataset of the analysis model, consisting of the processes that generate the ATC/\nATS file (P 1.1) and the ATM flight file that can be managed (P 1.2). Process P 1.1 generates the file of control and ATS agencies,\nbased on the information obtained through research carried out in the Internet Aisweb portal (DECEA 2019b) and in Chapter 5 of\nROTAER (DECEA 2019c). The P 1.2 process aims to create the ATM flights file, which correspond to the manageable fractions of the\noriginal flights file (ANAC 2019). For each of the records in the National Civil Aviation Agency (ANAC) flights file, the shortest route\nbetween the origin and destination airports is established, which makes it possible to identify, based on the analysis of this route in the\nrespective Aeronautical Route Chart (DECEA 2019b), all ATC/ATS agencies that will be responsible for managing the entire flight. As an example, during a flight from Fortaleza International Airport (SBFZ) to Manaus International Airport (SBEG), the aircraft \nreceives ATC service from six different ATC agencies, one at a time: Fortaleza Tower (TWR-FZ), Fortaleza Approach Control \n(APP-WZ), Recife Area Control Center (ACC-RE), Amazonian Area Control Center (ACC-AZ), Manaus Approach Control (APP-WN) \nand Eduardo Gomes Tower (TWR-EG). Transfers of control occur within the limits of operational responsibility between two \nadjacent ATC agencies. In this example, processing one single record in the file of active scheduled flights (ANAC 2019)—(SBFZ, J. Aerosp. Technol. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 General Analysis The Brazilian air traffic network was modeled having as nodes the ATC/ATS agencies (146), linked by fractions of flights \n(17,347) in which ATM, control and services are effectively performed. Figure 4 visually presents the weekly distribution of \nscheduled flights in Brazil, registered with ANAC on August 12, 2019, with a focus on the ATC and service agencies that exert \nthe greatest influence on the network Brazilian airline. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX 10 Source: Elaborated by the authors. Figure 4. Brazilian ATM network: weekly distribution of scheduled flights, highlight for the most \ninfluential ATC/ATS agencies classified by the volume of managed air movements. Source: Elaborated by the authors. Source: Elaborated by the authors. re 4. Brazilian ATM network: weekly distribution of scheduled flights, highlight for the most li yl\ng\ng\ng\ninfluential ATC/ATS agencies classified by the volume of managed air movements. yl\ng\ng\ng\ninfluential ATC/ATS agencies classified by the volume of managed air movements. Table 1 summarizes the global metrics of the network, where “Connected ATC/ATS agencies” represents the ability of an \nagency to connect to other agencies in the network, with values ranging from 0% (no connection possible) to 100% (all agencies \ncan connect to each other directly or via intermediary agencies); “Average clustering coefficient” represents a measure of the \ndensity of connections between adjacent agencies; “Average managed flights” expresses the average number of flights managed by \neach of the agencies in the network; “Average shortest path” translates the average of all paths connecting an origin agency to any \ndestination agency, with the fewest passes through other intermediary agencies; “Average degree” is the result of the ratio between \nthe sum of the degrees of all agencies (the total number of direct connections, links arriving or leaving each agency) and the total \nnumber of agencies in the network; “Diameter” is defined as the longest of the shortest paths between any pair of agencies in the \nnetwork; and finally “Density”, which is defined as the ratio of the number of flights transferred by all ATC agencies to the total \nnumber of possible transfers of flights across the entire network. Table 1. Brazilian ATM network: global metrics and parameters. Table 1. Brazilian ATM network: global metrics and parameters. In a comprehensive way, the degree of network nodes follows a power law, revealing the joint existence of a few operational \nATC agencies that manage many flights and many agencies that provide ATS to a few aircraft in flight. In this way, the \nBrazilian air traffic network can be characterized as a scale-free network. Far from being just an abstract classification, J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 General Analysis As for cohesion, that is, how close to having direct connections between all its vertices, the air traffic network has a low \ndensity (1.5%), reflecting the existing hierarchy in the Brazilian airspace control system with few direct connections between \nthe ATC/ATS agencies. Regarding the dispersion of the network—with reference to the definition of unitary distance as the one that separates two \ndirectly connected vertices—the flights that most need ATM involve the participation of seven ATC/ATS agencies with six transfers \nof control (network diameter). Regarding the dispersion of the network—with reference to the definition of unitary distance as the one that separates two \ndirectly connected vertices—the flights that most need ATM involve the participation of seven ATC/ATS agencies with six transfers \nof control (network diameter). The Brazilian network is also characterized by a low level of redundancy in the connections between the control agencies, since, \naccording to the metrics obtained, the average of clustering coefficients shows that each ATC/ATS unit has only 5.3% chance of \nbeing connected to a neighboring control on this network. In practice, however, this is not entirely correct to say, as the ACCs and some of the larger APPs (e.g., São Paulo and Rio de \nJaneiro) are subdivided into operational sectors, each operating as an independent ATC agency, that is, there is internal redundancy \nin these operational units, but it is not perceived by the calculated metrics due to the structuring of the dataset used in the modeling. It is also possible to connect the TWRs directly with the ACCs, in the event of an APP failure. The general characteristics of the Brazilian air traffic network, identified in this initial analysis, can be explained by the hierarchy \nof the structure of the control agencies in spatially restricted areas, since, in a situation of operational normality, the TWRs are \nconnected to the ACCs through the APPs. In situations of operational degradation of approach control, however, a TWR can \nbe linked directly to the ACC responsible for the FIR where it operates. Regarding the AFIS, the connection with the ACCs is \ncarried out directly. This differentiation is justified by the complexity of ATC compared to the flight information service provided by AFIS. Control agencies are responsible for controlled airspaces with a high volume of air traffic, while agencies that provide ATS are \nresponsible for uncontrolled airspaces, normally with low flight traffic. General Analysis Metric / Parameter\nValue\nATC/ATS agencies connected \n100%\nAverage clustering coefficient \n5.3%\nAverage managed flights \n407 managed flights\nAverage shortest path \n3.272 flight transfers\nAverage degree \n2.171 direct connections\nDiameter \n6 flight transfers\nDensity \n1.5%\nSource: Elaborated by the authors. Table 1. Brazilian ATM network: global metrics and parameters. Metric / Parameter\nValue\nATC/ATS agencies connected \n100%\nAverage clustering coefficient \n5.3%\nAverage managed flights \n407 managed flights\nAverage shortest path \n3.272 flight transfers\nAverage degree \n2.171 direct connections\nDiameter \n6 flight transfers\nDensity \n1.5%\nSource: Elaborated by the authors. Table 1. Brazilian ATM network: global metrics and parameters. In a comprehensive way, the degree of network nodes follows a power law, revealing the joint existence of a few operational \nATC agencies that manage many flights and many agencies that provide ATS to a few aircraft in flight. In this way, the \nBrazilian air traffic network can be characterized as a scale-free network. Far from being just an abstract classification, J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Modeling Resilience of Air Traffic Management Systems Based on Complex Networks identifying the type of network resulting from modeling a real air traffic system provides relevant information to rationalize \nit and mitigate its vulnerabilities. It is known from studies by Albert et al. (2000) that scale-free networks are intensely resilient to random failures, since \nthe vast majority of nodes have a small number of connections. Removing nodes of this type, therefore, would not affect \nthe overall functionality of the network too much. In the opposite direction, however, scale-free networks are particularly \nvulnerable to attacks targeting the most central and influential nodes in the network. Sun and Wandelt (2021) reinforce these \nideas by analyzing the different impacts of attacks, concluding that targeted attacks are more harmful to ATM systems than \nrandom failures. Regarding the connectivity of the Brazilian air traffic network, the graph generated by the modeling is fully connected, \nforming a single large component (Fig. 4), showing that it is possible to reach all operational ATC agencies from any of them. However, connectivity between control agencies is low, since each ATC/ATS agency is directly linked, on average, to 2.171 of \nthem, about 1.5% of the total operational agencies. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 General Analysis Turning to the quantitative analysis of the results, the Table 2 expresses the classification of Brazilian ATC/ATS agencies in \nrelation to relevance in the national network, based on the volume of air movements managed in a period of one week, betweenness \ncentrality and proximity centrality, respectively. While betweenness centrality allows assessing the ability of a given node to present \nitself as a necessary link for the connection between groups in the network, proximity centrality helps in the perception of which \nATC/ATS agencies are responsible for the initial management of flights with the lowest number of control transfers. The two \nmetrics together show the most relevant ATC agencies. The data presented show that the São Paulo approach control (APP-XP) concentrates the largest volume of managed air \nmovements (17,662), even surpassing all ACC. This result comes from the direct connection of this APP with three of the busiest \nTWRs in Brazil: Guarulhos (TWR-GR) with 4,336 movements; Congonhas (TWR-SP) with 2,964; and Campinas (TWR-KP) \nwith 1,514 weekly movements controlled. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX 12 Table 2. The 10 most relevant ATC/ATS agencies classified by number of flights managed \nduring a week of operation, betweenness centrality and proximity centrality. Rank \nFlights managed \nBetweenness centrality \nProximity centrality\n1\n17,662 APP São Paulo\n0.5499 ACC Curitiba\n0.52 ACC Curitiba\n2\n14,911 ACC Curitiba\n0.4538 ACC Amazonian\n0.49 ACC Amazonian\n3\n13,051 ACC Brasília\n0.3339 ACC Recife\n0.48 ACC Brasília\n4\n8,428 ACC Recife\n0.3030 ACC Brasília\n0.48 ACC Recife\n5\n6,236 APP Rio de Janeiro\n0.0680 APP São Paulo\n0.41 ACC Atlantic\n6\n4,336 TWR Guarulhos\n0.0410 APP Rio de Janeiro\n0.37 APP São Paulo\n7\n3,855 ACC Amazonian\n0.0275 APP Belo Horizonte\n0.35 APP Vitória\n8\n3,824 APP Brasília\n0.0275 APP Recife\n0.35 APP Rio de Janeiro\n9\n3,004 APP Belo Horizonte\n0.0275 APP Londrina\n0.34 APP Curitiba\n10\n2,964 TWR São Paulo\n0.0275 APP Bauru\n0.34 APP Florianópolis\nSource: Elaborated by the authors. Table 2. The 10 most relevant ATC/ATS agencies classified by number of flights managed \nduring a week of operation, betweenness centrality and proximity centrality. Table 2. The 10 most relevant ATC/ATS agencies classified by number of flights managed \nduring a week of operation, betweenness centrality and proximity centrality. General Analysis Likewise, the approach control of Rio de Janeiro (APP-WJ) ranks significantly in fifth place with 6,236 controlled flights, behind \nonly the approach control of São Paulo and the Curitiba, Brasília, and Recife ACCs. With an operational structure similar to that \nused in São Paulo, the APP-WJ manages the air flow of the TWRs at Santos Dumont (TWR-RJ) and Galeão (TWR-GL) airports. The establishment of these two regions of intense movement can be understood by the correlation of air traffic with economic \nactivity in the two largest Brazilian cities in terms of population and gross domestic product, resulting in the creation of the air \nbridge between Rio de Janeiro and São Paulo, one of the busiest routes around the world. Also noteworthy are the results of the approach control and the control tower in Brasília (APP-WR and TWRBR) with, \nrespectively, 3,824 and 1,917 movements, demonstrating the relevance of the federal capital in the Brazilian air network. Also noteworthy are the results of the approach control and the control tower in Brasília (APP-WR and TWRBR) with, \nrespectively, 3,824 and 1,917 movements, demonstrating the relevance of the federal capital in the Brazilian air network. Once again, the hierarchy of the ATC structure of the Brazilian network can be used to justify the results of centrality obtained, \nsince the ACCs are responsible for liaising with the APPs and with the AFIS at isolated aerodromes in their areas of operational \nresponsibility. The APPs act in the same way, intermediating the links between TWRs and ACCs. Once again, the hierarchy of the ATC structure of the Brazilian network can be used to justify the results of centrality obtained, \nsince the ACCs are responsible for liaising with the APPs and with the AFIS at isolated aerodromes in their areas of operational \nresponsibility. The APPs act in the same way, intermediating the links between TWRs and ACCs. Thus, the results presented are coherent, classifying the ACC in the first positions, followed by the APP. The TWR and AFIS, \nin the opposite direction, have the lowest centrality values, since they are positioned at the ends of the network. Thus, the results presented are coherent, classifying the ACC in the first positions, followed by the APP. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 General Analysis ATC/ATS agencies removed\n0\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\nATC/ATS agencies disconnected\n–\n106\n49\n118\n23\n18\n24\n15\n16\n10\n4\nFlights removed\n0\n20\n265\n12\n992\n1,504 916\n1,790 1,514 2,964 7,534\nFlights on the network (%)\n100.00 99.98 99.78 99.99 99.17 98.74 99.23 98.50 98.73 97.52 93,69\nLargest comp. – Qty of nodes connected (%)\n100.00 99.32 98.63 97.95 96.58 95.89 94.52 93.15 92.47 91.78 77,40\nConnec. ATC/ATS agencies groups\n1\n2\n3\n4\n6\n7\n9\n11\n12\n13\n28\nLargest connected ATC/ATS agency – comps. 146\n145\n144\n143\n141\n140\n138\n136\n135\n134\n113\nAverage managed flights\n407\n407\n407\n407\n400\n390\n383\n371\n361\n340\n289\nAverage degree\n2.171\n2.130 2.103 2.089 2.055 2.041 2.014 1.979 1.966 1.952 1.664\nDensity\n0.015\n0.015 0.015 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.011\nDiameter\n6\n6\n6\n6\n6\n6\n6\n6\n6\n6\n6\nAverage shortest ATC/ATS path\n3.272\n3.281 3.284 3.288 3.284 3.276 3.268 3.260 3.254 3.248 3.188\nSource: Elaborated by the authors. Table 3. Resilience analysis: random failures. In the opposite direction, however, the removal of a node with a high degree of distribution or centrality—targeted attack on a \ncritical node identified, according to Sun and Wandelt (2021)—usually has a devastating effect on the operational capacity of the \nnetwork, as can be seen in Table 4, which show the results obtained by the selective removal of ATC/ATS agencies with a higher \ndegree of betweenness centrality. The interdiction of the Curitiba ACC, the ATC agency with the highest degree of centrality in \nthe network, causes an immediate loss of 31% of network operability (IRe = 0.31). It is still possible to assess the resilience of the network in situations of simultaneous unavailability of ATC/ATS units. Checking the column corresponding to ACC-AZ in Table 4, it appears that the removal of ACC-CW and ACC-AZ together \nreduces the operational capacity of the network to 44.52%, that is, resilience index (IRe = 0.55). Finally, it is proved that the Brazilian ATM network presents a behavior similar to that of scale-free networks, being strongly resistant \nto random failures, but with a relevant loss of its operational capacity in situations of attacks directed at its most relevant ATC agencies. Table 4. Resilience analysis: targeted attacks. General Analysis The TWR and AFIS, \nin the opposite direction, have the lowest centrality values, since they are positioned at the ends of the network.ii Specifically in relation to the betweenness centrality, the classification of APPs is supported by direct connections with more \nthan one TWR/AFIS in their respective areas of operational responsibility and in the volume of managed flights. Resilience Analysis Specifically in relation to the betweenness centrality, the classification of APPs is supported by direct connections with more \nthan one TWR/AFIS in their respective areas of operational responsibility and in the volume of managed flights. Specifically in relation to the betweenness centrality, the classification of APPs is supported by direct connections with more \nthan one TWR/AFIS in their respective areas of operational responsibility and in the volume of managed flights. Resilience Analysis The resilience analysis of air traffic systems is relevant due to the need to know and understand the impacts caused on the functionality \nof the network in cases of unavailability of ATC agencies, especially the ACCs, which act as intermediaries in flights from different FIRs, \nlinking the APPs and connecting isolated AFIS. Air traffic systems have occasional failures in equipment and subsystems, but they \nrarely lead to loss of global network functionality. This stability is normally attributed to the redundant links present in its structure. In general, scale-free networks are little affected when their nodes are randomly removed, that is, this type of network has \nstrong resilience to undirected node loss. In the case of the Brazilian air traffic network, such behavior can be understood by the \npredominance of ATC/ATS agencies with a low number of managed flights, so the impact on the global operability of the network \nby the loss of this type of agency is not significant. The results recorded in Table 3 prove that the random removal of ATC/ATS agencies from the Brazilian network has a reduced \neffect on its operability, because even after the simultaneous unavailability (removal) of 10, the network resilience index remained \nat IRe = 1 − 77.40 / 100 = 0.226, where 77.40 is the is the percentage number of nodes connected in the largest component after \nthe random removal of 10 ATC/ATS agencies. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Modeling Resilience of Air Traffic Management Systems Based on Complex Networks Table 3. Resilience analysis: random failures. Metric / Parameter description\nValues\nN. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 General Analysis Metric / Parameter description \nValues\nATC/ATS agency removed\nComplete Network\nACC-CW\nACC-AZ\nACC-BS\nTotal number of ATC/ATS agencies removed\n0\n1\n2\n3\nFlights removed\n0\n14,911\n3,855\n13,051\nFlights on the network (%)\n100.00\n87.51\n84.28\n73.34\nLargest component – Qty of nodes connected (%)\n100.00\n69.18\n44.52\n20.55\nConnected ATC/ATS agencies groups\n1\n30\n58\n76\nLargest connected ATC/ATS agency – comps. 146\n101\n65\n30\nAverage managed flights\n407\n307\n279\n208\nAverage degree\n2.171\n1.644\n1.205\n0.945\nDensity\n0.015\n0.011\n0.008\n0.007\nDiameter\n6\n5\n5\n4\nAverage shortest ATC/ATS path\n3.272\n3.207\n3.115\n2.451\nSource: Elaborated by the authors. Table 4. Resilience analysis: targeted attacks. Table 4. Resilience analysis: targeted attacks. Table 4. Resilience analysis: targeted attacks. Sampaio FCG, Costa Filho RN, Guterres MX J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Community Analysish The analysis of communities allows the understanding of the relationships of dependence between air traffic agencies, whether \nstructural, organizational, or even economic, when carefully observing the areas of operational responsibility where these control \nagencies provide their specialized services. It is also possible to infer knowledge about the commercial interests of airlines, through \na critical look at the concentration of flights originating in or destined for a specific control area. Figure 5 depicts the Brazilian air \ntraffic network with the ATC/ATS agencies grouped into five identified communities, of which identification process was carried \nout using the Newman–Girvan algorithm. Source: Elaborated by the authors. Source: Elaborated by the authors. Figure 5. Brazilian ATM network: ATC/ATS agencies grouped into communities, by color, Figure 5. Brazilian ATM network: ATC/ATS agencies grouped into communities, by color, l Figure 5. Brazilian ATM network: ATC/ATS agencies grouped into communities, by color, \nwith the size of the nodes proportional to the volume of flights managed. with the size of the nodes proportional to the volume of flights managed. In general, these communities reflect the influence exerted by the ACC in their respective areas of operational responsibility, \nbeing approximately equivalent to the FIR in geographic terms. Thus, the region covered by the green community, for example, \nshows the Amazonian FIR; the light blue community reflects the distribution of FIR Recife’s control agencies; the orange one \ncovers the Brasília FIR; and the purple-colored community overlaps the Curitiba FIR. The exceptions are the dark blue community—which shows the influence of São Paulo APP, with Guarulhos, Congonhas, \nCampinas and São José dos Campos control towers—and the Atlantic ACC—located in the upper flights between Brazil and \nEurope, which has connections only with Amazonian, Recife, and Curitiba ACCs, absorbed by the Recife ACC community. CONCLUSION We developed a resilience analysis model of ATM systems based on complex networks and applied it to the Brazilian ATM \nnetwork. For that, we also gathered and integrated different data sources to build the complex network representation of the \nanalyzed system and, by using metrics and analysis techniques from CNT, evaluated the characteristics and resilience level of it. We developed a resilience analysis model of ATM systems based on complex networks and applied it to the Brazilian ATM \nnetwork. For that, we also gathered and integrated different data sources to build the complex network representation of the \nanalyzed system and, by using metrics and analysis techniques from CNT, evaluated the characteristics and resilience level of it. The Brazilian air traffic network is a scale-free network, that is, there are few control agencies that manage a large number \nof flights and many agencies that provide ATS to a few aircraft. Therefore, the Brazilian network is resilient to random failures; \nhowever, it is particularly vulnerable to targeted attacks to the nodes with the highest centrality.h The Brazilian air traffic network is a scale-free network, that is, there are few control agencies that manage a large number \nof flights and many agencies that provide ATS to a few aircraft. Therefore, the Brazilian network is resilient to random failures; \nhowever, it is particularly vulnerable to targeted attacks to the nodes with the highest centrality. The graph generated by the modeling is fully connected, demonstrating that it is possible to reach all operational ATC agencies \nfrom any of them. The network is also not very cohesive, with a low density (0.015), reflecting the existence of few direct links \nbetween the ATC/ATS agencies. The average shortest path is low (3.272), meaning that few transfers of control, three to four on \naverage, are required when managing flights in Brazilian airspace. The characteristics identified in the Brazilian air traffic network \ncan be explained by the hierarchy of the structure of control agencies in spatially restricted areas and justified by the complexity \nof the ATC system. From the quantitative analysis of the air movements managed by ATC/ATS agencies, the results prove that the area control \ncenters play a prominent role in the national context, however, the approach control of São Paulo (APPXP) concentrates the \nlargest volume of air movements (17,662), surpassing all area control centers in the country. Dynamics Analysis The results of the previous analysis are based on fixed information in relation to time, not reaching the effects caused by the \nduration of the flights, their distribution throughout the day or even on different days of the week. In this sense, in order to better \nunderstand these impacts on the Brazilian air traffic system, it is necessary to investigate the network metrics referenced in days \nand times when flights occur. Table 5 consolidates the information on the air movements of the Brazilian network, during the period of one week. The data \nis totaled by day of the week, divided into 4–6-h intervals. From the data analysis, it appears that the daily cycle of ATM has a \npractically constant volume of flights from Tuesday to Friday, regarding the daily total of movements. Mondays are the busiest \ndays of the week, as opposed to Saturdays and Sundays when air activity is reduced. With regard to the daily distribution of flights, \nTable 5 allows the inference that air activity is significantly intensified in the afternoon and at night on week-days. The movements, \nespecially the low air traffic on Saturday and Sunday, are not explained by the resilience analysis model, but may be related to \nexternal factors not evaluated in this study—whether financial or operational by the airlines - exceeding the scope of this research. Modeling Resilience of Air Traffic Management Systems Based on Complex Networks 15 Table 5. Brazilian ATM network: Dynamics analysis. Day of the week\n00–06 UTC\n06–12 UTC\n12–18 UTC\n18–00 UTC\nDaily subtotal\nSunday\n1,090\n2,169\n4,297\n4,994\n12,550\nMonday\n3,874\n4,332\n6,376\n6,545\n21,126\nTuesday\n3,194\n3,563\n5,249\n5,344\n17,349\nWednesday\n3,109\n3,891\n5,924\n6,071\n18,995\nThursday\n3,135\n3,768\n5,697\n5,979\n18,579\nFriday\n3,178\n3,858\n5,914\n5,800\n18,750\nSaturday\n3,146\n2,913\n3,224\n2,739\n12,023\nSource: Elaborated by the authors. Table 5. Brazilian ATM network: Dynamics analysis. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 CONCLUSION This expressive result comes from \nthe direct connection of this APP with three control towers with significant air movement in the country: Guarulhos (TWR-GR) \nwith 4,336 movements; Congonhas (TWR-SP) with 2,964; and Campinas (TWR-KP) with 1,514 controlled weekly movements. With an operational structure similar to the one used in São Paulo, the Rio de Janeiro approach control (APPWJ), which manages \nthe airflow from the control towers at Santos Dumont (TWR-RJ) and Galeão (TWR-GL) airports, it ranks significantly in fifth place \nwith 6,236 air movements, behind only the approach control of São Paulo and the control centers of Curitiba, Brasília, and Recife. The existence of these two regions of intense flight movement, established between the terminal control areas of São Paulo \nand Rio de Janeiro, can be understood by the correlation of air traffic with economic activity in the two largest Brazilian cities \nin population and economic terms, the which results in one of the busiest routes in the world, the Rio-São Paulo aerial bridge. The analysis of communities makes it possible to understand the dependency relationships between air traffic agencies. In general, the distribution of communities in the Brazilian air traffic network reflects the influence exerted by the control centers \nin their respective areas of operational responsibility, being approximately equivalent to the FIR in geographic terms. Thus, five \nlarge communities were identified with ATC/ATS agencies distributed, grouped, along the Amazonian, Recife, Brasília, and \nCuritiba FIRs, and a fifth highlighted community, which portrays the influence of the approach control of São Paulo and of its \nbusy control towers in Guarulhos, Congonhas and Campinas. J. Aerosp. Technol. Manag., São José dos Campos, v14, e2222, 2022 Sampaio FCG, Costa Filho RN, Guterres MX\n16 Sampaio FCG, Costa Filho RN, Guterres MX Regarding the dynamic analysis of data on the daily cycle of ATM, the results show that the volume of flights has a small \nvariation from Tuesday to Friday, regarding the daily movement total. Mondays are the days with the most air traffic during the \nweek—21,126 movements—as opposed to Saturdays and Sundays, when air activity is the least. As for the variation of movements \nduring the day, the air activity intensifies considerably in the afternoon and night periods, with the highest average movement \nclose to 6,000 in the night period from Monday to Friday. CONCLUSION The practical results that may come from the analysis of air traffic systems through modeling tools that employ the CNT showed \nhigh potential to identify bottlenecks in the networks, expand operational capabilities and raise the levels of operational safety of the air \noperations. Thus, there are great opportunities for future research in this area of knowledge, and some of the following can be glimpsed: \nto model and analyze an ATM system taking into account the sectorization of airspace under the responsibility of the ACC and APP, \ntreating each sector as an independent operational ATC agency; and to structure the network nodes based on the navigation points \nof the air routes, in order to identify bottlenecks in the airways, with the purpose of rationalizing the design of the airspace structure. Studies on complex networks have developed rapidly in recent years, as shown by the significant attention of scientists from \ndifferent areas to the field of complex systems, signaling the relevance of this knowledge area for the Academy and for society. 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W3202170127.txt	https://www.mdpi.com/2073-4395/11/10/1932/pdf?version=1632650591	en		Proposal for Integrated Management of Verticillium Wilt Disease in Avocado Cultivar Hass Crops	Agronomy	2,021	cc-by	10,192	agronomy Article Proposal for Integrated Management of Verticillium Wilt Disease in Avocado Cultivar Hass Crops Joaquín Guillermo Ramírez-Gil 1, * 1 2 * Citation: Ramírez-Gil, J.G.; Morales-Osorio, J.G. Proposal for Integrated Management of Verticillium Wilt Disease in Avocado Cultivar Hass Crops. Agronomy 2021, 11, 1932. https://doi.org/10.3390/ agronomy11101932 and Juan Gonzalo Morales-Osorio 2 Departamento de Agronomía, Facultad de Ciencias Agrarias, Universidad Nacional de Colombia sede Bogotá, Carrera 45 No 26-85, Edificio 500, Oficina 228, Bogotá 111321, Colombia Departamento de Ciencias Agronómicas, Facultad de Ciencias Agrarias, Universidad Nacional de Colombia sede Medellín, Carrera 65 No 59A-110, Bloque 11, Oficina 117-13, Núcleo El Volador, Medellín 050034, Colombia; jgmoraleso@unal.edu.co Correspondence: jgramireg@unal.edu.co Abstract: The area planted with avocado crops in Colombia has been growing rapidly in recent years, especially for export varieties such as Hass. The increase in planted area coincided with increased phytosanitary problems, where pathogens such as fungi of the genus Verticillium spp. are becoming of economic importance. The objective of this study was to evaluate different control strategies for avocado wilt disease caused by Verticillium spp., under in vitro, net house, and field conditions. Strategies tested included fungicides (benomyl, azoxystrobin, captan, and carbendazim), beneficial and antagonistic microorganisms (Trichoderma sp., and Rhizoglomus fasciculatum), and physical and cultural practices such as solarization, drainage and removal of diseased tissues. Treatments T7fi (pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage) and T8fi (fungicidepruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage), showed the greatest reduction in the area under disease progress curve and Verticillium dahliae inoculum in soil and plant tissues under field conditions. Fruit with extra quality increased 120.8% with T7fi and 108% with T8fi, compared to the control with diseased trees. The highest costs were identified for T7fi and T8fi; however, these treatments also showed the best cost/benefit relationship. Integrated approaches as in T7fi and T8fi showed the best results for Verticillium wilt control. As no fungicides of chemical synthesis are included in T7fi (pruning-solarisation-Trichoderma-mycorrhiza-sucrose-organic matterdrainage), it should be preferred to T8fi, which does include them, to avoid their negative impacts on avocado production. Academic Editors: Dora Batista and Marco Cristancho Received: 31 August 2021 Keywords: Verticillium wilt; integrated disease management; beneficial microorganism; cultural practice; disease reduction; Persea americana Accepted: 20 September 2021 Published: 26 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 1. Introduction The genus of fungi Verticillium Nees comprises a cosmopolitan group of Ascomycetes, within which are classified devastating plant pathogen species that cause vascular wilt and plant death in a number of crops [1,2]. Verticillium spp. may survive in the environment for long periods of time by resistant structures and present a wide range of host plants [1–5]. Arguably, Verticillium albo-atrum Reinke and Beerthold, and Verticillium dahliae Kleb., are the species in this genus that cause the largest losses in several crops worldwide [2,6]. Control of plant diseases caused by Verticillium spp. are a challenge because infections start through the roots, making it difficult to perform detection of this causal agent and limiting the effect of most fungicides. In addition, inoculum persists for long time periods under field conditions, the pathogen exhibits a wide host range and there is a lack of resistant varieties for most crops [7–10]. Most control measures of diseases caused by Verticillium spp. are based on application of synthetic chemicals to the soil, which usually increase costs, may induce resistance in pathogen populations, show toxicity or affect the 4.0/). Agronomy 2021, 11, 1932. https://doi.org/10.3390/agronomy11101932 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 1932 2 of 16 ozone layer as methyl bromide, and present other adverse effects on the environment and human health [1,2,5]. V. albo-atrum and V. dahliae have been reported causing wilt disease on Persea americana Mill. in several countries around the world [8,9,11,12]. There are examples of integrated disease management programs for Verticillium wilt in crops such as tomato, olive, and many others [1,13]. However, information for avocado is scarce, and most integrated management proposals have been focused in the root rot disease caused by the oomycete Phytophthora cinnamomi Rands [9,14]. In general, it has been reported that pruning and other cultural practices, selection of Mexican rather than Guatemalan tolerant rootstocks, to avoid planting avocado crops on lands previously used for other crops susceptible to Verticillium spp. and to prevent the use of affected trees as a source of budwood or seeds, are agronomical practices recommended to reduce Verticillium wilt in avocado crops [15–17]. However, despite its high incidence and wide distribution, no efficient management practices are readily available [12,18]. Efficient programs for integrated disease management usually include several strategies of pathogen control. Beneficial microorganisms have proven useful for agriculture around the world. Mycorrhizas and Trichoderma spp. have been extensively studied for various aspects useful in crop production, such as fertilization, plant growth promotion and adaptation to abiotic and biotic stresses [19,20]. There are several reports about the effect of biological control on Verticillium spp., but in general, the results are not conclusive about the effective reduction of disease incidence and severity and on yield increase in crop fields [1,6,21,22]. Other practices reported as successful in reducing Verticillium spp. inoculum in avocado and others hosts are heat treatments [10], fungicide applications, plant resistance inducers, peroxygen-based disinfectants [7,13], use of manures or compost of different origins [23–26], combination of biological and chemical control measures [27], and tolerant rootstocks with different levels to avocado disease response [15,16]. In recent publications, the concept of “next-generation green composts” is proposed, which relates to the use of multiple sources of agricultural residues with great potential in the biocontrol and suppressive effect on soilborne plant pathogenic fungi such as V. dahliae [25,26]. The area growing avocados in Colombia expanded 212% during the decade 2008–2018 [28]. Comparing the period from January to June of the year 2019 with the year 2020, the area growing avocado cv. Hass for export increased 34%, with a change in value from 52 to 72 million USD of FOB price [29]. Currently, Colombia is third in area grown and fourth in avocado fruit production in the world [30]. Avocado wilt caused by Verticillium spp. has gained growing importance in Colombia during the last years up to reaching second place in diseases of biotic origin after the root rot wilt caused by P. cinnamomi [12,18]. Verticillium wilt has been detected in Colombia in the departments of Antioquia, Caldas, Cundinamarca, Risaralda, Quindío and Valle del Cauca [31]. However, studies about basic biology, epidemiology, incidence, severity, prevention, diagnosis, integrated management and economic losses of this disease are scarce [17]. As most available management strategies of avocado wilt diseases are focused on root rot caused by P. cinnamomi, but little is known about practices for other pathogens such as Verticillium spp., the present work aimed to test the in vitro activity of antagonists against this fungal pathogen and to evaluate different strategies as tools for integrated management of the avocado wilt disease caused by Verticillium spp. at net-house and field conditions. Integrated approaches, including pruning-solarization-Trichodermamycorrhiza-sucrose-organic matter-drainage, showed the best results for Verticillium wilt control, and also showed the best cost/benefit relationship. 2. Materials and Methods 2.1. Experimental Site The present work was carried out between the years 2011–2014. Net-house average environmental conditions were temperature of 18–24 ◦ C, relative humidity of 75–95% and photosynthetically active radiation (PAR) of 650–1920 µmol photons m−2 s−1 . Research Agronomy 2021, 11, 1932 3 of 16 under field conditions was performed in two plots planted with avocado cv. Hass trees of 6 years old at a distance of 7 × 7 m. Seedlings of the genotype West Indian of high altitude were used as rootstock. As farmers use plants from sexual seeds instead of a clonal rootstock, variability may be present in avocado fields, including sometimes plants of cv. Hass. Plots were located in the Department of Antioquia, Colombia in the municipalities of La Ceja, in the eastern region (5.95931 latitude–75.41777 longitude, 2387 masl), and Donmatias (6.496961 latitude–75.412118 longitude, 2213 masl) in the Northern region. Plots localization corresponded to the ecological life zone of lower montane very humid forest (vhf-LM) sensu Holdridge. Edaphoclimatic details are shown in Supplementary Material (Table S1 and Figure S1A). 2.2. Plant Material for Evaluation under Net-House Conditions Avocado seeds of cv. Hass were collected from a healthy tree and disinfested; then, a pre-germination treatment was applied, and they were sown in autoclaved quartz. When seedlings exhibited five fully expanded leaves and the secondary root system was well developed, cotyledons were excised to promote root growth. Seedlings were then transferred to plastic pots containing 2 kg of soil (Andisol from the municipality of El Peñol, Antioquia, Colombia, autoclaved at 0.1 MPa and 121 ◦ C for 2 cycles of 1 h each). Details of soil parameters are shown in supporting information 1. Avocado seedlings were kept under net-house conditions at 50% of the maximum soil moisture retention capacity until further use. 2.3. Microorganism Isolation and Identification V. dahliae strains were obtained from the collection kept at “Universidad Nacional de Colombia branch Medellín” (Vert1, Vert2, Vert3, and Vert4), which were morphologically and molecularly identified [12]. V. dahliae strains were used because they were the prevalent species under field conditions. Trichoderma sp. TAF22, TC2 and TC3, morphotypes were isolated from the rhizosphere of healthy and well developed avocado trees [16] Rhizoglomus fasciculatum (Thaxt.) C. Walker and A. Schüßler (syn. of Rh. fasciculatum (Thaxt.) Sieverd., G.A. Silva and Oehl.) mycorrhizal fungal strain was kindly provided by professor Dr. Walter Osorio from the microorganisms bank at Laboratorio of Microbiología Ambiental del Suelo, from “Universidad Nacional de Colombia branch Medellín”. 2.4. In Vitro Assays In vitro assays were performed to determine the inhibitory capacity of fungicides of chemical synthesis and Trichoderma sp. Morphotypes. An r-mycelium plug taken from the first 5 mm of the edge of an actively growing V. dahliae strain in sterile potato dextrose agar (PDA) medium culture (PDA, Difco, Lawrence, KS, USA) was transferred to fresh PDA in a side of a Petri dish, and on the other side, a plug of 5 mm2 taken from an actively growing Trichoderma sp. strain in PDA was placed. The doses of fungicides under in vitro condition were designed based on previous tests associated with the use of different molecules to control of Verticillium species. Fungicides of chemical synthesis tested were Benomyl (0.05% of 500 g of active ingredient (a.i.) kg−1 , Dupont, Bogotá, Colombia) [32], Azoxystrobin (40 mL L −1 of 23.2 a.i. kg−1 , Syngenta, Bogotá, Colombia) [13], Captan (10 mg L−1 of 500 g a.i. kg−1 , Adama, Barranquilla, Colombia), and Carbendazim (20 mL L−1 of 500 g a.i. kg−1 , Vecol, Bogotá, Colombia) [33]. Tests were developed on PDA, supplemented with the different fungicides. In each treatment, a plug of mycelium of 5 mm2 taken from an actively growing V. dahliae strain in PDA was transferred to fresh PDA amended with each corresponding fungicide. Radial growth rate was measured as the response variable of the V. dahliae strain tested. It was measured daily for 20 days using a caliper (Mitutoyo Digimatic Caliper, Sendai, Japan). Experiments were repeated twice through time. Agronomy 2021, 11, 1932 4 of 16 2.5. Net-House Tests 2.5.1. Pathogenicity Tests on Avocado cv. Hass Plants V. dahliae strains (Vert1, Vert2, Vert3, and Vert4), were grown on PDA medium as described before. Conidia were collected in sterile distilled water and concentration adjusted to 1 × 105 conidia mL−1 . Then, 100 mL of conidia suspension were added, distributed in four equidistant points to each pot containing an avocado plant. Soil humidity was increased to 70% of the maximum retention capacity to favor disease development. Aggressiveness of each isolate was determined as the area under disease progress curve (AUDPC) [34] using a specific disease scale developed and calibrated for Verticillium spp. infecting avocado in Colombia [35]. 2.5.2. Effect of Fungicides, Solarization, and Inoculation with Beneficial Microorganism (Trichoderma sp. and R. fasciculatum) on V. dahliae inoculum) Two experiments were designed with the objective to know the effect on soil infected with V. dahliae. of the addition of fungicides and solarization. First, 100 mL of a suspension of 1 × 105 conidia × mL−1 of V. dahliae (Vert 2) were added to each pot containing 2 kg of soil. Then, avocado plants were planted into inoculated pots and were maintained until the plants died to produce inoculum. Afterward, 500 mL of a solution with each fungicide, diluted in sterile distilled water at doses described previously, were applied into the infested soil, and distributed through time in three applications at days 5, 10, and 15. For solarization, a layer of 10 cm thick soil was covered with plastic film (transparent, caliber 4) for 40 days. In addition, temperature during solarization was measured using a digital sensor coupled with communication and storage of information, assembled in an Arduino Uno plate [36]. Solarized soil was then used to fill the pots for performing the experiments. Each experimental unit consisted of a pot with 2 kg of soil, three experimental units were used per treatment in a completely randomized experimental design. All experiments were repeated twice through time. In each experiment, quantification of V. dahliae inoculum was performed by serial dilutions in sterile distilled water obtained from 15 random samples of 10 g of soil each, to recover structures of V. dahliae (Vert 2) that were quantified in PDA medium acidified with lactic acid (PDA-A) supplemented with streptomycin (100 µg L−1 ). Inoculum quantification was performed every five days after treatment application and during 40 days. Based on previous tests, a new experiment was proposed to be implemented for plants under net-house conditions. Treatments were (where “nh” stands for net-house conditions): a non-inoculated control of healthy plants (nh—healthy control); a control of plants inoculated with V. dahliae (Vert 2) (nh—diseased control); T1nh (fungicide): Verticilliuminoculated plants + benomyl applied in drench; T2nh (Trichoderma): Verticillium-inoculated plants + inoculation with Trichoderma sp. TC2; T3nh (mycorrhiza): Verticillium-inoculated plants + inoculation with R. fasciculatum; T4nh (fungicide-Trichoderma-mycorrhiza): Verticillium-inoculated plants + simultaneous application of T1nh + T2nh + T3nh; and T5nh (Trichoderma-mycorrhiza): Verticillium-inoculated plants + simultaneous application of T2nh + T3nh. In each treatment, V. dahliae inoculum were added to the soil as described before, and Trichoderma sp. and R. fasciculatum were applied following the method described in [14]. Fungicide was diluted in sterile distilled water at the dose recommended by the manufacturer and 200 mL of fungicide solution × plant−1 were applied directly to the soil when plants showed the degree one of disease scale developed for Verticillium spp. [35]. Aall treatments under net-house conditions may be observed in Supplementary Material Table S2. Disease severity was measured weekly during 120 days using the aforementioned scale of severity [35], and with the obtained data, the AUDPC was calculated for each treatment [34]. At the end of the experiment, V. dahliae inoculum were quantified in plant tissues and soil. For plant tissues, 15 random samples of 10 g of root and stem pieces were processed according to assay reported to this microorganisms [12,36]. Briefly, tissues were macerated and serial dilutions were prepared in sterile distilled water. Dilutions Agronomy 2021, 11, 1932 5 of 16 were placed and dispersed in Petri dishes containing PDA media, incubated, and colony forming units (UFC) were quantified. The procedure described before was followed for quantification of V. dahliae in soil. Total inocula of V. dahliae were considered as the sum of inocula from soil plus inocula from plant tissues. Mycorrhizal colonization was calculated using the line interception method [37]. Avocado roots were collected and washed with tap water, then were bleached in KOH (10%) for 24 h [38], followed by alkaline immersion (0.5% NH4 OH and 0.5% H2 O2 v/v, in aqueous solution) for 30 min. Mycorrhizal hyphae were visualized by staining with trypan blue (0.025%) [39]. Additionally, to verify the effect of fungicides on Trichoderma sp. and R. fasciculatum populations, an experiment was developed in pots using the same procedure described before. Plants of Leucaena leucocephala Lam, which is an indicator of mycorrhization, were germinated on sterile quartz until three fully developed leaves were observed and then were transferred to previously prepared pots. R. fasciculatum and Trichoderma sp. were inoculated as described before. Tested fungicides were applied at each dose at 10 and 30 days after microbe inoculation into the soil. Mycorrhizal colonization and quantification of AVM propagules and Trichoderma sp. CFUs, were evaluated 40 days after the inoculation of microorganisms following procedures already described. Pots containing plants of L. leucocephala inoculated with both microorganisms were used as a negative control. All results may be observed in Figure S2. 2.6. Assays under Field Conditions Based on data obtained from experiments performed under in vitro and net-house conditions described before, the following treatments were evaluated under field conditions (where “fi” stands for field conditions): fi—healthy control: non-inoculated control of healthy plants; fi—diseased control: plants infected with V. dahliae; T1fi (fungicide): Verticillium-infected plants + application of fungicide in drench (3000 mL of benomyl solution (0.05% of 500 g a.i. kg−1 )) in rotation with azoxystrobin (3000 mL of 40 mL L−1 , 23.2 a.i.), sprayed around a diameter of 2 m from the base of the stem of the avocado trees; T2fi (pruning): Verticillium-infected plants + sanitary pruning (trimming all symptomatic and necrosed tissues); T3fi (solarization): Verticillium-infected plants + solarization covering a diameter of two meters around the base of the stem with a plastic film (caliber # 4); T4fi (Trichoderma-mycorrhiza-sucrose): Verticillium-infected plants + soil inoculation with the following mixture: Trichoderma sp. morphotype TC2 (1000 mL of a solution in sterile distilled water at a concentration of 1 × 105 conidia per plant) plus R. fasciculatum (1 kg of roots and soil with R. fasciculatum, prepared from mycorrhiza-associated maize plants, adjusted at a concentration of 45 propagules per g of soil), and the addition of 1000 g of sucrose diluted in sterile distilled water (1:1 p:v) as a source of energy [40,41]; T5fi (organic matter): Verticillium-infected plants + application of 10 kg of compost in a diameter of two meters around of the base of the stem, made with bovine manure, mineral amendment (P2 O5 , 13%; CaO, 25%; MgO, 7%; S, 10%; SiO2 , 6%) and a layer of 15 cm thick of homogenized plant residues and mushroom residues (1:1) [14]; T6fi (drainage): Verticillium-infected plants + a drainage network around the base of the stem consisting of a crescent-shape ditch in the upper zone of the slope (10 cm wide and 10 cm depth) and a drain in the lower zone of the slope (20 cm length, 10 cm wide and 30 cm depth); T7fi (pruning- solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + integrated management proposal without fungicide consisting of simultaneous application of T2fi + T3fi + T4fi + T5fi + T6fi; and T8fi (fungicidepruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + integrated management proposal with fungicide consisting of simultaneous application of T1fi + T2fi + T3fi + T4fi + T5fi + T6fi. A scheme summarizing drainage treatment under field conditions may be observed in Figure S3. Treatments were applied in 7 year old plants showing symptoms corresponding to scale 2 of wilt disease under field conditions, caused by V. dahliae [35]. V. dahliae was Agronomy 2021, 11, 1932 6 of 16 verified using a polyphasic approach, which included plant symptomatology, morphology and molecular characterization [12]. Treatments for soil solarization and drainage were performed at the initial stage of the experiment. Soil solarization was applied during 40 days. Fungicides were applied when plants showed symptoms corresponding to a value above 2 of the scale of the wilt disease. In T1fi, three applications of fungicide were performed. For T8fi, one application of fungicide was made before solarization and inoculation with R. fasciculatum and Trichoderma sp. Incorporation of compost, mineral amendment and plant and mushroom residues were performed every six months for two years. For pruning of affected plants, permanent monitoring for symptomatic and necrosed tissues was carried out. Benefic microorganisms and the energy source were applied at the beginning of the experiment and one year later. In treatments in which microorganism application was combined with solarization, microbes were applied after soil solarization. Disease severity was measured, and the AUDPC calculated every two months for two years [34]. At the end of the experiment, inoculum of Trichoderma sp., mycorrhizal colonization and inoculum of V. dahliae were quantified following procedures described above. During the whole period of evaluation, the accumulated fruit production, the percentage of fruit of extra quality and the unitary cost of each treatment were determined. Using these parameters, the cost/benefit ratio was calculated [14]. All treatments under field conditions may be observed in Table S3. 2.7. Experimental Design and Statistical Analyses An in vitro experimental unit was a Petri dish, 10 replicates were performed per treatment and the experiments were repeated twice through time using a completely randomized experimental design. At net-house, three plants were an experimental unit, five replicates were performed per each treatment and the experiments were repeated twice through time using a completely randomized experimental design. Under field conditions, three 7 year old plants were used as an experimental unit, with three replicates per treatment and using a randomized block experimental design (consisting of each plot), where the primary factor were the treatments and the secondary factor was the terrain slope classified in three arbitrary categories (0–10, 10.1–25 and >25.1). Under Colombian conditions there are two harvesting seasons per year; therefore, each year tested corresponded to two harvesting seasons for a total of four harvesting seasons. Data homoscedasticity and normality were determined using the Levene and Kolmogorov– Smirnov tests, respectively. Means were compared by analysis of variance (ANOVA) followed by the Tukey test (p ≤ 0.05). All statistical analyses were performed using the computational package R. 3. Results 3.1. In Vitro Assays All fungicides tested inhibited more than 95% the growth of V. dahliae (p < 0.05) (Figure 1A). Similarly, but to a lesser extent, all isolates of Trichoderma sp. significantly reduced the growth of all V. dahliae isolates tested (p < 0.05), when compared to the control (Figure 1B). Isolate TC2 showed the best effect in decreasing the growth of V. dahliae isolates Vert1 (47.7%), Vert2 (44.5%), Vert3 (55.1%) and Vert4 (41.19%) (p < 0.05) (Figure 1B). my 2021, 11,Agronomy x FOR PEER REVIEW 2021, 11, 1932 7 of 17 7 of 16 Figureof1.different Effect oftreatments different treatments in vitroofgrowth of Verticillium dahliae. (A): of differFigure 1. Effect on in vitroongrowth Verticillium dahliae. (A): effect ofeffect different chemical fungient chemical fungicides on growth of Verticillium dahliae. (B): effect of different strains of Trichoderma cides on growth of Verticillium dahliae. (B): effect of different strains of Trichoderma sp., on growth of Verticillium dahliae. sp., on growth Verticillium dahliae. Vert1-4 = code strain of Verticillium dahliae.ofTAF22, TC2 and Vert1–4 = code of strain of of Verticillium dahliae. TAF22, TC2 and of TC3 = code of morphotypes Trichoderma sp. Error bars TC3 = code of morphotypes of Trichoderma sp. Error bars represent the confidence interval of the represent the confidence interval of the mean, validated by Tukey mean separation test. Equal letters indicate that there are mean, validated by Tukey mean separation test. Equal letters indicate that there are no significant no significant differences (p > 0.05). differences (p > 0.05). 3.2. Net-House Assays 3.2. Net-House Assays Isolate Vert2 showed the highest aggressiveness of all V. dahliae isolates (p < 0.05), Isolate Vert2 showed the highest aggressiveness of all V. dahliae isolates (p < 0.05), exhibiting AUDPC values higher than isolates Vert3 (19.6%) and Vert1 (24.4%) (Figure 2A). exhibiting AUDPC values higher than isolates Vert3 (19.6%) and Vert1 (24.4%) (Figure All fungicides evaluated and solarization decreased V. dahliae inoculum in soil, compared to 2A). All fungicides evaluated and solarization decreased V. dahliae inoculum in soil, comthe control (p < 0.05); however, none completely eradicated this fungus. Benomyl showed pared to the control (p < 0.05); however, none completely eradicated this fungus. Benomyl the best effect followed by Azoxystrobin, Carbendazim and Captan (p < 0.05) (Figure 2B). showed the best effect followed by Azoxystrobin, Carbendazim and Captan (p < 0.05) (FigThe effect of solarization at 5, 10 and 15 days after application of treatments, was inferior to ure 2B). The effect of solarization at 5, 10 and 15 days after application of treatments, was reduction observed for fungicides (p < 0.05); nevertheless, for a longer period of time (i.e., inferior to reduction observed for fungicides (P < 0.05); nevertheless, for a longer period >15 days), solarization exhibited a higher reduction of V. dahliae inoculum than Azoxysof time (i.e., >15 days), solarizationorexhibited a higher reduction of V. inoculum trobin, Carbendazim Captan, and similar to Benomyl (p >dahliae 0.05) (Figure 2B). It is apparent than Azoxystrobin, Carbendazim or Captan, and similar to Benomyl (p > 0.05) (Figure 2B). that under solarization, fluctuations of V. dahliae inoculum through time, coincided with It is apparent that under solarization, fluctuations of V. dahliae inoculum through fluctuations in the maximum temperature, with lower quantities of time, inoculum observed coincided withwhen fluctuations in the maximum temperature, with lower quantities of ◦ the temperature was above 35 C (Figure 2B and Figure S1B). inoculum observed when temperature was abovereduced 35 °C (Figure 2B and S1B2B). Allthe treatments significantly the AUDPC caused by V. dahliae (p < 0.05). Benomyl, when applied in drench alone (T1nh) or in combination with R. fasciculatum and Trichoderma sp. (T4nh), showed the best reduction of AUDPC values: 65.5% (T1nh) and 68% (T4nh), respectively, compared to the control inoculated with V. dahliae. T5nh (R. fasciculatum plus Trichoderma sp.,), T2nh (Trichoderma sp.,) and T3nh (R. fasciculatum), exhibited reductions in the AUDPC values of 51.4, 44.5 and 30.62%, respectively (Figure 3A). V. dahliae inoculum levels measured for each treatment coincided with AUDPC values (p < 0.05). For T1nh, T4nh, T5nh and T2nh, reduction in AUDPC values of 55.1, 51.7, 41.3 and 24.1%, respectively, was quantified compared to the control inoculated with V. dahliae. For T3nh, no significant differences were observed when compared to the control inoculated with V. dahliae (p > 0.05) (Figure 3B). Agronomy 2021, 2021, 11, 11, 1932 x FOR PEER REVIEW Agronomy of 16 17 88 of Figure 2. 2. AUDPC and inoculum inoculum quantification for management practices of Verticillium dahliae in Hass Hass avocado avocado under under Figure net-house conditions. (A): evaluation of strain aggressiveness of V. dahliae on Hass avocado. (B): effect of chemical funginet-house conditions. (A): evaluation of strain aggressiveness of V. dahliae on Hass avocado. (B): effect of chemical fungicide Agronomy x FOR PEER cide2021, and11, solarization onREVIEW soil inoculum of V. dahliae. bars represent the confidence interval of the mean, validated by9 of 17 and solarization on soil inoculum of V. dahliae. Error Error bars represent the confidence interval of the mean, validated by Tukey Tukey mean separation test. Equal letters indicate that there are no significant differences (p > 0.05). mean separation test. Equal letters indicate that there are no significant differences (p > 0.05). All treatments significantly reduced the AUDPC caused by V. dahliae (p < 0.05). Benomyl, when applied in drench alone (T1nh) or in combination with R. fasciculatum and Trichoderma sp. (T4nh), showed the best reduction of AUDPC values: 65.5% (T1nh) and 68% (T4nh), respectively, compared to the control inoculated with V. dahliae. T5nh (R. fasciculatum plus Trichoderma sp.,), T2nh (Trichoderma sp.,) and T3nh (R. fasciculatum), exhibited reductions in the AUDPC values of 51.4, 44.5 and 30.62%, respectively (Figure 3A). V. dahliae inoculum levels measured for each treatment coincided with AUDPC values (p < 0.05). For T1nh, T4nh, T5nh and T2nh, reduction in AUDPC values of 55.1, 51.7, 41.3 and 24.1%, respectively, was quantified compared to the control inoculated with V. dahliae. For T3nh, no significant differences were observed when compared to the control inoculated with V. dahliae (p > 0.05) (Figure 3B). Figure Figure 3. 3. Effect Effect of of management management practices practices of of Verticillium Verticillium dahliae dahliae in in Hass Hass avocado avocado under under net-house net-house conditions conditions on on beneficial beneficial population population of of soil soil microorganism microorganism and and reduction reduction of of AUDPC. AUDPC. (A): (A): area area under under disease disease progress progress curve curve (AUDPC) (AUDPC) of of wilt wilt disease under different treatments. (B): mycorrhizal colonization and inoculum of Trichoderma sp. quantified under differdisease under different treatments. (B): mycorrhizal colonization and inoculum of Trichoderma sp. quantified under different ent treatments for wilt disease. bars represent the confidence interval of thevalidated mean, validated bymean Tukey mean sepatreatments for wilt disease. ErrorError bars represent the confidence interval of the mean, by Tukey separation test. ration test. Equal letters indicate that there are no significant differences (p > 0.05). Treatments were (where “nh” stands Equal letters indicate that there are no significant differences (p > 0.05). Treatments were (where “nh” stands for net-house for net-house conditions): a non-inoculated control of healthy plants (nh—healthy control); a control of plants inoculated conditions): a non-inoculated control of healthy plants (nh—healthy control); a control of plants inoculated with V. dahliae with V. dahliae (Vert 2) (nh—diseased control); T1nh (fungicide): Verticillium-inoculated plants + benomyl; T2nh (Tricho(Vert 2) (nh—diseased control); plants T1nh (fungicide): Verticillium-inoculated benomyl; T2nh (Trichoderma): Verticilliumderma): Verticillium-inoculated + inoculation with Trichoderma sp.;plants T3nh + (mycorrhiza): Verticillium-inoculated plants plants with Trichoderma sp.; T3nh (mycorrhiza): Verticillium-inoculated plantsplants + inoculation with +inoculated inoculation with+R.inoculation fasciculatum; T4nh (fungicide-Trichoderma-mycorrhiza): Verticillium-inoculated + simultaneR. fasciculatum; (fungicide-Trichoderma-mycorrhiza): Verticillium-inoculated plants + simultaneous of ous application T4nh of T1nh+T2nh+T3nh; and T5nh (Trichoderma-mycorrhiza): Verticillium-inoculated plants + application simultaneous application of+T2nh+T3nh. T1nh + T2nh T3nh; and T5nh (Trichoderma-mycorrhiza): Verticillium-inoculated plants + simultaneous application of T2nh + T3nh. As expected, Trichoderma sp. and mycorrhizal colonization values were higher (p < 0.05) in the treatments where individual application of these microorganisms was performed (T2nh and T3nh, respectively). In contrast, combined application of these microorganisms with a fungicide (T4nh) exhibited reduction of these variables 47.8% and 52.7%, respectively, when compared to T2nh (Figure S2). Similarly, application of Trichoderma sp. and mycorrhiza without fungicide (T5nh), reduced values of these variables 34.4% and Agronomy 2021, 11, 1932 9 of 16 As expected, Trichoderma sp. and mycorrhizal colonization values were higher (p < 0.05) in the treatments where individual application of these microorganisms was performed (T2nh and T3nh, respectively). In contrast, combined application of these microorganisms with a fungicide (T4nh) exhibited reduction of these variables 47.8% and 52.7%, respectively, when compared to T2nh (Figure S2). Similarly, application of Trichoderma sp. and mycorrhiza without fungicide (T5nh), reduced values of these variables 34.4% and 23%, respectively, compared to values obtained in T3nh (Figure 3B). 3.3. Assays under Field Conditions V. dahliae caused plant death when no control measures were applied as evidenced in the diseased control treatment, confirming pathogenicity of isolates. A similar effect was observed in the treatment where only drainages were made (T6fi). In the other treatments, no plant death was observed and their effects were classified into two groups. In the first group, composed by T1fi (fungicides Benomyl and Azoxystrobin) and integrated management proposals without (T7fi) or with fungicides (T8nh), disease severity was always below a value of 3 in the scale used (Figure 4A). In the second group, composed by treatments T5fi (mineral amendment, bovine manure and plant and mushroom residues), T4fi (Trichoderma sp., R. fasciculatum and energy source), T3fi (solarization) and T2fi (pruning), disease severity was between values of 3 and 4 according to the scale used (Figure 4A). All treatments evaluated, with the exception of drainage alone (T6fi), showed significant reduction of AUDPC and V. dahliae inoculum in soil and plant tissues (p < 0.05), compared to the diseased control. Integrated management proposals (T7fi and T8fi) showed the greatest reduction in AUDPC and V. dahliae inoculum in soil and plant tissues, followed by treatments T1fi, T5fi, T2fi, T3fi and T4fi, which reduced the AUDPC value by 80.3, 79.1, 62.3, 50.3, 42.2, 35.7 and 27.7%, respectively; and reduced V. dahliae inoculum by 76.9, 69.2, 56.4, 53.8, 48.7, 51.28 and 41.0%, respectively (Figure 4B). Accumulated production of fruit and the amount of fruit classified in quality “extra”, significantly varied for all treatments applied (p > 0.05). As expected, the highest values for these variables were found in the healthy control, and the lowest values were observed in the inoculated and not treated control (p < 0.05). Diseased plants showed a reduction in fruit production of 54.2% and a reduction in the extra quality of fruit of 59.6%, when compared to healthy plants. In the integrated management treatments (T7fi and T8fi), fruit production increased 26.9% (T7fi) and 25.7% (T8fi), and fruit with extra quality increased 120.8% (T7fi) and 108% by (T8fi), compared to the control with diseased trees. In treatments T1fi, T5fi, T2fi, T3fi and T4fi, avocado trees increased fruit production in 20.1, 15.9, 14.5, 12.3 and 9.9%, respectively, and increased the percentage of fruit classified as extra quality in 82.4, 64, 71.2, 52 and 36%, respectively, compared to the diseased control (Figure 5A). Similar results to those observed in the net-house assays for mycorrhizal colonization and the amount of inoculum of Trichoderma sp., were observed in field conditions (p < 0.05) (Figure 5B). The highest costs among all treatments tested for V. dahliae management were identified for the integrated management proposals T7fi and T8fi; however, these treatments also showed the best cost/benefit relationship (Table 1). Likewise, in the evaluated treatments, there were large differences in production and losses per year (Table 1). In percentage terms, the treatment with infected plants (Control +) presented a reduction of 212.9% with respect to the non-diseased control (Control-), contrasting with the integrated management proposal (T8fi), with a reduction of 18%. Agronomy 2021, 11, 1932 10 of 16 Table 1. Costs associated with Verticillium dahliae in avocado cv. Hass crops in Antioquia department, Colombia. Treatment Control+ T1fi f T2fi T3fi T4fi T5fi T6fi T7fi T8fi Control− Cost of Production of 1 kg c Yield and Reduction Variable Unit Cost for Tree/Two Years a,b Fixed a,b Variable a,b Yield (kg/tree) Reduction (%) d 0.0 5.2 2.5 4.5 4.2 6.5 2.2 19.9 25.1 0.0 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.32 0.65 0.50 0.45 0.40 0.42 0.40 0.90 0.95 0.32 20.9 39.5 28.2 25.0 28.1 31.4 25.9 50.3 55.4 65.4 212.9 65.5 131.9 161.6 132.7 108.2 152.5 30.0 18.0 na Cost/Benefit Ratio Cost/Benefit Ratio as % Respect to Control e 0.95 3.30 2.00 1.50 1.65 2.2 1 4.5 4.7 na 1 347 210 157 175 231 1.05 473 494 na a Variable cost associated with the management treatment of diseased plants infected with Verticillium dahliae. b Value given in constant USD for 23 December of 2014 with an exchange rate of COP 2392.4 per USD 1. c The absorption costing method was used for calculating the cost of each treatment. d The production reduction was determined based on the yield of each treatment compared to the control—(healthy plants). e The cost/benefit ratio method was applied to calculate farm income related to each treatment applied, quantified by dividing the cost/benefit ratio of each treatment by the cost/benefit ratio of T0, multiplied by 100. f fi stands for treatments under field conditions. The following treatments were evaluated under field conditions (where “fi” stands for field conditions): fi—healthy control: non-inoculated control of Agronomy 2021, 11, x FOR PEER REVIEW 10 of 17 healthy plants; fi—diseased control: plants infected with V. dahliae; T1fi (fungicide): Verticillium-infected plants + application of fungicide in drench (benomyl in rotation with azoxystrobin); T2fi (pruning): Verticillium-infected plants + sanitary pruning; T3fi (solarization): Verticilliuminfected plants + solarization; T4fi (Trichoderma-mycorrhiza-sucrose): Verticillium-infected plants + soil inoculation with Trichoderma sp. and R. fasciculatum; T5fi (organic matter): Verticillium-infected plants +inapplication compost; T6fi (drainage): + afoldrainage the greatest reduction AUDPC of and V. dahliae inoculumVerticillium-infected in soil and plant plants tissues, network around the base of the stem; T7fi (pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected lowed by treatments T1fi, T5fi, T2fi, T3fi and T4fi, which reduced the AUDPC value by plants + integrated management proposal without fungicide consisting of simultaneous application of T2fi + T3fi + T4fi + T5fi + T6fi; and T8fi 80.3, 79.1, 62.3, 50.3, 42.2, 35.7 and 27.7%, respectively; and reduced V.+dahliae inoculum (fungicide-pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants integrated management 76.9, 69.2,application 56.4, 53.8, of 48.7, 41.0%, proposal with fungicide consisting of by simultaneous T1fi51.28 + T2fiand + T3fi + T4firespectively + T5fi + T6fi.(Figure 4B). Area under disease progress curve (AUDPC), and inoculum of Verticillium dahliae under management practices Figure 4.Figure Area 4. under disease progress curve (AUDPC), and inoculum of Verticillium dahliae under management practices of of disease on field conditions. (A): Dynamics of the severity of V. dahliae under different treatments. (B): AUDPC of V. disease ondahliae field under conditions. (A):treatments. DynamicsError of the severity of V. underinterval different treatments. (B): AUDPC of V. dahliae under different bars represent thedahliae confidence of the mean, validated by Tukey mean test. Equal letters indicatethe that there are no significant differences (p > 0.05). by TheTukey following treatments weretest. Equal different separation treatments. Error bars represent confidence interval of the mean, validated mean separation evaluated conditions (where “fi” stands fieldThe conditions): fi—healthy control: control letters indicate thatunder there field are no significant differences (p >for 0.05). following treatments werenon-inoculated evaluated under fieldofconditions healthy plants; fi-diseased control: plants infected with V. dahliae; T1fi (fungicide): Verticillium-infected plants + application (where “fi” stands for conditions): non-inoculated control of healthy plants; fi-diseased control: plants of fungicide in field drench (benomyl in fi—healthy rotation withcontrol: azoxystrobin); T2fi (pruning): Verticillium-infected plants + sanitary pruninfected with V. dahliae; T1fi (fungicide): Verticillium-infected plantsT4fi + application of fungicide in drench (benomyl in rotation ing; T3fi (solarization): Verticillium-infected plants + solarization; (Trichoderma-mycorrhiza-sucrose): Verticillium-infected plantsT2fi + soil inoculation with Trichoderma sp. plants and R. + fasciculatum; T5fi (organic Verticillium-infected plants with azoxystrobin); (pruning): Verticillium-infected sanitary pruning; T3fimatter): (solarization): Verticillium-infected plants + application of compost; T6fi (drainage): Verticillium-infected plants + a drainage network around the base of the stem; + solarization; T4fi (Trichoderma-mycorrhiza-sucrose): Verticillium-infected plants + soil inoculation with Trichoderma sp. and T7fi (pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + inteR. fasciculatum; (organicproposal matter):without Verticillium-infected plants applicationapplication of compost; T6fi (drainage): Verticillium-infected grated T5fi management fungicide consisting of +simultaneous of T2fi+T3fi+T4fi+T5fi+T6fi; and (fungicide-pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + aT8fi drainage network around the base of the stem; T7fi (pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic plants + integrated management proposal with fungicide consisting of simultaneous application of matter-drainage): Verticillium-infected plants + integrated management proposal without fungicide consisting of simultaneous T1fi+T2fi+T3fi+T4fi+T5fi+T6fi. application of T2fi + T3fi + T4fi + T5fi + T6fi; and T8fi (fungicide-pruning-solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infectedAccumulated plants + integrated management with consisting of simultaneous production of fruit andproposal the amount offungicide fruit classified in quality “extra”, significantly varied for all treatments applied (p > 0.05). As expected, the highest values application of T1fi + T2fi + T3fi + T4fi + T5fi + T6fi. for these variables were found in the healthy control, and the lowest values were observed in the inoculated and not treated control (p < 0.05). Diseased plants showed a reduction in fruit production of 54.2% and a reduction in the extra quality of fruit of 59.6%, when com- Agronomy 2021, x FOR PEER REVIEW Agronomy 2021, 11, 11, 1932 11 of1117of 16 Figure 5. Quantity andquality qualityofoffruit fruitproduction, production, mycorrhizal and in management Figure 5. Quantity and mycorrhizalcolonization, colonization,Trichoderma Trichodermasp. sp.inoculum inoculum and in management practices dahliae underfield fieldconditions. conditions.(A): (A): Production Production and crops under different treatments practices of of V. V. dahliae under andquality qualityofofHass Hassavocado avocado crops under different treatments for V. dahliae. (B): mycorrhizal colonization and inoculum of Trichoderma sp. quantified under different treatments of wilt for V. dahliae. (B): mycorrhizal colonization and inoculum of Trichoderma sp. quantified under different treatments of wilt disease. Error bars represent the confidence interval of the mean, validated by Tukey mean separation test. Equal letters disease. Error bars represent the confidence interval of the mean, validated by Tukey mean separation test. Equal letters indicate that there are no significant differences (p > 0.05). The following treatments were evaluated under field conditions indicate that arefor nofield significant differences (p > control: 0.05). The following treatments evaluated under field control: conditions (where “fi”there stands conditions): fi—healthy non-inoculated control ofwere healthy plants; fi—diseased (where “fi” standswith for field conditions): fi—healthy control: non-inoculated of healthy plants; control: plants infected V. dahliae; T1fi (fungicide): Verticillium-infected plants + control application of fungicide infi—diseased drench (benomyl in rotation T2fi (pruning):Verticillium-infected Verticillium-infected plants plants + + application sanitary pruning; T3fi (solarization): Verticil- in plants infectedwith withazoxystrobin); V. dahliae; T1fi (fungicide): of fungicide in drench (benomyl lium-infected plants + solarization; T4fi (Trichoderma-mycorrhiza-sucrose): Verticillium-infected + soil inoculation rotation with azoxystrobin); T2fi (pruning): Verticillium-infected plants + sanitary pruning; T3fi plants (solarization): Verticilliumwith Trichoderma sp. and R. fasciculatum; T5fi (organic matter): Verticillium-infected plants + application of compost; T6fi infected plants + solarization; T4fi (Trichoderma-mycorrhiza-sucrose): Verticillium-infected plants + soil inoculation with (drainage): Verticillium-infected plants + a drainage network around the base of the stem; T7fi (pruning-solarizationTrichoderma sp. and R. fasciculatum; T5fi (organic matter): Verticillium-infected plantsplants + application of compost; T6fi (drainage): Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected + integrated management proVerticillium-infected plants + a drainage network around the base of the stem; T7fi (pruning-solarization-Trichodermaposal without fungicide consisting of simultaneous application of T2fi+T3fi+T4fi+T5fi+T6fi; and T8fi (fungicide-pruningmycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + integrated management proposal without solarization-Trichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + integrated management proposal with consisting of simultaneous application T1fi+T2fi+T3fi+T4fi+T5fi+T6fi. fungicide consisting offungicide simultaneous application of T2fi + T3fi + T4fi +ofT5fi + T6fi; and T8fi (fungicide-pruning-solarizationTrichoderma-mycorrhiza-sucrose-organic matter-drainage): Verticillium-infected plants + integrated management proposal The highest costs among treatments tested for +V.T6fi. dahliae management were idenwith fungicide consisting of simultaneous application of T1fi all + T2fi + T3fi + T4fi + T5fi tified for the integrated management proposals T7fi and T8fi; however, these treatments showed the best cost/benefit relationship (Table 1). Likewise, in the evaluated treat4.also Discussion ments, there were large differences in production and losses per year (Table 1). In percentAdequate programs of integrated management for Verticillium wilt in avocado are age terms, the treatment with infected plants (Control +) presented a reduction of 212.9% scarce and not readily available [9,15]. Here, we identified treatments for avocado wilt with respect to the non-diseased control (Control-), contrasting with the integrated mancaused by proposal V. dahliae,(T8fi), which reduced disease, the percentage of the high value “exagement with a reduction of increased 18%. tra” fruit category and showed a good cost/benefit relationship (Table 1). Integrated disease management proposals T7fi (sanitary pruning, solarization, Trichoderma sp., R. fasciculatum, a source of energy in the form of sucrose, compost, and drainage without fungicides) and T8fi (same as T7fi, but including benomyl and azoxystrobin fungicides in rotation), consisting of combined strategies, exhibited the best effect in controlling the wilt disease caused by V. dahliae in avocado crops (Figures 4 and 5, and Table 1). Individual control methods as fungicide applications, application of one biocontroller, addition of organic matter or other amendments, usually reduce some level of disease. However, when all those strategies are combined they can act in synergy reducing or suppressing disease development to an effective threshold for farmers. Each strategy has its own advantages and constraints (Figures 4 and 5). Fungicide effectiveness in wilt control have been shown in other crops such as Capsicum annuum L. cv. Soroksari, where benomyl application reduced Verticillium wilt in 88.2 and 94.6% when applied after and before infection with the pathogen, respectively [32]. A negative side effect of benomyl application observed in the present work was the reduction of other microorganisms that may be beneficial for avocado crops such as Agronomy 2021, 11, 1932 12 of 16 Trichoderma sp. and R. fasciculatum (Figure S2). Similarly, the total number of fungi in soil was reduced after several applications of metalxyl + mancozeb, for controlling P. cinnamomi in avocado crops [16,17]. In addition, continuous application of chemical fungicides may select resistant isolates in the pathogen population, causing ineffectiveness of the molecule, as has been widely reported for benomyl [42]. Therefore, the use of chemical fungicides may be useful as part of an integrated disease management program, but caution should be taken to prevent aforementioned problems. As T7fi was effective to manage Verticillium wilt disease, it should be preferred to T8fi because no fungicides are included in T7fi (Figures 4 and 5). The use of microorganisms such as Trichoderma sp. and mycorrhizas for the biological control of diseases caused by Verticillium sp. have proven useful in several crops [21,22]. In addition, mycorrhizas such as R. fasciculatum, have shown positive effects, improving the nutritional status of the host plant, particularly the phosphorus uptake [19,20]. However, despite the large amount of literature available about biological control agents against Verticillium, few products effective at the field-level have been developed [1]. An apparent antagonism between Trichoderma sp., and R. fasciculatum was identified in the present work. Antagonism between Trichoderma spp. and mycorrhizas has been observed previously in many plants, including avocado [43,44]. To lessen this problem, the addition of an energy source readily available such as sucrose has been reported as having a positive effect in reducing such antagonism [40]. Further research is required to solve this challenge. Pruning may help in controlling disease by eliminating inoculum sources and by stimulating regrowth of new disease-free tissues as observed in our research. Besides, solarization reduced the inoculum amount in an apparent temperature-dependent manner (Figure S1B). The temperature for controlling effectively Verticillium sp. has been reported to be above 42 ◦ C [45]. This finding suggests that it is important to closely monitor temperature during the solarization process and to measure viable pathogen populations, looking for an adequate control of V. dahliae inoculum in soil, before plant establishment in nursery or in field conditions. Solarization and heat treatments have been tested in the management of Verticillium with encouraging results [5,10]; therefore, their effects should not be neglected as a component of an integrated management of Verticillium wilt. Although the drainage itself did not show a clear effect on disease management, its implementation in avocado crops should not be ignored because plant roots are sensitive to oxygen reduction and the water to air ratio and availability of oxygen significantly influences growth and mineral nutrition of avocado plants [46]. In general, it is widely accepted that hypoxia and anoxia are of high importance as abiotic causal agents of the avocado wilt disease complex and the oxygen level in soil plays an important role in the dynamics of populations of pathogenic/beneficial microorganisms in the soil profile [16,17]. Specifically, severity of root rot caused by P. cinnamomi is increased under soil flooding/low oxygen conditions [17]. However, it was observed that viable Verticillium microsclerotia was reduced in soil under flooding conditions and severity of Verticillium disease was increased under non-flooding conditions compared to flooding conditions [47], suggesting an additional role of other factors such as edaphoclimatic conditions and the plant genotype on the infection outcome. Therefore, as avocado production may be significantly affected by poor soil oxygen availability, much more research is needed to make accurate conclusions about the relationship between avocado, soil oxygen levels and management of Verticillium wilt. Climate change is expected to strongly influence plant diseases; therefore, further research using precise measurements of available contents of water and oxygen using different drainage methods and edaphoclimatic conditions is a necessary step towards clarification of the role of air–water ratio in the Verticillium avocado wilt disease. Organic matter is crucial for good soil health, and its beneficial effects for avocado and others crops have been widely reported [6,14,48]. Some studied effects include increase in beneficial microbe populations, reduction of pathogenic microbe populations and disease incidence and severity, in addition to reduction of the stress caused by climate change and many others [6,14,47]. Currently, the addition of green compost from agricultural Agronomy 2021, 11, 1932 13 of 16 and industrial waste is reported as one of the most viable economic and environmental alternatives for increasing the beneficial microbiota in soils [25,26]. The addition of different sources of organic matter reduced disease severity caused by Verticillium spp. in several crops [23–26,48]. It has been shown that the addition of green compost to soils with high inoculum pressure of soilborne pathogens generates a suppression effect, becoming an excellent strategy to incorporate into an integrated plant management, which include fungi genus Verticillium [25,26]. However, organic amendments significantly varied in type, source, chemical components, crop yield, disease control and many other factors that may affect results [1,25,26]. Even more, it has been found that in some cases, organic matter reduce Phytophthora root rot, and in others may even increase disease severity [14,49]. Organic amendments are a valuable tool for Verticillium wilt control, but time consuming and expensive efforts on research should be made to find appropriate combinations for each specific planting sites and edaphoclimatic conditions where avocado orchards are grown. However, they significantly reward the effort, as observed in the present research. Each individual control strategy has shown some extent of effective reduction of Verticilium wilt in several crop systems [1]. Integrated management systems, which include several strategies have been implemented for crops as potato, olive, lettuce, cotton and others [1,2,5]. In avocado, research about integrated management of Verticillium is limited compared to other pathogens such as P. cinnamomi [14,31]. The integrated management proposed in the present research proved to be effective in the conditions performed; however, results may vary from location to location due to factors already mentioned, so it is important to make local adaptations to each specific condition because the benefit may exceed the investment on research. Other emerging strategies for Verticillium control as new sources of plant resistance, disinfectants, heat treatments, ways of biological control delivery, and others, may be implemented as they are developed, together with our proposal of integrated management of Verticillium wilt [7,10,23]. Integrated management practices proposed in T7fi and T8fi are economically viable, showing a good cost/benefit relationship, where the increase in the production of fruit in the extra quality category plays an important role because of its high price in the market that compensates and overcomes a lower net production. These results indicate that implementation of an appropriate integrated management for this disease may increase the profit of the avocado plantation. As mentioned above, T7fi should be preferred to T8fi where possible because T7fi does not include fungicides of chemical synthesis. Even more, a similar approach has proved to be effective for the control of P. cinnamomi [14], suggesting that it is possible to design a combined strategy against the two most important pathogens causing avocado wilt. 5. Conclusions The avocado wilt disease caused by V. dahliae is economically important in Colombian conditions. Integrated management may reduce the negative effects and economic losses caused by this pathogen by increasing the quality of fruits that get the higher prices. No single control method for Verticillium is completely effective, so implementation of several compatible strategies adapted to each local condition, together with a careful economic analysis, should be performed to satisfactorily manage this disease while keeping reasonable profits. The present work is an important contribution to the avocado industry because wilt symptoms caused by V. dahliae are easily confused with those caused by other causal agents, inducing erroneous diagnostics and implementation of ineffective control methods that may induce an increase in costs of production and even larger losses. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11101932/s1, Table S1: Edaphic variables of the soil in the crop plot and laboratory tests, Table S2: Description of experiments under net-house conditions, Table S3: Supporting information 65. Description of experiments under field conditions, Figure S1: (A): climatic conditions in the plots evaluated during the years 2012–2014. (B): dynamics of temperature associated with Agronomy 2021, 11, 1932 14 of 16 solarization under net-house and field conditions, Figure S2: Effect of application of fungicides to soil in microbial beneficial populations, Figure S3: Scheme of drainage treatment under field conditions. Author Contributions: Conceptualization, J.G.R.-G. and J.G.M.-O.; formal analysis, J.G.R.-G.; investigation, J.G.R.-G. and J.G.M.-O.; writing—original draft preparation, J.G.M.-O. and J.G.R.-G.; writing—review and editing, J.G.R.-G. and J.G.M.-O.; supervision, J.G.R.-G. and J.G.M.-O. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable, our study did not involve humans or animals. Informed Consent Statement: Not applicable, our study did not involve humans. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments: To “Universidad Nacional de Colombia sede Medellín” that provided partial funding, and Colciencias that provided the Ph. D. scholarship funding of J.G.R.-M. We are grateful with avocado producers for valuable information and help during research, Guillermo Noreña in North region, and Fernando Gomez in East region of Antioquia, Colombia. All authors have approved the final article. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Conflicts of Interest: The authors declare no conflict of interest. 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